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6
Stationary Phases and Their
Performance
O U T L I N E
6.1. Solid Supports for Stationary Phases 192 Silica Covered with a Bonded Active
General Comments 192 Polymer 225
Silica Support for Stationary Phases 193
6.3. Properties of Stationary Phases and
Organic/Inorganic Silica Supports 201
Columns 226
Coated or Immobilized Polymeric
General Comments 226
Stationary Phases on Silica 203
Packing of the Stationary Phase in the
Hydride-based Silica 203
Column and Flow Direction 226
Other Inorganic Support Materials 204
Physical Properties of the Stationary Phase 227
Inorganic Monolithic Supports
Physical Dimensions of the
(Silica-based Monoliths) 206
Chromatographic Column 227
Organic Polymers Used as Support
Types of Stationary Phases and USP
for Stationary Phases 207
Classification 228
6.2. Reactions Used for Obtaining Active Characterization of the Polarity of a
Groups of Stationary Phases 211 Column Using Kow for Models of
General Comments 211 Stationary Phase 235
Derivatizations to Generate Bonded Silica- Performance of Stationary Phases
based Stationary Phases 211 and Columns 237
Direct Synthesis of Silica Materials with an Selection of a Column for an HPLC
Active Bonded Phase Surface 220 Separation 242
Derivatization of Silica Hydride Supports 221 The Use of Guard Columns and
Derivatization of Presynthesized Organic Cartridges 244
Polymers 222 Column Protection, Cleaning,
Synthesis of Organic Polymers with Active Regeneration, and Storing 245
Groups 223 Selection of Columns for Orthogonal
Synthesis of Organic Polymeric Monoliths Separations 246
with Active Functionalities 224 Physicochemical Characterization
of Stationary Phases 247
6.4. Hydrophobic Stationary Phases and 6.6. Stationary Phases and Columns
Columns 249 for Ion-Exchange, Ion-Moderated,
Types of Hydrophobic Stationary Phases 249 and Ligand-Exchange
End-capping of Free Silanols 250 Chromatography 318
Preparation of Polar-Embedded General Aspects 318
Hydrophobic Phases 253 Silica-based Ion Exchangers 319
Physical Characteristics of Hydrophobic Synthesis of Organic Polymeric Ion
Stationary Phases 254 Exchangers 320
Retention and Separation Properties Latex-Agglomerated Ion Exchangers 325
of Hydrophobic Stationary Phases 255 Commercial Polymeric Stationary Phases
Methylene Selectivity 262 Used in Ion-Exchange HPLC 326
Hydrophobic Subtraction Model 264 Ion-Moderated Phases 326
Other Tests for Comparing Hydrophobic Ligand-Exchange and Immobilized Metal
Columns 279 Affinity Phases 330
Other Properties of Reversed-Phase
6.7. Stationary Phases and Columns for
Columns Critical for Separation 281
Chiral Chromatography 331
Common Hydrophobic Stationary Phases 285
General Comments 331
Hydrophobic Stationary Phases with Some
Procedures for the Synthesis of Phases
Polarity 289
with Chiral Properties 331
Other Types of Hydrophobic Phases 293
Main Types of Stationary Phases Used
Organic Polymer-based Hydrophobic
in Chiral HPLC 336
Stationary Phases 296
Special Hydrophobic Stationary Phases 296 6.8. Stationary Phases and Columns for
Selection of a Column for RP-HPLC Size-Exclusion Chromatography 344
Separations 299 General Comments 344
Silica-based SEC Phases and Glass
6.5. Polar Stationary Phases and Columns 300
Phases 346
General Aspects 300
Polymeric-based SEC Phases 346
Specific Procedures for Polar-Phase
Synthesis 301 6.9. Stationary Phases and Columns in
Retention and Separation Properties Immunoaffinity Chromatography 350
of Polar Stationary Phases 304 General Aspects 350
Bare Silica Stationary Phase 311 Synthesis of Stationary Phases Used
HILIC Stationary Phases with a Bonded in Affinity and Immunoaffinity
Surface 312 Chromatography 350
Silica Hydride-based Phases 316
ortosilicic acid (H4SiO4), disilicic acid (H2Si2O5), obtain silica gels varies considerably, depending
and pyrosilicic acid (H6Si2O7). All these acids on the intended use of a specific material [2].
are very weak acids (e.g., for ortosilicic acid, The reactions for silicic acid generation can be
pK1 9.84, pK2 13.2). In more concentrated written as follows:
solutions, polysilicic acids with the general
formula [SiOx(OH)4-2x]n are rapidly formed Na2 SiO3 HX H2 O / 2NaX
from the condensation reaction between the sila-
H4 SiO4 or SiOH4 or H2 SiO3 H2 O
nol groups that give rise to tridimensional struc-
tures (precipitates in gel form) containing (6.1.1)
siloxane (Si-O-Si) groups. The reaction of forma-
tion of silicic acid may start with acidic hydro- SiOR4 4 H2 O H / SiOH4
lysis of salts such as Na2SiO3, Na4SiO4, and (6.1.2)
4 ROH H
K4SiO4, or with the hydrolysis in the presence
of an acid or a base (as catalysts) of several
silicon compounds such as SiH4, SiCl4, or of SiOR4 4 H2 O B: /SiOH4 4 ROH B:
alkoxysilanes (Si(OR)4 where R CH3, C2H5, (6.1.3)
C3H7, etc.). Tetraethyl orthosilicate Si(OC2H5)4,
for example, is a common alkoxysilane used The formation of polysilicic acids from the
for the preparation of silica gels. The choice of initial silicic acid can be described by the (ideal-
the reactions and of the conditions used to ized) reactions 6.1.4:
OH HO OH OH OH
2 - H2O HO OH schematized in
Si OH Si O Si HO Si O Si OH
planar form as
HO
OH HO OH OH OH
OH OH
+ HO Si OH + HO Si OH
OH OH OH OH OH
OH OH
HO Si O Si OH HO Si O Si O Si OH .....
- H2O - H 2O
OH OH OH OH OH
H O H O H O
H OH H OH OH H OH
H O
O Si OH HO Si O Si O Si O
H
OH O OH O OH OH
HO Si O Si O Si O Si O H (6.1.4)
H OH O OH O OH H O
H O O Si OH HO Si O Si O
H OH H OH OH H
H O H O O H
6.1. SOLID SUPPORTS FOR STATIONARY PHASES 195
When the polysilicic acid is obtained by Growth of sol particles
hydrolysis of alkoxysilanes, part of the alkoxy
groups may remain attached to the silicon
Growth of reticulation
atom and further participate in the elimination
reaction to form siloxane bonds.
After their formation, the initial smaller pol-
ysilicic acids are still present in solution form
(sol form), and as suggested in reaction 6.1.4,
a large number of water molecules are bound
through hydrogen bonds or mechanically
retained in the structure. As the molecules
increase in size, the sol particles are growing FIGURE 6.1.1 Growth of sol particles and of reticulation
and further changing in a system containing from the initial small units of polysilicic acid.
both a liquid phase and a solid phase with
a morphology ranging from discrete particles
to a continuous polymeric network filled with syneresis or pore narrowing (the shrinkage of
a large number of pores of different dimen- the gel network with the elimination of liquid
sions. This material generates a gel (hydrogel from the pores), and coursing (the dissolution
or alcogel depending on the groups still left of small particles and the growth of larger
unreacted) where most of the structures are ones).
crosslinked. The distinction between a material Following the gelling and ageing process, the
in sol form and that of a gel is not precise, and hydrogel (or alcogel) is typically washed, and it
as the amount of water in the structure is dried (converted into a xerogel) for obtaining
decreases and the polymeric network increases, a solid material. A silica gel, as first precipitated
the gel form becomes more obvious. The from water, may contain up to 300 moles of H2O
control of the size of sol particles and the elim- to 1 mole of SiO2. The drying process consists of
ination of water with the increase in the cross- three stages. In the first stage the gel loses water,
linking of the polymeric structure can be and its volume decreases with the volume of
achieved by selecting the concentration of water that was evaporated. In this stage, the
reagents that form the polysilicic acid and pore volume decreases, and the stiffness of the
also by controlling other parameters such as dried gel increases. In stage two, the liquid
temperature, pH, gelling time, addition of elec- from the pores of the gel is eliminated by migra-
trolytes for flocculation, and addition of deter- tion to the surface of the material where it is
gents. The process is very complex, and as evaporated. In stage three, the liquid escapes
a function of selected parameters the growth from the pores directly by evaporation. When
of sol particles or of the reticulation can be dried below 150 C, surfaces containing a large
favored, such that the properties of the final number of silanol groups (^Si-OH) are main-
material may be different. This process is sche- tained. When heated at 300 to 1000 C, the
matically suggested in Figure 6.1.1 (see also silanol-covered surfaces dehydroxylate to form
[3]). When the gel continues to be kept in the siloxane (Si-O-Si) surfaces. The final properties
initial liquid, its structure changes in a process of the dried material, such as density (porosity),
known as ageing. Ageing affects the properties hardness, active surface area, pore volume,
of the final material by several mechanisms and pore-size distribution, depend on a consid-
known as polycondensation (further reactions erable number of parameters such as reagents
of silanol groups to form siloxane bonds), choice and concentration, electrolyte addition,
196 6. STATIONARY PHASES AND THEIR PERFORMANCE
Type B (common
Property Type A (fine pore) in HPLC) Type C (large pore)
* Note: Some confusion in the nomenclature of silicas may be encountered, when the terms Type A and Type B are related to silica purity and not to pore
size. Related to purity, Type A refers to less pure material while Type B refers to highly purified homogenous spherical silica.
A and Type B is also used for the caharacteriza- affect the retention process, usually causing
tion of purity, it is preferable to use other terms peak tailing in HPLC, owing to complexation
for the pore-size characterization. For example, with the functional groups from analyte mole-
Davisil silica is characterized by Grade, which cules. The acidity of the surface silanols may
describes both pore size and particle size as indi- also be increased in the presence of these metal
cated in Table 6.1.2 (all these silicas are of high impurities. For this reason, very high purity
purity). silica is desired. More recently, procedures for
The silica materials may be contaminated purification of silica have been developed, and
with traces of metallic ions such as Al3, Fe3 a silica that has a less acidic surface, lower metal
and Ni2, depending on the synthesis of the content, and a more homogeneous distribution
silica gel or the manufacturing process. These of silanol groups was developed. It leads by
metallic ions may be present either in the form derivatization to a denser and more reproduc-
of isolated oxides and hydrated oxides in silica ible bonding of the attached groups and is
or connected through oxygen bonds attached frequently utilized for the manufacturing of
to a Si atom. These metallic ion impurities may stationary phases. This type of silica is also
termed Type B (not to be confused with Type
B related to pore size) to differentiate it from
TABLE 6.1.2 Classification of several high purity silicas the lower purity silica that has higher metal
using Grade description. content and more acidity that is termed Type
A silica (a term not related to pore size).
Grade (Davisil)
Pore size A Particle size mesh Besides the chemical properties of the surface,
633 60 200e425 its physical properties are also extremely impor-
tant in HPLC. These physical characteristics
635 60 60e100
include: (1) particle surface area, (2) pore size,
636 60 35e60 (3) pore volume, (4) pore size distribution, (5)
643 150 200e425 particle shape, (6) particle size, (7) particle size
distribution, and (8) structural rigidity. These
646 150 35e60
properties play a major role in the retention
12 22 28e200 process of the stationary phase, even after
62 150 60e200 surface derivatization. For example, one param-
eter used for the characterization of column
923 30 100e200
retention is the phase ratio J that characterizes
198 6. STATIONARY PHASES AND THEIR PERFORMANCE
the volume of immobilized liquid on the solid particles and also of particles with similar sizes
support surface (the definition of J is given by [6]. By these procedures the desired particle
rel. 2.1.14). Phase ratio J depends on the total dimensions can be obtained. One example is
pore volume and is indirectly related to particle the production of microamorphous silica gels
surface area, smaller pores leading to larger made of particles with diameters less than
surface area. Although a large surface area 1 mm. These xerogels have very high surface
obtained when the pores are small is desirable, areas, usually greater than 300 m2/g.
smaller pores are useful up to a point since too The physical parameters of a stationary phase
small pores may block some larger molecules are related mainly to the extent of eddy diffusion
to enter them and therefore may not allow the in the chromatographic process. Eddy diffusion
derivatization process inside these pores or do is determined by the difference in the path
not allow larger molecules to interact with the length taken by individual molecules of the
surface. Although the lowest pore size may same species when they migrate across the
increase the phase ratio J, a small pore size porous solid-phase material. The difference in
lower than about four times the hydrodynamic the path length and therefore the eddy diffusion
diameter of the molecule (diameter of a sphere can be significantly higher when a wider range
that diffuses at the same rate as the molecule) of long and short paths is possible. This is the
can also hinder the diffusion and leads to lower case when the particles are of different sizes or
N than expected for the calculated particle when the particle shape is irregular. Figure 6.1.3
surface. For this reason, for the separation of ana- suggests this difference in length of the path for
lytes with molecules having the molecular an irregular particle shape, a spherical particle,
weight up to about 10,000 Dalton, the pore size and a spherical smaller particle. For reducing
typically used is between 4 and 20 nm, leading eddy diffusion, considerable technological effort
to surface areas between about 300 m2/g up to was involved in generating particles with
about 600 m2/g (the weight refers to the final a controlled size and shape [7, 8].
stationary phase). For the separation of proteins A reduction in the differences in potential
and of other polymers with the molecular weight path length of molecules of the same species
above 10,000 Dalton, the typical pore size is when they go across a stationary-phase particle
about 30 nm or larger. can be achieved by using uniformly shaped
Also of considerable interest for HPLC use is smaller particles, as suggested in Figure 6.1.3.
the control of the xerogel particle size distribu- The first step in achieving the goal of reduced
tion and shape. Several technologies have been eddy diffusion was the use of spherical particles
developed for controlling these parameters, instead of particles of irregular shape, with all
mainly with the purpose of production of spher- particles having (about) the same diameter. The
ical particles in situ. This can be achieved by: 1) other step was the use of particles of smaller
the choice of the reagents used to be hydrolyzed, diameter. Spherical particles with 3 to 5 mm
2) choice of the medium of hydrolysis (use of diameter are common for many analytical
ammonia as a morphological catalyst), 3) control columns. On the other hand, the required pres-
of the way the gel particles are growing [4], 4) the sure to pass the mobile phase increases for
use of seeding (procedure also used for the columns filled with small particles. In the past,
production of core-shell [5]). Another procedure when the pressure generated by the pumps
for determining the size distribution and shape was more limited, particles with a diameter
of particles is the use of a special spray-drying around 5 mm were common, and the backpres-
procedure that leads to the formation of spher- sure due to the column was not higher than
ical particles rather than irregular-shaped about 240 bar (3500 psi), this depending on the
6.1. SOLID SUPPORTS FOR STATIONARY PHASES 199
Molecule path 1
Molecule path 1
Molecule path 1
Molecule path 2
Molecule path 2 Molecule path 2
FIGURE 6.1.3 Three particles, one with an irregular shape, one spherical, and one spherical but smaller. The potential
differences in the molecular path for each shape are also suggested.
solvent, column length, and column diameter. In particles or even pellicular particles. Superfi-
modern HPLC, the tendency is to reduce the cially porous particles (core-shell) have a solid
particle size (3 mm, 2.1 mm, or even lower); these nonporous core surrounded by a porous outer
columns require higher working pressure up to shell. For particles of about 3 mm diameter, the
600 bar (9000 psi) or even higher. The decrease porous shell may have 0.3e0.4 mm in depth. Pel-
in particle size is also related to other properties licular particles are solid spheres covered with
of the column such as column lifetime. Particle a thin layer of stationary phase. The shape and
size influences the interstitial volume, which is structure of fully porous particles, core-shell
the space between particles. Interstitial volume ones, and pellicular particles are schematically
also contributes to the peak broadening in shown in Figure 6.1.5. The hypothetical paths
HPLC. This volume is typically around 70% of of two molecules in a fully porous particle,
the total internal volume of the column. core-shell particle, and a non-porous particle
Columns with particles around 2 mm and are also shown in the same figure. The differ-
lower may generate pressures up to 15,000 psi, ence in path is shorter in core-shell particles,
and typically they are used with lower flow and the path lengths are very close to each other
rates and narrow columns [9]. Several SEM for pellicular particles [10].
pictures at different magnifications of the parti- Since the interaction between the molecules
cles used in a C18 column with 5 mm diameter in the mobile phase with the stationary phase
particles are shown in Figure 6.1.4. takes place at the active surface of the particles,
The same effect of paths with less variable it is expected that porous particles will have the
length as achieved with small porous particles largest available surface (large J values for the
can be achieved using superficially porous same other characteristics), while the nonporous
200 6. STATIONARY PHASES AND THEIR PERFORMANCE
FIGURE 6.1.4 SEM pictures at different magnifications of 5 mm diameter particles used in a C18 column (the scale
100 mm, 50 mm, 10 mm and 5 mm is indicated on pictures).
6.1. SOLID SUPPORTS FOR STATIONARY PHASES 201
Molecule path 1
Molecule path 1 Molecule path 1
FIGURE 6.1.5 Schematic aspect of fully porous, core-shell, and pellicular (non porous) particles. Hypothetical paths of
two molecules in each particle type showing length differences are also illustrated.
ones will have a very low available surface 8,000 and 15,000 psi (depending also on the
(small J and resulting lower capacity particle dimension). As smaller particles are
factors k). On the other hand, the fully porous used for making stationary phases, higher pres-
particles will have the largest eddy diffusion. sures of the mobile phase must be used for
Pellicular particles will show very little eddy performing the chromatography. When the
diffusion, but the low J may not be sufficient particle strength is exceeded, these may
for some separations. The core-shell particles mechanically collapse and this affects the
may be the optimum choice between fully column performance. Organic polymeric phases
porous and pellicular ones. Typical core-shell have a lower structural rigidity as compared
particles have similar standard diameters as with silica; they also swell and shrink depending
porous particles (e.g., 5 mm, 3 mm, 2.7 mm, and on the mobile-phase solvents, these being consid-
around 2 mm). The pore size for the porous shell erable disadvantages for the polymeric phases.
of core-shell particles is chosen similar to that
for porous particles, and the resulting surface
Organic/Inorganic Silica Supports
areas are typically around 150 m2/g.
Structural rigidity is another important The hydrolytic instability of pure silica-based
parameter of the stationary phase. Silica-based supports, even after a derivatization that covers
particles typically offer a good structural rigidity the surface with desired groups (ligands)
(particle strength), standing values between such as long hydrocarbon chains, represents
202 6. STATIONARY PHASES AND THEIR PERFORMANCE
chemical reaction for this process is the crystallographic and amorphous forms and
following: possesses both acidic and basic properties
(amphoter properties). Some physical properties
O O O
(mechanical stability, high surface area, control
O Si OH OC2H5 O Si O Si H of average pore diameter and of particle diam-
O + H5C2O Si H O O eter, swelling capability) as well as chemical
- C2H5OH properties (chemical stability over a large pH
O Si OH OC2H5 O Si O Si H
range 1e14) recommend this compound as
O O O
substrate for stationary phases in HPLC. ZrO2
can be prepared by precipitation from zirconium
(6.1.6)
salts, zirconyl salts (ZrOX2), or zirconium alkox-
Hydride-based silica can also be obtained by ides. The method of precipitation, pH, tempera-
different reactions such as reduction of a mate- ture, and the other parameters discussed at the
rial containing chlorine attached to a silica formation of silica xerogels also influence the
surface, as shown in reaction 6.1.7: formation of zirconia xerogels that may consist
O
of small (2e25 mm) spherical and rigid particles.
O
Sol -gel derived zirconia xerogels prepared via
O Si Cl O Si H the hydrolysis of zirconium n-propoxide in
O + LiAlH4 O methanol, ethanol, or 2-propanol is one of the
- HCl O Si H most common approaches to preparing this
O Si Cl
support [23]. Other procedures to prepare
O
O zirconia supports can use zirconium oxide that
is further treated with a base for hydration and
(6.1.7) reprecipitated in the presence of a surfactant
It has been shown that the Si-H group on the and in specific physical conditions (temperature,
silica surface is stable in aqueous solutions in stirring) that allow control of the particle proper-
the range of pH from about 2 up to about 9, and ties. It is also possible to embed hydrated zirco-
it is not hydrolyzable as would be a small mole- nium oxide in colloidal form in a mass of an
cule of silane [19]. Unbonded silica hydride can organic polymer that is further destroyed by
be used as a normal phase (similar to a bare silica combustion. Similar to hydrated zirconium
material), but further derivatization of the silica dioxide, hydrated titanium dioxide (TiO2
surface can be done using hydrosilation reactions H2O) or titania can be used as solid support
for the attachment of desired bonded phases (see for stationary phases in HPLC [24].
Section 6.2). The silica hydride surface can be The surface of zirconia xerogels is formed from
populated with hydride material up to 95%. A Zr atoms bound to O atoms and also to OH
number of studies have been published in the groups, similar to the case of amorphous silica
literature regarding the preparation and use of surfaces. The average concentration of Zr atoms
silica hydride stationary phases [20e22]. on zirconia surface is about 12 mmole/m2.
The surface is covered with hydroxyl groups in
a concentration of about 20e25 mmole/m2 and
Other Inorganic Support Materials
is the site for further reactions when the derivati-
Another inorganic material used as solid zation is desired. The specific surface area of
support for stationary phases in HPLC is zirconia supports is dependent on the thermal
zirconia (hydrated). Zirconium dioxide (ZrO2), treatment in the drying process, and this area
also known as zirconia, may exist in several decreases sharply when the drying is performed
6.1. SOLID SUPPORTS FOR STATIONARY PHASES 205
at temperatures higher than 300 C. Above this ionic interactions. For this reason, deactivation
temperature two processes are responsible for of the zirconia surface, in particular the elimina-
the decrease of area surface: microcrystallite tion of Lewis acid sites using a chelating agent,
growth and intercrystallite sintering. Tempera- is applied to the newly developed zirconia
ture treatment influences the porous structure stationary phases. The pore structure of zirconia
of zirconia. When subjected to heat treatment of is also somewhat different from that of silica and
150e300 C, a transitionally microporous mate- leads in general to lower chromatographic
rial is obtained; at temperatures of 300e600 C performance compared to silica. Phases coated
a transitionally macroporous material is on zirconia are also known.
obtained; and above 700 C macroporous adsor- Attempts have also been made to use other
bents with pores of 20e500 A are obtained. These materials as solid support, which would have
materials obtained at higher temperatures do not higher stability to high pH and to heating.
have enough OH groups necessary for derivati- Among these materials are Al2O3 (alumina), as
zation and surface modification. Generally, the well as ThO2 and CeO2. Although ThO2 and
pore volume of various zirconia-based materials CeO2 have been studied, columns made with
is much lower than that of silica materials, which these materials are not commercially available.
makes silica continue to be the preferred solid The surface of alumina and its corresponding
support used in manufacturing stationary phases chemistry is more complex than silica. The basic
for HPLC. spinel structure of Al2O3 often possesses defects
For the use of HPLC support, zirconia that result in various arrangements of aluminum
stationary phases pose a number of challenges ions. Therefore, the hydration of its surface as
that are different from those of silica. The OH well as the number of hydroxyl groups per unit
groups bound to zirconium atom are much area is determined by the specific three-dimen-
less acidic as compared to the silanol groups, sional structure of the oxide [25]. The advantage
although they have the capability to act as of Al2O3-based stationary phases is their stability
a Bronsted acid. The lower acidity of zirconia up to pH 12. Al2O3 is mostly used as support
makes this phase resilient to a wider pH range, for phases containing Al2O3 covered on the
between pH 1 to about pH 10. Zirconia surface with a polymeric layer (e.g., polysty-
surface may also act as a Bronsted base, and rene-divinylbenzene copolymer). These columns
also as Lewis acid due to the d electrons of zirco- are more stable in acidic medium, but they pose
nium atoms that can interact with various problems during the analysis of acidic
ligands. The simplified surface of zirconia compounds due to the mixed types of interac-
surface (showing empty electronic levels, OH tions they offer, one with the polymer and the
groups, and water) is as follows: other with the alumina surface. Alumina support
H H has also been evaluated for reaction with trie-
OH OH OH O thoxysilane (catalyzed by HCl) to obtain a silica
Zr Zr Zr Zr Zr hydride-covered alumina.
Zr O O O O O
O O
Among other inorganic compounds that are
O O O O O
used in chromatography are specific materials
that have ion-exchange properties. These
Besides the behavior determined by the materials include several groups of natural sili-
substituents attached to the zirconia surface, for cates such as zeolites. Zeolites are hydrated
example, that of a reverse phase when the aluminosilicates with the general formula
surface is covered with hydrophobic substituents MxO $ Al2O3 $ nSiO2 $ mH2O, where M is Na,
(e.g. C18), zirconia also exhibits polar and K, Ca, or other similar metals. These compounds
206 6. STATIONARY PHASES AND THEIR PERFORMANCE
act as selective cation exchangers, the exchange permeability, a large number of channels (the
process being associated with a molecular macropores are typically interconnected, the
sieving mechanism. Several hydrated oxides reason for which they are also indicated as
also act as cation exchangers. Among these through pores), and a high surface area (gener-
oxides are hydrated oxides of Si, Al, Zr, Fe, ated mainly by mesopores) [26]. The backbone
and Sn. These hydrated oxides have both of a monolithic column can easily be chemically
adsorbing and ion-exchange properties. Other altered for specific applications. Figure 6.1.6
inorganic compounds, such as polymeric shows the SEM picture of the typical porous
hydrated zirconium phosphate and hydroxyap- structure of a monolithic silica column. The
atite, also have ion-exchange properties. monoliths unique structure gives them several
physicomechanical properties that enable them
Inorganic Monolithic Supports to perform competitively against traditionally
packed columns. Monolithic columns show an
(Silica-based Monoliths)
efficiency equivalent to 3e5 mm i.d. silica parti-
Monolithic columns have been more recently cles, but with a 30e40% lower pressure drop.
introduced in practice as compared to those In general, the monolithic materials are prepared
having the stationary phase made from small in macro- and microscopic formats. The first
particles. Modified silica rods are probably the category includes disks, rods (continuous
most common type of monolithic columns, but columns), and tubes, whereas the second cate-
organic polymers are also used as monoliths. gory comprises capillary columns in fused-silica
The columns based on a silica rod are made of or silica steel tubing and in microfluidic chips.
a single piece of porous silica. These rods are In the case of particle-packed columns, the
prepared by a polymerization or polycondensa- plate height H is roughly proportional to the
tion process, either in situ in a column tube, such particle diameter, whereas the backpressure is
as in glass tubes or fused silica capillaries, or in inversely proportional to the particle diameter.
a column mold. In situ preparation of mono- Usually, a linear increase in column efficiency
lithic columns has the advantage that no further due to the use of smaller particles is accompanied
encapsulation of the porous monolith in a tube by a quadratic increase in column backpressure.
resistant to the pressure of the solvent is needed. In this respect, the monoliths have a major advan-
However, this approach is not compatible with tage over the particle-packed columns. The flow
monolithic silica columns having larger diame- through monolithic channels is closer to laminar,
ters (4.6 mm or more) due to the shrinkage and thus they allow less eddy diffusion. The
that occurs during the sol-gel preparation transport of an analyte through the monolithic
process. When the monolithic silica rod is bed is based mainly on perfusion, and in the
made in a mold, it has to be further clad with monolithic media with a low proportion of
a suitable material such as PEEK (polyether- mesopores, the analyte diffusion into and out of
ether-ketone), to which the column end fittings the pores does not significantly contribute to
can be attached for use in the HPLC process [26]. the band broadening. Due to the reduced diffu-
Monoliths have a porous structure character- sion in solute transfer through the monolithic
ized by mesopores (pores between 2 and 50 nm bed, the peak efficiency does not decrease as
in diameter) and macropores (about 4,000 to sharply as in particle-packed columns when
20,000 nm in diameter), with silica skeleton of increasing the linear flow rate of the mobile
approximately 1e2 mm thick and a void volume phase. This allows the application of higher
of almost 80% of the entire column volume. flow rates and shorter analysis time, without
These pores provide monoliths with high a significant loss of plate numbers [27e29].
6.1. SOLID SUPPORTS FOR STATIONARY PHASES 207
FIGURE 6.1.6 SEM picture of the typical porous structure of monolithic silica columns.
For the production of silica-based monoliths, together to form an infinite network. Xerogels
the basic sol-gel process is similar to that used in are dried by evaporation of the liquid, and aero-
the preparation of solid supports for porous gels are usually obtained by removal of solvents
silica materials. The process is conducted as under hypercritical conditions. The free silanols
a sequential hydrolysis followed by a polycon- resulting from polycondensation are further
densation of silane derivatives. Used for this derivatized with reagents generally used for
purpose are compounds such as tetraethoxysi- derivatization of silica-based particles.
lane (TEOS), or tetramethoxysilane (TMOS) in However, monoliths having the surface covered
aqueous acid or basic medium with an appro- with the desired functionalities can be directly
priate porogenic solvent (e.g., polyethylenegli- obtained by hydrolysis of the appropriate
col (PEG)). The hydrolysis conditions and the compounds such as trimethoxyoctylsilane or tri-
choice of the porogenic solvent are essential methoxyoctadecylsilane. Nonsilica aerogels are
for obtaining mesopores and macropores [30, notably weak and fragile in monolithic form;
31]. Similarly to the preparation of porous silica however, the synthesis of high-porosity mono-
particles, either hydrogels or alcogels can be lithic alumina aerogels has been recently
obtained. The first step in the process is the described.
formation of a sol (a colloidal suspension of
solid species in a liquid). The sol is converted Organic Polymers Used as Support
into a gel through polycondensation of the sol,
for Stationary Phases
leading to a wet structure. The process of gela-
tion starts with the aggregation of particles Porous polymers are frequently used as
into fractal clusters, and then the clusters inter- stationary phases for size-exclusion chromatog-
penetrate to some extent and finally link raphy, and some are used for ion-exchange
208 6. STATIONARY PHASES AND THEIR PERFORMANCE
chromatography. Some polymers are also used and contain specific functionalities can be
for reversed-phase chromatography. For size- obtained either by derivatization or directly
exclusion chromatography, the polymers do not by polymerization of a monomer having
need specific functionalities, and the main charac- already attached the desired functionality. For
teristic of those phases is their tridimensional RP-HPLC columns having C18, NH2 or CN
structure. Perfusion particles usually contain groups are commercially available, and those
very large pores (e.g., 600 to 1000 nm diameter) having COOH, SO3H, NH2, and NR 3 are
connected to a network of smaller pores (30 to commercially available for ion-exchange
100 nm diameter). The flow of the mobile phase chromatography.
through a column filled with perfusion particles A particular direction where efforts have
takes place through the particles and participates been made to develop polymeric stationary
in a diffusion process. Polymers with controlled phases is that of polymeric monoliths [33e36].
pore dimensions are frequently used as the Monoliths were obtained from a variety of
stationary phase for size-exclusion chromatog- monomers, using or not using a crosslinker.
raphy. Also, the size of the polymer particles is Initial monoliths were simply obtained from
important to control (similar to the case of silica styrene and divinylbenzene that were copoly-
particles). This can be achieved using monosized merized in a porogenic solvent and in the pres-
polymer seed particles [32]. ence of a radical initiator. A new convenient
One of the most common polymeric supports procedure starts with glycidyl methacrylate,
used as a stationary phase is polystyrene cross- which is polymerized by UV, thermal, or
linked with divinylbenzene (PS-DVB). This g-radiation initiation, in the presence of a cross-
material is obtained by suspension polymeriza- linker such as ethylenedimethacrylate, and
tion using a two-phase organic/aqueous using a porogenic solvent and an initiator
system. The crosslinking polymerization is per- (e.g., 2,20 -azobis-isobutyronitrile) [37]. Solvents
formed in the presence of inert diluents that are typically used as porogens are mixtures of
miscible with the starting monomers but must cyclohexanol and dodecanol, a higher content
not dissolve in the aqueous phase. Submicrom- of dodecanol leading to monoliths with larger
eter particles (microbeads) form as the styrene- pores [38]. The reactions taking place during
divinylbenzene polymerizes and precipitates polymerization are of the type described
out of solution. The formed microbeads fuse by 6.1.8:
together to form macroporous particles.
Initially, a network of microporosity may be
present in the microbeads, and polymerization O CH3
H2C CH3
conditions must be controlled to minimize this C
+ H2C O
type of porosity because it results in a soft poly- C O CH2
mer that has poor mechanical strength and high O O
CH3
O O
propensity of swelling. After the crosslinked PS-
DVB porous particles are formed, any residual O
reactants, diluents, and surfactants must be
removed by thorough washing. H3C
C O
Other polymers such as methacrylates and H2C
polyvinyl alcohol were also used. Organic
O
polymers were utilized for preparation of
both particles and monolithic columns. Also, crosslinked
(Continued)
210 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.1.3 Methacrylate polymers evaluated for producing monolith columns for HPLC [39]. (contd)
Type of polymer Crosslinking agent
Reactive
Phase Handle Phase group Phase
Silica O Si Silica Silica
Phase 1
Phase 1 Phase 2 Handle Phase 1 Phase 2
Silica Silica Phase 2 Silica
phase on the silica xerogel surfaces. More The problem with this procedure is that the
recently, the same procedure has been success- Si-O-C bond obtained in a reaction of the type
fully applied on organic/inorganic silica struc- shown in 6.2.1 is not very stable, and proce-
tures such as those having ethylene bridges dures to obtain more stable derivatized
(a bridged ethane-silicon hybrid or BEH-type surfaces were considered. One important desir-
stationary phase) or methyl groups already able characteristic of HPLC chromatographic
present in the silica structure. columns is the absence of the bleed from the
For a successful reaction with the silanol bonded phase. This characteristic affects the
groups from the silica surface, it is important column stability in time and matters during
that these groups are reactive. Silica surface measurements, in particular when the detec-
can be activated either by physical treatment tion is performed using a MS-type detector.
(thermal or hydrothermal) that changes the ratio The increased hydrolytic stability is one of the
of silanol and siloxane concentration of the silica main features that allow minimum bleed from
surface or by chemical treatment that leads to the column. The stability of the bonds in the
changes in chemical characteristics of silica stationary phase is a factor to be considered
surface. One such procedure is the boiling of when using a specific column in a separation
silica with acidic solutions that can generate on method. One procedure to obtain stable bonds
silica surface an adequate and dense layer of is the use of reagents that can generate Si-O-Si
silanols and consequently of the bonded groups. bonds in which one silicon is part of the silica
A thermal treatment of the silica consists of heat- surface and the other is further connected to
ing at 250e400 C before the derivatization [41]. a hydrophobic moiety.
The most common type of HPLC is reversed- A stable Si-O-Si bond can be obtained, for
phase HPLC (RP-HPLC) where the stationary example, using a monofunctional silanization
phase is nonpolar. Nonpolar phases are frequen- reagent. One common type of reaction applied
tly based on a derivatized silica support, for the generation of covalently bonded hydro-
although columns with a hydrophobic stationary phobic groups to the silica surface through
phase can be made using other procedures. Long a Si-O-Si bond is indicated in 6.2.2 [1]:
alkyl chains are most commonly used to form
a hydrophobic surface on the porous silica skel- CH3 CH3
eton. The first attempt to attach alkyl chains to Si OH + X Si R Si O Si R
a silica matrix was made using the reaction CH3
- HX
CH3
between the silanol groups on the silica surface
and 1-octanol, leading to a Si-O-C bond. This (6.2.2)
reaction is indicated in 6.2.1 [42]:
where X is most commonly eCl, but other
groups such as eOCH3, eOCH2CH3, or even
Si OH eNH2 can be used, and R is typically a hydro-
- H 2O phobic n-hydrocarbon chain such as octyl
+
(C8) or octadecyl (C18). The derivatization
HO CH2 CH2 CH2 CH3
CH2
of a silica surface with a monofunctional
CH2 CH2 CH2
silanization reagent is typically referred to
as monomeric functionalization. Monomeric
Si O CH2 CH2 CH2 CH3
functionalization attaches a monomolecular
CH2 CH2 CH2 CH2
layer of active phase on the silica surface
(6.2.1) [43e45].
6.2. REACTIONS USED FOR OBTAINING ACTIVE GROUPS OF STATIONARY PHASES 213
A variety of other R groups can be attached to silica surface is not a homogeneous one. It
the silica surface using this procedure, including may contain a significant number of underivat-
a variety of hydrophobic groups (linear alkyl, ized silanol groups, which will further partici-
cycloalkyl, phenyl, etc.) when reversed stationary pate in the retention mechanism. The presence
phases are desired. However, the procedure is of free silanols after derivatization represents
also applied to obtain phases containing ligands one of the major challenges in obtaining fully
such as e(CH2)3-NH2, e(CH2)3-CN, e(CH2)3- derivatized silica surfaces. Further derivatiza-
NO2, and eCH2-CH(OH)-CH2(OH), which are tion is necessary to reduce the silanols number.
polar. The common reaction based on chlorosi- A schematic description of a portion of a silica
lanes as derivatization reagents needs the surface partly derivatized using a monofunc-
presence of organic bases (pyridine or triethyl- tional reagent is shown in Figure 6.2.2. The struc-
amine) in the reaction medium to remove ture shows the Si-O-Si bonds, the
HCl formed as a by-product. When alkoxydi- dimethylsilyloctyl groups, the OH groups (iso-
methyl-R-silane is used as a silanization reagent, lated, vicinal, geminal), as well as the crosslink-
toluene is a proper solvent for the reaction. ing bonds.
In this case, the alcohol formed as a reaction The calculated point charges on a hypothetic
by-product should be removed from the surface molecule where the octyldimethylsilyloxy
because it can subsequently react with silanol group is attached to a Si(OH)3 group is shown
groups leading to alkoxy-derivatized silica in Figure 6.2.3. This molecule simulates a silica
surface, which is unstable toward aqueous structure partly derivatized with dimethyloc-
mobile phases. In both cases, the modified tylsilyl groups. Figure 6.2.3 shows that the
CH3
OH
CH3 CH3
O
Si Si O
O O Si
O O Si O
Si Si O Si O OH
CH3
Si Si
O O
O OH O Si CH3
O O OH O OH O Si CH3 CH
3
Si Si O
Si CH 3 O Si
HO Si O O
O O O Si
O H3C Si Si Si
O O O O O O Si
Si Si O Si O O Si
O O
Si Si O OH OH
O O O Si
Si O Si OH Si OH
O O O
Si O OH
Si O
O Si
Si O
O
OH Si O
O
Si
OH
FIGURE 6.2.2 Schematic description of a portion of a silica structure partly derivatized with a monofunctional reagent,
showing the Si-O-Si bonds, the dimethyloctylsilyl groups, the OH groups (isolated, vicinal, geminal), as well as the
crosslinking bonds ().
214 6. STATIONARY PHASES AND THEIR PERFORMANCE
+0.21 +0.02
H H +0.02
-0.35 H
O +0.02 +0.03 +0.03 +0.02
H C -0.05 +0.03 H +0.03 H +0.03 H
+0.03 H
H H H +0.02
+0.21 -0.35 +0.58 H
O +0.19 -0.05
H Si Si C -0.05 C -0.05 C C -0.07
-0.37
+0.02
O C -0.04 C -0.05 C -0.05 C -0.06 H
+0.21 -0.35 +0.02 -0.05 +0.02 +0.03 +0.03 +0.03
H O H C H +0.02 H H +0.03 H +0.03 H H +0.03
H H
+0.02 +0.02
H H
FIGURE 6.2.3 Point charges on a hypothetic molecule with octyldimethylsilyloxy group attached to a Si(OH)3 unit.
long alkyl chain (C8) in the model molecule has stationary phases. This is performed by a sepa-
point charges very similar to those in a long- rate procedure of blocking residual silanols by
chain hydrocarbon (see Figure 4.1.6), and very derivatization with small groups, such as tri-
likely the charge distribution on a real bonded methylsilyl, using trimethylchlorosilane or
phase is similar to that in a long-chain hydro- hexamethyldisilazane as reagents. This proce-
carbon. On the other hand, the hydrogens dure is known as end-capping. The process is
from the silanols of the Si(OH)3 group have applied after the derivatization with the
a point charge of 0.21, indicating a quite desired bonded phase is performed. Besides
strong acidic character typical for the silanols trimethylchlorosilane or hexamethyldisilazane,
on the silica surface. The residual silanols difunctional derivatization reagents are also
remaining in the silica structure offer used for end-capping. The reaction is typically
a secondary place of interactions besides the performed at elevated temperatures to achieve
hydrophobic group. They produce peak tailing a derivatization that is as complete as possible
for highly polar compounds due to the interac- [46e48]. By end-capping, the carbon content
tion of the type H-bonding, dipole-dipole, or of packing material does not significantly
ion-dipole with different functional groups in change, and thus its hydrophobic character
analyte molecule. Above pH 5, a pronounced for the reversed phase phases is kept almost
tailing for basic compounds is observable owing constant (the carbon load % C is determined
to the dissociation of silanol groups and their by elemental analysis).
involvment in ion-exchange-type interactions The lack of reactivity of some silanol
with polar or dissociated groups from analyte groups with a specific derivatization reagent
molecule. Even when the silanol group presence is thought to be caused mainly by steric
on the bonded phases can be useful for partic- hindrance. When attempting to attach, for
ular separations, the problem that remains is example, two long aliphatic chains on two
the reproducibility of their polar interactions, vicinal or germinal silanol groups, the reaction
since the silanol groups retain water from the may not occur because the reagent cannot
mobile phase and their polarity may vary penetrate to the reactive site. For this reason,
depending on the composition of the mobile end-capping is done with reagents having
phase recently utilized. a small molecule. Another possibility of deri-
The effort to reduce the number of residual vatizing more silanols from silica surface is
silanols on the silica matrix is a common proce- through the use of reactions with di- or tri-
dure in preparing chemically bonded functional reagents. These reagents have two
6.2. REACTIONS USED FOR OBTAINING ACTIVE GROUPS OF STATIONARY PHASES 215
or three reactive functional groups such as A reaction between vicinal silanols and a di-
ethoxy or chloro. A schematic description of functional reagent is shown in 6.2.4.
the reactions of a di-functional reagent with
two ethoxy groups is shown in reactions Si OH C2H5O CH3 Si O CH3
6.2.3 for an isolated silanol. The reaction takes O + Si O Si
place by means of three-step sequences. In the Si OH C2H5O R - 2 C2H5OH
Si O R
first step the di-functional reagent reacts with di-functional
silica to link methylalkylethoxysilyl or methyl- reagent
alkylchlorosilyl groups, which in the second
step are hydrolyzed in order to substitute (6.2.4)
the chlorine atom or the ethoxy group with As seen from reactions 6.2.3 and 6.2.4, a di-
Si-OH, and then subjected to another derivati- functional reagent has better capability to react
zation reaction with di-functional silane. This with vicinal silanols and also to give a good
sequence is repeated several times (8 to 10 density of substituents for isolated silanols.
times) and is schematically shown in reaction A model chain of reactions with a tri-functional
6.2.3. reagent (triethoxysilane) is given in 6.2.5:
(6.2.5)
O Si O
C2H5O OC2H5 Si Si
C2H5O + O Si
+ Si OC2H5 OH OH
Si O Si O O Si Si O O Si O
OH C2H5O R
O Si O Si R O Si R
R Si O R - 2 C 2H5OH Si O R
Si O
216 6. STATIONARY PHASES AND THEIR PERFORMANCE
The possibility that a tri-functional reagent reagent with small-volume substituents and
connects with the silica surface with all three use of more intense reaction conditions (e.g.,
reactive substituents is also possible, as shown higher temperatures).
in the following schematic reaction: In contrast to vertical polymerization, hori-
zontal polymerization can also be practiced with
Si Si
di- or tri-functional silanes. In this procedure,
O OH Cl O O the tri-functional reagent is reacted with silica
Si OH + Cl Si R Si O Si R in anhydrous conditions. This procedure
- HCl
Cl O O
requires the use of a mixture of reagents with
O OH
Si Si long R substituents (e.g., octadecyl) and short
R (e.g., propyl) in order to obtain a more
(6.2.6) complete cover of the silica surface. Horizontal
derivatization seems to be associated with
Derivatization with difunctional or trifunc- a self-assembling process of the hydrocarbon
tional silanes typically leading to multiple chains that leads to a more uniform coverage
bonds and tridimensional bonding on the silica of the silica surface [50]. Since in this case the
surface is known as polymeric functionalization. layer of molecules covering the silica surface is
Since reactions of the type 6.2.5 and 6.2.6 take closer to monomolecular, the carbon load
place in an aqueous medium, they lead to a poly- achieved by horizontal polymerization is typi-
condensation process such that besides the cally lower than that for vertical polymerization.
direct bonding of groups O-Si-R to the silica Horizontal polymerization is less frequently
surface, there are also aggregates of pre-reacted utilized to obtain stationary phases. The result-
reagent that are attached, forming a polymeric ing structures obtained by the two derivatiza-
thin layer of stationary phase bonded to the tion techniques are schematically shown in
silica and containing a tridimensional structure. Figure 6.2.4.
The polymeric functionalization attaches In summary, according to the type of reagent
a multimolecular layer of bonded phase on the used in the derivatization procedure (mono-,
silica surface, differently from monomeric func- di-, and tri-functional silanes) and the condi-
tionalization that attaches a monomolecular tions applied for derivatization (addition of
layer of active phase. Polymeric derivatization water with the derivatization reagent or use of
leading to a multimolecular layer of bonded anhydrous conditions), the hydrophobic
phase is called vertical bonding (or vertical poly- bonded phases can be classified in three cate-
merization) [49]. The process is rather complex, gories: brush phases (monomeric), oligomeric
particularly when two vicinal silanols are phases, and bulk phases (polymeric). The brush
involved. Although this approach leads to phases are obtained using monochlorsilanes,
a better derivatization yield, a significant such as octyldimethylchlorsilane (to attach
number of silanols remain on the silica surface. dimethyloctylsilyl chains), or octadecyldime-
Further derivatization by repeating the process thylchlorsilane (to attach dimethyloctadecylsilyl
or using reagents containing less bulky moieties chains). A brush phase is also obtained when
can be performed, using, for example, tri- using horizontal polymerization with reagents
methyl-chlorosilane or hexamethyldisilazane. such as alkyltrichlorsilanes or alkyltrialcoxysi-
Even so, not all the silanols are removed due lanes in anhydrous conditions. The alkyl chains
to problems related to steric hindrance. These in these cases are considered to stand out from
residual hydroxyl groups are made more inert the silica surface like bristles of a brush, a struc-
by repeated derivatization with the reactive ture that is the result of a self-assembling
6.2. REACTIONS USED FOR OBTAINING ACTIVE GROUPS OF STATIONARY PHASES 217
O Si O
O
O Si
O
OH Si O
Si O OH
OH Si O O
O O O
Si Si O Si Si Si
O O
OH O OH O O O O O O O
Si Si Si Si Si Si Si Si Si Si Si Si
O O O O O O O O O O O O O O
O O O O O O O O O O O O
Vertical polymerization Horizontal polymerization
FIGURE 6.2.4 Schematic illustration of vertical (polymeric) derivatization that generates a bonded polymer layer on
silica surface and horizontal derivatization of silica.
process. The di-functional silanes (with limited phase on the silica surface. Among these groups
amounts of water in the reaction) produce olig- are the alkyl, cyanopropyl, and phenyl. Other
omeric bonded materials. The bulk phases are groups can be used as the active bonded phase,
obtained from alkyltrichlorsilanes or alkyltrial- and also as intermediates for further derivatiza-
coxysilanes in vertical polymerization, which tions. Among such groups are glycidyl, amino,
takes place in the presence of some water and and azido.
leads to a complex crosslinking process that In addition to the simple reagents used to
finally generates a polymeric layer covering derivatize the silica surface, more complicated
the silica surface. Crosslinking is achieved not compounds can be made reactive and used for
only involving the silanol groups from the silica reaction with the silanol groups. For example,
surface, but also from reactions between the cholesterol can react with allyl bromide to
reagent molecules. The carbon load on the silica form a 3-allyl ether. In the presence of a catalyst
is in this case the highest, and bulk polymeric (H2PtCl6), the double bond of the allyl ether
phases are very common. reacts with the silanol groups attaching the
A number of common reagents used for the cholesterol fragment to the silica surface (with
derivatization of silica surfaces are shown in a C3 spacer) [51].
Table 6.2.1. These reagents may be used for Another method that has been reported for
attaching on the silica surface either hydrophilic the modification of silica supports is based on
or hydrophobic functionalities. Also, the same a chlorination/organometalation two-step reac-
reagents used for the derivatization of silica tion sequence. In the first step, the silanols on
gel particles can be used for the derivatization the silica surface are converted to chlorides
of silica monoliths. A much wider range of with thionyl chloride as shown in reaction 6.2.7:
compounds than is indicated in Table 6.2.1 can
be attached to the silica surface by direct reac- Si OH + SOCl 2 Si Cl + SO2 + HCl
tion with the silanol groups. Some of the
attached groups may act as the active bonded (6.2.7)
218 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.2.1 Examples of reagents used for the derivatization of silica surface.
This step must be performed under extremely Those structures that have R directly bonded
dry conditions (usually in a closed vessel to the siloxane matrix are more stable than struc-
purged with a dry gas such as nitrogen) tures based on substitution of H from silanol,
because the presence of water traces leads to which could be an advantage for the phases
the reversal of the reaction, with hydroxyl bonded by this procedure. However, due to
groups replacing the chloride bonded to the specific reaction condition from the first
the silica and regeneration of silanols. Then step and the formation of salts as by-products,
the substitution chain R can be attached to the the materials obtained using silica chlorination
surface using a Grignards reagent or an orga- are less known as commercial packing materials
nolithium compound: for HPLC applications.
A common procedure for preparing more
complex desired stationary phases is to further
react a molecular fragment already attached to
Si Cl + RMgCl Si R + MgCl2 (6.2.8)
the silica surface with another reagent. The
initially attached functionality is either itself
transformed into the desired functionality or it
can be used as a reactive anchor to attach
Si Cl + RLi Si R + LiCl (6.2.9) a desired fragment that will form the stationary
phase.
6.2. REACTIONS USED FOR OBTAINING ACTIVE GROUPS OF STATIONARY PHASES 219
One example of changing one attached group
OC2H5
into another group is shown in 6.2.10 for the
case of obtaining a strong cation exchange Si OH + H5C2O Si
NH2
stationary phase: OC2H5
O
Si OH
O + (CH3O)3Si (CH2)3-SH Si O Si
NH2
Si - x CH3OH O
(6.2.11)
Si O Si (CH2)3-SH
The amino silica is further reacted with an
O
acid chloride or an isocyanate of a long carbon
Si chain carboxylic acid (e.g., with R C8, C18,
etc.), as exemplified in the reaction 6.2.12 for
(6.2.10)
an acid chloride:
Si O Si (CH2)3-SH
O + H2O2
O
Si
O
Si O Si
NH2 + C R
O Cl
Si O Si (CH2)3 SO3H
O
Si O O
Si O Si C
NH R
O
The use of fragments that are already
attached to the phase and have a reactive group (6.2.12)
able to continue a coupling for obtaining more
complicated stationary phases is much more The reaction with an isocyanate is given by
widespread than the functionality change. The 6.3.13:
procedure can be used, for example, to obtain
hydrophobic phases with embedded polar
groups, phases having macrocyles attached to O
silica, special chiral phases (this procedure will
Si O Si
be discussed and exemplified later in this NH2 + O=C=N R
chapter for particular types of stationary O
phases). The two-step approach for the
synthesis of polar-embedded phases is common O O
[52e54]. This approach is based on derivatizing
Si O Si C R
the silica surface with a group such as amino- NH NH
propyl, followed by further reactions of the O
amino silica. A typical reaction for preparation
of amino silica is shown in reaction 6.2.11: (6.2.13)
220 6. STATIONARY PHASES AND THEIR PERFORMANCE
The preparation of octadecyl phases with appropriate reagents, without having bare
polar-embedded groups was obtained, for active silica prepared as a first step, followed
example by a reaction similar to 6.2.12 where by further derivatization. For example, the
R is octadecyl [55]. hydrolysis of tetramethyl orthosilicate (tetrame-
The two-step derivatization can also start thoxysilane or TMOS) in the presence of octyltri-
with a glycidyl group on silica surface. An methoxysilane in an acidic medium generates
example of such a reaction is given by 6.2.14: a silica gel that contains octyl groups attached
to the silica structure. The same procedure can
be applied to obtain silica having C18 chains
O attached on its surface. Another example of
Si O Si direct reaction for obtaining a functionalized
O + H2N R silica surface is the preparation of propylsul-
O O fonic acid functionalized mesoporous silica.
R
This material can be prepared in a unique step
O NH starting with tetraethyl orthosilicate, 3-mercap-
topropyltrimethoxysilane, and H2O2, in strong
Si O Si acidic conditions [57].
O
O
OH
Direct hydrolysis of a mixture of silane deriv-
atives (co-hydrolysis) may generate the desired
(6.2.14) functional groups on the silica surface, but it
Silica derivatized with azido groups can can also make the process more difficult to
participate in Huisgen cycloadditions (Click control particularly in relation to the size and
chemistry) as shown in reaction 6.2.15: shape of stationary phase particles [58]. For
this reason, co-hydrolysis has been used more
frequently for preparation of monolith-type
O materials where the control of particle size is
R not a critical step. The procedure leads to so-
Si O Si Cu(I)
N N N + HC C called organic-inorganic monoliths. One
O example of co-hydrolysis reaction is given in
6.2.16:
O
Si O Si N OCH3 R
N + n H 2O
N
O R x H3CO Si OCH3 + y H3CO Si OCH3
- n CH3OH
OCH3 OCH3
(6.2.15)
Numerous other phases obtained from silica O R
surface derivatization are reported in the litera- ( Si O )x ( Si O )y
ture [56].
O O
Si Si Si Si Si Si Si Si
O O O O O O O O
O O O O O O O O
FIGURE 6.2.5 Schematic structure of a mixed mode phase with octyl and amino groups and a phase with octyl and
cyano groups.
in the hydrosilation as shown in the reaction order to obtain the desired functionalities
6.2.18 [60]: on the surface of material forming the
stationary phase. Specific derivatizations
were performed on vinyl-type polymers that
Si have attached on the polymeric backbone
O H
specific reactive groups, such as glycidy or
Si H + HC C C16H33
alkyne. Such reactions were applied for the
(catalyst)
O H synthesis of particle-type phases as well as
Si
monoliths. One example of synthesis starts
with the polymerization of ethylene dimetha-
crylate with propargyl acrylate as shown in
Si 6.2.19:
O
Si H CH CH2 C16H33
O O
Si
H2C
O CH
(6.2.18)
CH2 + O
The same procedure can be used to attach other O CH2
functional groups, such as cyano, amino, and H3C O
fluorinated hydrocarbons [61e64]. Silica hydride O CH3
phases can also be the subject of end-capping.
The functionalities bound to silica hydride
O
are not limited to simple groups. For example,
a very stable structure based on Si-C bonds CH
Polymer O
can be obtained from the reaction of hydride
silica with vinylbenzo-18-crown-6, in presence
of H2[PtCl6], leading to the following structure (6.2.19)
of the stationary phase:
The polymer with alkyne groups is further
O derivatized using a copper(I)-catalyzed (3 2)
azide alkyne cycloaddition that is shown sche-
O O
matically in reaction 6.2.20:
O
O Si CH2 CH2 O O
O
O O
Cu(I)
O - +
Polymer CH + N N N R
This type of phase can be used in ion-
exclusion separations.
O
Derivatization of Presynthesized Organic N
R
Polymers Polymer O
N N
Various procedures were applied for the
derivatization of a preformed polymer in (6.2.20)
6.2. REACTIONS USED FOR OBTAINING ACTIVE GROUPS OF STATIONARY PHASES 223
The radical R in reaction 6.2.20 can be, for leads to a tridimensional thermorigid polymer.
example, a long hydrocarbon chain such as The inert resin can be modified by direct sulfona-
C18 [65]. tion into a strong cation exchanger.
Other examples include the synthesis of The polycondensation in the presence of acid
cation-exchange resins, or anion-exchange catalysts may leave a significant number of free
resins can be obtained by such derivatizations. eCH2OH groups. These can be further derivat-
The polymeric backbone of organic ion ized with SOCl2, and the eCH2OH groups are
exchangers is usually obtained either by poly- changed into eCH2Cl groups. Upon treatment
condensation or by polymerization. A with N(CH3)3, the resin can be changed into
common polycondensation reaction used to a strong anion-exchange material as shown in
obtain polymeric resins is that between phenol the following reaction:
OH OH OH OH
OH OH
CH2OH CH2Cl
CH2N(CH3)3+
SOCl2 N(CH3)3
OH OH
OH
and formaldehyde. The reaction is written in Various other organic polymer-based sta-
6.2.21: tionary phases were synthesized. However,
silica-based stationary phases have several
advantages and are much more commonly
OH OH used in HPLC [66].
CH2OH
+ HCHO
- nH 2O Synthesis of Organic Polymers
with Active Groups
OH OH
Direct synthesis of polymers with active
CH2OH groups present in their molecular structure is
a convenient procedure for obtaining poly-
mer-based stationary phases. For this purpose,
OH
the polymerization is done starting with
specific monomers that have the desired
group. Sulfonated styrene can be used, for
example, in a copolymerization reaction with
(6.2.21) divinylbenzene (usually in the presence of
benzoyl peroxide) to form a polymer with
Both acid and basic catalysts can be used in this sulfonic groups. Copolymerization between
reaction. In the presence of basic conditions, the acrylic acid or methacrylic acid and divinyl-
reaction takes place more completely and usually benzene generates a resin with carboxyl
224 6. STATIONARY PHASES AND THEIR PERFORMANCE
C2H5
HO O N
O H5C2
OH OH
+ H 2O H3C + NH(C2H5)2
H3C C O
C O H3C H2C H3C
NaOH C O NaOH C O
H2C
H2C O H2C
O hydrolysis amination
O O
C2H5
H5C2 + -
O N Cl O
H5C2 O HO3S
OH OH
H3C + N(C2H5)3HCl + SO3/H2SO4
C O H3C
H2C H3C C O H3C
NaOH C O O
H2C C
O H2C H2C
amination O
O sulfonation O
HOOCCH2 O H5C6 S
O O O OH
OH
C6H5 O
+ ClCH2COOH
H3C H3C H3C SH H3C
C O O O C O
C C
H2C H2C H2C H2C
O O O O
carboxymethylation
NH Ligand
O
H2N Ligand OH
H3C
H3C C O
C O H2C
H2C
O
O
FIGURE 6.2.6 Various coupling reactions for polymers having glycidyl groups.
groups, as shown in reaction 6.2.22 (for meth- This resin can be used as a weak cation-
acrylic acid): exchange resin.
CH3
Synthesis of Organic Polymeric
CH=CH2 Monoliths with Active Functionalities
C CH2 CH CH2
CH3
COOH
A number of polymeric monoliths that can be
C CH2 + further derivatized with desired functional
COOH groups were synthesized and utilized as
stationary phases in HPLC [37]. A convenient
CH=CH2
CH CH2 procedure to perform such derivatizations starts
with a polymer having glycidyl groups. Such
(6.2.22) polymers can be further derivatized using
6.2. REACTIONS USED FOR OBTAINING ACTIVE GROUPS OF STATIONARY PHASES 225
various reactions including hydrolysis, amina- with the mobile phase. This type of phase
tion, alkylation, and sulfonation. Some of these can be obtained, for example, when the
reactions are shown in Figure 6.2.6. Other polymer covering the silica particles is
reagents besides those indicated in this figure poly(methyloctyl-siloxane) or poly(methylte-
were used to react with the glycidyl groups tradecylsiloxane). Covalent bonding on the
bonded to a polymeric backbone. Bio- silica surface can be achieved by using
functionalization of the epoxy or hydroxyl- a derivatization procedure with a reactive
bearing monoliths was also reported in the liter- polymer. In this case, the silica is first deriv-
ature [67]. Reactions involving intermediate atized, for example, with g-aminopropyltrie-
derivatization using a diamine and glutaralde- thoxysilane. This reaction is followed by
hyde, carbonyldiimidazole, disuccinimidyl derivatization with a polymer containing
carbonate, and the like, were used to bind specific reactive groups. As an example, silica
ligands on the polymeric monolith. can be coated with poly(N-isopropylacryla-
mide), following the chain of reactions indi-
Silica Covered with a Bonded Active cated in 6.2.23:
Polymer
The stationary phases made with a poly-
mer covering silica particles and at the
Property Range
Purity (for silica) High purity (Type B purity), medium (Type A purity)
Surface area (for silica) 50 m2/g to 500 m2/g
228 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.3.2 Classification of HPLC columns based on dimensions and purpose [71].
of analyte that can be injected. The upper limit classification given in Table 1.2.1 (the principle
of the amount of sample to be loaded on the of separation except that the classification of the
column is limited by the amount of stationary HPLC types also included the type of mobile
phase and its capacity to retain the solute. phase). A few examples of columns are also given
Approximate formulas were developed for in Table 6.3.3.
estimating maximum column load (see rel. Although the stationary phases can be classi-
9.2.8). Other factors that determine the dimen- fied based on their main characteristics, the
sion of the column include available equipment concept of type is broad, and a range of prop-
and required column resilience. A classification erties can be present for phases of the same
of columns based on their dimension is indi- type. As an example, the columns used in
cated in Table 6.3.2 [71]. The table also notes reversed-phase chromatography (RP-HPLC)
the approximate sample loading for the are characterized by their hydrophobic character.
column. Except for preparative and semipre- Columns with the hydrophobicity within
parative columns (used e.g. in flash chromatog- a wide range are available, starting with cyano-
raphy), the other types of columns listed in propyl stationary phases that have an interme-
Table 6.3.2 are used (mainly) for analytical diate character between polar and nonpolar
purposes. Industrial preparative columns can (hydrophobic), and ending with C30 phases
be very large (compared to the dimension sug- (having attached to the silica a hydrocarbon
gested in this table), and sample loading can be chain with 30 carbon atoms) that are highly
significantly higher than mg. hydrophobic. This range of a specific character
(such as hydrophobicity) is essential in selecting
Types of Stationary Phases and USP a column for obtaining an optimum separation.
The choice of a specific type of column (from
Classification
a range of columns available), combined with
Depending on the type of chromatography, the selection of a solvent with certain properties
a considerable number of stationary phases types (e.g., polar character of the solvent used in RP-
were differentiated. Along with the classification HPLC), allow the fine tuning of a separation.
of HPLC types given in Table 1.2.1, a similar clas- Other, more detailed classifications of
sification can be indicated for the stationary stationary phases are reported in the literature.
phases. This classification is given in Table 6.3.3 One example is the classification proposed by
and follows the same principles as the US Pharmacopeia (USP) [72]. This classification
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 229
TABLE 6.3.3 List of several types of stationary phases.
1) Hydrophobic (non polar, Reversed phase bonded on silica C4, C8, C18, endcapped C18, other n-alkyl
widely used in RP-HPLC) non-polar (e.g. C30), mixed alkyl, phenyl, diphenyl,
mixed alkyl-aryl, cyclohexyl, fullerene,
carbon (graphytized), etc.
1) Hydrophobic Reversed phase polymeric Divinylbenzene (DVB), polystyrene-
divinylbenzene (PS-DVB), PS-DVB-
methacrylate, DVB/methacrylate,
polymethacrylate, etc.
1) Hydrophobic Reversed phase polymer-coated Polybutadiene on silica, polybutadiene on
alumina.
1) Hydrophobic Reversed phase with polar C18-carbamate, C8-carbamate, alkylamide,
embedded on silica C8-amide, C14-amide, C18-amide, C16-
sulfonamide, phenyl ether
L1 Octadecyl silane (C18) chemically bonded to porous Over 250 columns such as: Luna C18(2), Luna
silica or ceramic microparticles, 1.5 to 10 mm in C18(2)-HST, Gemini C18, Synergi Hydro-RP, Onyx
diameter, or a monolithic rod. C18 (M), Aquity UPLC BEH C18, Aquity UPLC
Shield RP 18, Atlantis T3, mBondapack C18,
Nova-Pak C18, Symmetry C18, ABridge C18,
XTerra MS C18
L2 Octadecyl silane (C18) chemically bonded to silica gel Bondapak Prep C18, etc.
of a controlled surface porosity that has been bonded
to a solid spherical core, 30 to 50 mm in diameter.
L3 Porous silica particles, 1.5 to 10 mm in diameter, or Luna Silica(2), Aquity UPLC BEH HILIC, Atlantis
a monolithic silica rod. HILIC Silica, Onyx Si (M), SunFire Silica, XBridge
HILIC, Zorbax SIL, etc.
L4 Silica gel of controlled surface porosity bonded to Porasil Prep Silica
a solid spherical core, 30 to 50 mm in diameter
L5 Alumina of controlled surface porosity bonded to
a solid spherical core, 30 to 50 mm in diameter.
L6 Strong cation-exchange packing: sulfonated
fluorocarbon polymer-coated on a solid spherical core,
30 to 50 mm in diameter.
L7 Octyl silane (C8) chemically bonded to totally porous Luna C8(2), Aquity UPLC BEH C8, Nova-Pak C8,
silica particles, 1.5 to 10 mm in diameter, or a monolithic Resolve C8, SunFire C8, Symmetry C8, XBridge C8,
silica rod. XTerra MS C8, XTerra RP8, Onyx C8 (M), Nucleosil
C8, Zorbax C8, etc.
L8 An essentially monomolecular layer of Luna 10 mm NH2, mBondapak NH2, Waters
aminopropylsilane (NH2) chemically bonded to totally Spherisorb NH2, Zorbax NH2, etc.
porous silica gel support, 3 to 10 mm in diameter.
L9 Irregular or spherical, totally porous silica gel having Partisil 10 mm SCX (I), Spherisorb SCX, Luna 10 mm
a chemically bonded, strongly acidic cation-exchange SCX, Zorbax SCX
coating, 3 to 10 mm in diameter.
L10 Nitrile groups (CN) chemically bonded to porous silica , Capcell CN UG, mBondapak CN,
Luna CN 100 A
particles, 3 to 10 mm in diameter. Nova-Pak CN, Resolve CN, Waters Spherisorb CN
L11 Phenyl groups (C6H5) chemically bonded to porous Synergi Polar-RP, Luna Phenyl-Hexyl, Gemini C6-
silica particles, 1.5 to 10 mm in diameter. Phenyl, Prodigy PH-3 , Aquity UPLC BEH Phenyl,
mBondapak Phenyl, XBridge Phenyl, XTerra
Phenyl, etc.
L12 Strong anion-exchange packing made by chemically AccelPlus QMA
bonding a quaternary amine to a solid silica spherical
core, 30 to 50 mm in diameter
L13 Trimethylsilane (C1) chemically bonded to porous , TSKgel TMS-250,
Develosil TMS-UG (C1) 130 A
silica particles, 3 to 10 mm in diameter. Waters Spherisorb C1
(Continued)
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 231
TABLE 6.3.4 USP classification of HPLC chromatographic columns [73]*. (contd)
Code Description Examples
L14 Silica gel having a chemically bonded, strongly basic Partisil 10 mm SAX (I), PartiSphere 5 mm SAX,
quaternary ammonium anion-exchange coating, 5 to Waters Spherisorb SAX
10 mm in diameter.
L15 Hexylsilane (C6) chemically bonded to totally porous PhenoSphere C6, Waters Spherisorb C6
silica particles, 3 to 10 mm in diameter.
L20 Dihydroxypropyl groups chemically bonded to porous Luna HILIC Shodex PROTEIN KW-800 series,
silica particles, 3 to 10 mm in diameter. TSKgel QC-PAK 200 and 300, BioSuite 125, Insulin
HMWP (I), Protein-Pak (I)
L21 A rigid, spherical styrene-divinylbenzene copolymer, 3 Polymerx RP-1, Phenogel 100 A , IC-Pak Ion
to 10 mm in diameter. Exclusion, Shodex SP-0810, etc.
L22 A cation exchange resin made of porous polystyrene Rezex ROA
gel with sulfonic acid groups, about 10 mm in size.
L23 An anion exchange resin made of porous Shodex IEC QA-825, TSKgel BioAssist Q, TSKgel
polymethacrylate or polyacrylate gel with quaternary SuperQ-5PW, BioSuite Q AXC, BioSuite DEAE,
ammonium groups, about 10 mm in size. Protein-Pak Q 8HR
L24 A semi-rigid hydrophilic gel consisting of vinyl YMC-Pac PVA-Sil, Toyopearl HW-type
polymers with numerous hydroxyl groups on the
matrix surface, 32 to 63 mm in diameter.
L25 Packing having the capacity to separate compounds PolySep-GFC-P2000, Shodex OHpak SB-802.5HQ,
with a MW range from 100 to 5000 Daltons (as Ultrahydrogel DP +120, TSK-gel G1000PW
determined by polyethylene oxide), applied to neutral,
anionic, and cationic water-soluble polymers. A
polymethacrylate resin base, crosslinked with poly-
hydroxylated ether (surface contained some residual
carboxyl functional groups) was found suitable.
L26 Butyl silane (C4) chemically bonded to totally porous Jupiter 300 C4, Aquity UPLC BEH300 C4, Delta-
silica particles, 1.5 to 10 mm in diameter. Pak C4, Symmetry300 C4, XBridge BEH300 C4
L27 Porous silica particles, 30 to 50 mm in diameter. Sepra (I), Porasil (I), Nucleodur, YMS-Pack Silica
L28 A multifunctional support, which consists of a high Altech mixed mode C8/anion, Generik C8/Amino,
, spherical silica substrate that has been
purity, 100 A ProTec C8
bonded with anionic (amine) functionality in addition
to a conventional reversed phase C8 functionality.
(Continued)
232 6. STATIONARY PHASES AND THEIR PERFORMANCE
L29 Gamma alumina, reversed phase, low carbon Gamabond ARP-1, Gamabond Alumina Potency
percentage by weight, alumina-based polybutadiene
spherical particles, 5 mm diameter with a pore diameter
.
of 80 A
L30 Ethyl silane (C2) chemically bonded to a totally porous (I), Nucleosuil C2, APEX Prepsil
Maxsil RP2 60 A
silica particle, 3 to 10 mm in diameter. C2
L31 A strong anion-exchange resin-quaternary amine Ion Pac AS 10, Ion Pack AS 16
bonded on latex particles attached to a core of 8.5 mm
macroporous particles having a pore size of 2000 A and
consisting of ethylvinylbenzene cross-linked with 55 %
divinyl benzene.
L32 A chiral ligand-exchange packing- L-proline copper CHIRACEL WH, Astec CLD-D (or eL), Nucleosil
complex covalently bonded to irregularly shaped silica Chiral-1
particles, 5 to 10 mm in diameter.
L33 Packing having the capacity to separate proteins of BioSep-SEC-S2000, BioSep-SEC-S3000 BioBasic
4,000 to 400,000 daltons. It is spherical, silica-based and SEC 120, Nucleosil 125-5 GFC, Shodex KW-404
processed to provide pH stability.
L34 Strong cation-exchange resin consisting of sulfonated Aminex Fast Carbohydrate, Rezex RPM
cross-linked styrene-divinylbenzene copolymer in the Monosaccharide, Shodex Sugar SP0810, Nucleogel
lead form, about 9 mm in diameter. Sugar Pb
L35 A zirconium-stabilized spherical silica packing with Bio-Sep-SEC-S2000, Zorbax GF-250, Zorbax GF-450
a hydrophilic (diol-type) molecular monolayer bonded
phase having a pore size of 150 A.
(Continued)
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 233
TABLE 6.3.4 USP classification of HPLC chromatographic columns [73]*. (contd)
Code Description Examples
L44 A multifunctional support, which consists of a high Chromegabond RP-SCX, Generik C8/SCX
, spherical silica substrate that has been
purity, 60 A
bonded with a cationic exchanger, sulfonic acid
functionality in addition to a conventional reversed
phase C8 functionality.
L45 Beta cyclodextrin bonded to porous silica particles, Astec Cyclobond I, II or II ser., Chiral CD-Ph,
5 to 10 mm in diameter Nucleodex Beta-PM, ChiralDex,
L46 Polystyrene/divinylbenzene substrate agglomerated CarboPac PA1, Transgenomic AN1
with quaternary amine functionalized latex beads,
10 mm in diameter.
L47 High capacity anion-exchange microporous substrate, CarboPac MA1, Hamilton PRP-X100, X110,
fully functionalized with a trimethylamine group, Hamilton RCX-10
8 mm in diameter.
L48 Sulfonated, cross-linked polystyrene with an outer IonPac AS5, IonPac AS7
layer of submicron, porous, anion-exchange
microbeads, 15 mm in diameter.
L49 A reversed-phase packing made by coating a thin layer Zirchrom PBD, Discovery ZR-PBD
of polybutadiene on to spherical porous zirconia
particles, 3 to 10 mm in diameter.
L50 Multifunction resin with reversed-phase retention OmniPac PAX-500, Proteomix SAX-POR
and strong anion-exchange functionalities. The resin
consists of ethylvinylbenzene, 55 % cross-linked with
divinylbenzene copolymer, 3 to 15 mm in diameter, and
a surface area of not less than 350 m2/g, substrate is
coated with quaternary ammonium functionalized
latex particles consisting of styrene cross-linked with
divinylbenzene.
L51 Amylose tris-3,5-dimethylphenylcarbamate-coated, Chiralpak ADS, Nucleocel Alpha
porous, spherical, silica particles, 5 to 10 mm in
diameter.
L52 A strong cation exchange resin made of porous silica TSKgel SP-2SW, BioBasic SCX, Supecosil LC-SCX
with sulfopropyl groups, 5 to 10 mm in diameter.
L53 Weak cation-exchange resin consisting of IonPac CS14
ethylvinylbenzene, 55 % cross-linked with
divinylbenzene copolymer, 3 to 15 mm diameter.
Substrate is surface grafted with carboxylic acid
and/or phosphoric acid functionalized monomers.
Capacity not less than 500 mEq per column.
L54 A size exclusion medium made of covalent bonding of Superdex peptide HR 10/30
dextran to highly cross-linked porous agarose beads,
about 13 mm in diameter.
L55 A strong cation exchange resin made of porous silica IC-Pak C M/D, Waters Spherisorb SCX, Universal
coated with polybutadiene-maleic acid copolymer, Cation
about 5 mm in diameter.
(Continued)
234 6. STATIONARY PHASES AND THEIR PERFORMANCE
L58 Strong cation-exchange resin consisting of sulfonated Rezex RNM-Carbohydrate, Aminex HPX-87N,
cross-linked styrene- divinylbenzene copolymer in the Shodex SUGAR KS-801, -802, etc.
sodium form, about 7 to 11 mm diameter.
L59 Packing with the capacity to separate proteins by BioSep-SEC-S3000, Biosuite 125, Nanofilm SEC-
molecular weight over the range of 10 to 500 kDa. 150, TSK-GEL G2000SW, G3000SW, etc
Spherical 10 mm, silica-based, and processed to
provide hydrophilic characteristics and pH stability
L60 Spherical, porous silica gel, 3 to 10 mm in diameter, Acclaim polar Advantage, Ascentis RP-Amide,
surface has been covalently modified with HALO RP-Amide, Prism RP, Supecosil LC-ABZ,
palmitamidopropyl groups and endcapped. etc.
L61 Hydroxide-selective, strong anion-exchange resin Ion Pac AS-11, Ion Pac AG-11
consisting of a highly cross-linked core of 13 mm
microporous particles, pore size less than 10 A , and
consisting of ethylvinylbenzene cross-linked with 55 %
divinylbenzene with a latex coating composed of
85 nm diameter microbeads bonded with alkanol
quarternary ammonium ions (6 %).
L62 C30 silane bonded phase on a fully porous spherical Develosil Combi-RP, Develosil RP-Aqueous,
silica, 3 to 15 mm in diameter. Develosil RP-Aqueous-AR, ProntoSil c30, YMC-
Pack Carotenoid, Zodiac 120 C30
L63 Glicopeptide teicoplanin linked to spherical silica CHIROBIOTIC V, T, TAG, R.
(chiral phase)
L64 Strongly basic anion exchange resin consisting of 8% AG 1-X8
crosslinked styrene-divinylbenzene copolymer with
a quaternary ammonium group in the chloride form,
45 to 180 mm in diameter
(Continued)
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 235
TABLE 6.3.4 USP classification of HPLC chromatographic columns [73]*. (contd)
Code Description Examples
L## (Ethylhexyl triazone, FluoFix)-Fluorocarbon chains Wakopak FluoFix-II 120E, 120E, 120N
chemically bonded to 5 mm spherical silica particles
* Note: All particles are spherical unless differently indicated (M) for monolith, (I) for irregular shape.
as code L1, L2, and so on, is shown in Table from column to column because it refers to a solid
6.3.4, including several examples of columns phase that is not even uniform, containing groups
for each category [73]. In spite of its complexity, belonging to both the support and the bonded
the USP code is not always appropriate for moieties. Characterization of columns using
a column classification. For example, new a set of parameters obtained from their behavior
core-shell type columns such as Kinetex XB- toward a set of "test" compounds has been devel-
C18, Poroshell 120 SB-C18, or certain zirconia- oped for a considerable number of RP columns
based columns such as ZirChrom-EZ, are not (see Section 6.4) as well as for HILIC columns
easily classifiable using the criteria shown in (see Section 6.5). However, a simplified character-
Table 6.3.4. ization can be obtained using Kow (log Kow) values
for simplified models representing different
Characterization of the Polarity of stationary phases. This type of characterization
a Column Using Kow for Models is given in Table 6.3.5. The models from this table
were selected only to illustrate the variation in the
of Stationary Phase polarity of different columns. However, other
The polarity of the stationary phase is an models can be used for the same purpose. In the
important parameter for predicting its behavior models from Table 6.3.5, the terminal -Si(CH3)3
in a separation. As previously discussed in group was selected to simulate an end-capped
Section 5.1, this polarity is difficult to assess silica structure with hydrophobic character. This
236 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.3.5 Simplified models for the stationary phase and the corresponding calculated log Kow
Fluorinated F F F F F F 8.38
CH3
O
F 3C Si Si CH3
F F F F H 3C CH3 CH3
Amide O 7.86
embedded CH 3
C O
H 3C NH Si Si CH 3
H 3C CH 3 OH
Butyl-phenyl 5.81
CH 3
O
Si Si CH 3
H 3C CH 3 CH 3
Cyanopropyl CH 3 2.80
O
C Si Si CH 3
N CH 3
H 3C CH 3
(Continued)
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 237
TABLE 6.3.5 Simplified models for the stationary phase and the corresponding calculated log Kow (contd)
Column Model structure log Kow*
Zwitterionic CH 3 0.43
CH 3
HO O
C N Si Si CH 3
H 3C CH 3 CH 3
O CH 3
* Note: The Kow values for the model compounds were calculated using a computer program from MarvinSketch 5.4.0.1, ChemAxon Ltd., [74].
group was replaced with -Si(CH3)2OH for for assessing the selectivity of the column. A
surfaces that were not end-capped. hydrophobic selectivity parameter is particu-
larly useful for characterization of hydrophobic
Performance of Stationary Phases stationary phases (see Section 6.4). Selectivity
a is also the parameter to which the resolution R
and Columns
is very sensitive; therefore the choice of a column
The criteria for comparing the performance with large a for the specific separation is very
of a stationary phase include a number of char- important. The resolution is also a function of k
acteristics, some related to the chemical and and N (as given by rel. 2.1.57, repeated in 6.3.1).
physical properties of the phase and others
related to external factors [75e77]. Among these 1 a1 k
R N 1=2 (6.3.1)
criteria are those listed in Table 6.3.6. 4 a 1k
The separation capability is typically character-
ized by the selectivity a, which is dependent, The choice of a column for which the capacity
besides the column characteristics, on the nature factor k value is higher can also be advantageous
of the compounds to be separated and on the for achieving a specific separation. Higher k
mobile phase. A variety of tests were developed values help the separation but also indicate
238 6. STATIONARY PHASES AND THEIR PERFORMANCE
No. Criterion *
1 Efficiency and ability to obtain separation of sample analytes, between them and from the sample V
matrix
2 Low skew/low asymmetry/low tailing C
3 Large values for theoretical plate number N I
4 Achieve separation within relatively low tR (low tR being related to low k values) C
5 No bleed of the stationary phase I
6 Stability to extreme operating conditions such as low pH, high pH, range of solvents (e.g. high D
water content for hydrophobic columns), various components in the sample matrix
7 Capability to maintain identical separation characteristics for a large number of injections I
8 Stability under varying operating conditions D
9 Rapid regeneration capability D
10 Acceptable values for the backpressure and the capability to maintain this back-pressure constant in C
time when identical conditions of a separation are used
11 Ability to tolerate high pressure and a rapid change in pressure D
12 Reproducibility of the separation from column to column of the same type but from different D
batches
13 Capability to achieve analytes separation within a wide range of experimental conditions (have D
versatility)
14 Availability in different formats that can be chosen to fit a wide range of available HPLC D
instruments, and ease of transferability from format to format of separation conditions
15 Low price and extensive information regarding the column characteristics D
longer retention times, which is not always desir- particles significantly increases the number of
able. Because the k values are both compound theoretical plates as compared to porous particles
dependent and mobile phase dependent, the of the same dimensions. For example, in
comparison of columns regarding the k value a comparison to a traditional 3 mm particle C18
should be performed under similar conditions column (150 mm length and 4.6 mm i.d.) that
[78, 79]. had N 166,502 theoretical plate per m, a core-
The increase in resolution R is also dependent shell column with the same dimensions (Kinet-
on the increase in the number of theoretical ex from Phenomenex) had N 295,343 per m.
plates N (and n). This indicates that columns A traditional 1.7 mm particle C18 column (50
with high N values are preferable, as they mm length and 2.1 mm i.d.) had N 272,080
produce narrower peaks. Higher N (and n) per m, while a core-shell column with the same
values can be achieved through specific proper- dimensions (Kinetex from Phenomenex) had
ties of the stationary phase such as small parti- N 318,680 per m. On the other hand, core-shell
cles, uniform shape (spherical), and narrow columns may have lower k. The use of monolith
particle size distribution. The use of core-shell columns may also reduce peak broadening.
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 239
The peak shape is another parameter of consid- mobile phase significantly decreases the ioniza-
erable importance in HPLC. Besides the decrease tion of the silanol groups still existent in the
in the column resolution due to tailing or other stationary phase, diminishing interactions and
effects seen in a column with high skew, the preci- leading to better peak shape. The stability of
sion of the peak area integration is also affected by different columns at the pH of the mobile phase
the peak shape. Correct measurement of peak is typically reported by the column manufacturer
area is essential in obtaining quantitative informa- and/or is reported in the literature. Table 6.3.7
tion in HPLC. For this reason, columns with lower shows some of these reported results [75].
skew/asymmetry are preferable to those gener- The range of accepted solvents is another
ating peaks far from a Gaussian shape. The important factor regarding the quality of
skew/asymmetry values are compound depen- a stationary phase / chromatographic column.
dent, and a specific column may be perfectly suit- For the stationary phases used in size exclusion,
able for the separation and measurement of some ligand exchange, displacement chromatography,
compounds and may not be good for others. Peak bioaffinity chromatography, and chiral separa-
tailing is frequently caused by multiple types of tions, the columns are much more sensitive,
interactions of the stationary phase with the ana- and special care must be taken regarding the
lytes, such as hydrophobic interactions and polar nature of the mobile phase. The phases consist-
interactions, which may take place together, for ing of bonded organic moieties on a silica surface
example, in a column with a hydrophobic phase or on organic polymers, such as those used in
on silica that also has numerous free silanol RP-HPLC, are in general resistant to a large
groups [80]. A variety of phases were designed number of solvents. However, in some cases,
to reduce this problem, for example, those using the solvent may sufficiently modify the surface
end-capping. Also, the very high purity of the of the stationary phase to affect its properties
silica base (low in trace metals) contributes to regarding the separation. One such solvent is
the reduction of tailing. pure water (with no organic addition), which
The pH range acceptable for the mobile phase can affect conventional reversed stationary
used with a specific column is another parameter phases by a process known as dewetting and
that characterizes the performance of a specific also phase collapse (not mechanically). Most
stationary phase. The separation of some solutes separations on hydrophobic stationary phases
does not require mobile phases with the pH lower take place using a water-organic solvent with
than 2 or higher than 8. Most HPLC columns with a solvent ratio between 20 and 80%. Phases that
the stationary phase based on a silica support can can be used at higher water content (even in
be used without any problems in this range of 100% water) have been developed and are pref-
pH, and the organic polymeric stationary phases erable in numerous applications, in particular
are stable in a wider pH range. However, specific when the analytes are poorly retained by the
applications may require lower or higher pH for hydrophobic stationary phase.
the mobile phase than the range 2 to 8. For this The stability in time of the stationary phase is
reason, columns that are stable in a wider pH another important characteristic. This character-
range are preferred. The problem with the need istic can be estimated by the number of injections
for a wide pH range is encountered in particular n that can be made on a column, with unaltered
for basic compounds that can be partially ionized results for the chromatography. This number of
in the pH range 4 to 8. The ionized components of injections depends, however, on numerous other
the analyte interact with the free or ionized silanol factors besides the column quality. The type of
groups of the stationary phase, producing tailing sample, the nature of the matrix, the degree of
(undesirable peak shape). Lower pH of the cleanup performed on the sample before
240 6. STATIONARY PHASES AND THEIR PERFORMANCE
Column Manufacturer pH 2 pH 3 pH 6 pH 7 pH 7 pH 8 pH 9 pH 10 pH 11
ammon./ tri-
Medum** TFA phosph. phosph. acetat. phosph. bicarb. bicarb ammon. phosph.
* Note: 1 Stable to more than 500 injections, 2 Stable to more than 200 but less than 500 injections, 3 Stable only to less than 200 injections,
? no data available.
** Note: TFA trifluoroacetic acid, phosph. phosphate buffer, acetat. acetate buffer, bicarb. bicarbonate buffer, ammon. ammonia, tri-phosph.
sodium triphosphate.
injection, the injection volume, the sample for the chromatographic column is, however,
concentration, and the like, are factors influ- associated with a slightly lower value for the
encing n. Also, in order to protect the column number of theoretical plates N, and sometimes
from undesirable materials that may reach the a compromise should be chosen between
stationary phase, filters and pre-columns are a more robust column and a column offering
commonly utilized in the sample path. The effi- a better separation. One additional factor that
ciency of the pre-column and the frequency must be considered when changing from
with which this pre-column is changed also influ- a column with one diameter d1 to a column with
ence the number of injections n, as does the nature a different diameter d2 is the change in linear
of the mobile phase . When the column is utilized flow rate in the column. Considering that the
at extreme pH values (very close to the limits of volumetric linear flow rate U is related to the
the acceptable range), the lifetime of the column linear flow rate by the formula U A u, where
is diminished. The typical problems associated A is the area of the channel in which the flow takes
with columns that become old are loss of reten- place, in order to maintain the same linear flow
tion, peak broadening, increase in backpressure, rate u in two columns with different diameters,
and bleeding (observable especially in MS detec- the following condition must be fulfilled:
tion). Columns with predicted high n values are
those resistant to a wider pH range, have a lower A1 d2
U1 U2 12 U2 (6.3.2)
backpressure characterized as new, and/or have A2 d2
a high purity of the silica material. In practice, it
can also be noted that columns with a larger Relation 6.3.2 indicates that for narrower
amount of stationary phase appear to be more columns, a lower volumetric flow rate U is
resilient in time and lead to larger n. Larger i.d. necessary to achieve the same linear flow rate
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 241
u in the two columns. Since the height of theo- between the choice of smaller particles with
retical plate H is dependent on u (see van higher backpressure or better separation [81,
Deemter equation 2.2.8), the efficiency of the 82]. Since the viscosity of liquids typically
separation can be affected when changing decreases as the temperature increases,
columns with different diameters, even if all a higher temperature for the column typically
the other parameters are kept identical but the leads to a lower backpressure. However, limi-
flow rate is not adjusted properly. tations on the temperature are imposed by
The acceptable value for the backpressure of other factors such as the effect of temperature
a column when working in typical conditions on the separation and stability of the analytes,
is a very important parameter. This parameter solvents, and stationary phase. Columns with
is frequently limited not by the column but by core-shell particles and monolithic columns
the maximum possible pressure generated by characterized by the same number of theoret-
the HPLC pumps. In UPLC, where smaller ical plates N compared to traditional columns
particles and column diameters are selected have lower backpressure for similar flow
for achieving higher N values, the maximum conditions. For example, for a traditional C18
pressure of the pumps is higher than in stan- column, 1.7 mm particles, 50 mm long, 2.1
dard HPLC. The value of a column backpres- mm i.d, a typical working backpressure in
sure is determined by the particle size, the CH3CN/H2O 50/50 is higher than 400 bar at
flow rate, the nature of the mobile phase, 0.6 mL/min flow rate (N z 270,000 per m).
temperature, the column dimension, and the A core-shell C18 column with 2.6 mm particles
like. A formula that governs backpressure in that has a similar N, and identical length and
a packed bad column (difference between the i.d., develops pressures below 300 bar.
pressure at the column inlet and that at the From rel. 6.3.3, it can be seen that the specific
outlet) is the following: permeability K0 for a column can be obtained
hUL from the expression:
Dp (6.3.3)
pK0 r2 d2p hUL
K0 (6.3.4)
where Dp is the pressure necessary for a liquid to pDp r2 d2p
flow through the column with the volumetric
flow rate U. In rel. 6.3.3 h is the mobile phase For practical purposes, instead of the specific
viscosity (typically given in mPa s), K0 is specific permeability K0, a column permeability K* that
permeability for the phase (adimensional), r is can be directly measured and is defined by the
the column radius, L is its length, and dp is the formula:
diameter of the particles in the bed (in mm) K
uL
(6.3.5)
(see Darcy equation, rel. 2.2.17). Dp
As expected, the backpressure for a chro-
where u is the linear flow rate. Since u L / t0
matographic column is higher at higher
the expression for K* can be written in the form:
viscosity of the mobile phase, higher flow
rates, and longer columns. However, the L2
column diameter and the diameter of the K (6.3.6)
Dpt0
particles of the stationary phase are even
more sensitive parameters affecting Dp since The value for K* can be measured for a given
there are values at power 2. Since dp is also column based on Dp, t0 and L and provides
related to the number of theoretical plates of information on the column characteristics.
the column, a compromise must be found From rel. 2.2.3 that connects linear flow rate u
242 6. STATIONARY PHASES AND THEIR PERFORMANCE
with volumetric flow rate U, the relation column is very different from that for silica-
between K0 and K* is the following: based columns. For the silica-based columns,
the rigidity of the silica support and the pressure
h
K 0 K (6.3.7) at which the column was packed are important
d2p factors regarding the pressure limit for a specific
column. This working pressure must be lower
(where * is the column packing porosity). This than the specified maximum pressure, a wider gap
formula allows the evaluation of K0 for between the two values being desirable.
a column, when the solvent viscosity h, particle The reproducibility of the separation from
dimensions dp and * are known. column to column of the same type, but from
Holding the backpressure of a column in different batches, is another important param-
time, after repeated injections, is dependent on eter. By using high-purity silica, novel derivati-
the operating conditions, as well as on the zation procedures, standardized conditions for
quality of the stationary phase. Plugging the column packing, and rigorous column testing
frits of the column with sample components, before being put on the market, the requirement
use of extreme pH values for the mobile phase to be reproducible is met by most modern HPLC
that degrade the stationary phase, use of unfil- columns.
tered solvents that contain small particles, and The availability of the columns in different
plugging the column with noneluted compo- formats for the same stationary phase and for
nents are among the practices that must be ease of transferring a separation method from
avoided but are not directly related to column one column to another is also desirable. Longer
quality. The extreme case of column plugging columns for the same phase lead to higher
such as the use of a mobile phase that generates values for N (and n) as shown by rel. 2.1.42.
a precipitate in the column (e.g., use of an inor- Also, the decrease in the particle size (for
ganic buffer that precipitates during a gradient phases of the same nature) has a significant
run) also must be avoided. However, the impact on the N value. Since R is proportional
strength of the silica particles (for the column with N1/2, longer columns and columns with
with a packed particle bed) as well as the absence lower particle size are sometimes necessary
of very small dust particles in the stationary for better resolution. Increasing the column
phase are characteristics of the stationary phase length and decreasing the particle size have
necessary to avoid plugging in time of the frit the effect of increasing the backpressure. For
that retains the stationary phase in the column, this reason, the availability of equivalent
which can cause a significant increase in the phases in different formats that can accommo-
backpressure. date different requirements such as separation
One other aspect of column backpressure characteristics, column backpressure, and
(when the pumps are able to deliver a specified sample size is very useful.
one) is the ability of the stationary phase to
tolerate that high pressure and a rapid change Selection of a Column for an HPLC
in pressure (e.g., during a gradient run when
Separation
the mobile phase viscosity changes). This factor
is highly dependent on the nature of the The main criterion in the choice of a chromato-
stationary phase. For size-exclusion chromatog- graphic column is the adequacy for performing
raphy, for example, where the stationary phase the proper separation intended. This separation
is typically made of highly porous organic poly- must produce well-defined peaks (narrow, with
mers, the recommended working pressure of the no tailing) such that the peak processing can be
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 243
done accurately. Peak recognition, peak area can be achieved using higher flow rates, and
measurement, data averaging, and other data narrower and shorter chromatographic
processing operations done by the computer columns. This type of columns is used, for
programs controlling the HPLC instruments are example, in UPLC-type techniques.
always performed more accurately and repro- 2) The main property of the column that is
ducibly when the peak shape is good. basically independent of a specific
In general, the selection of a column is deter- compound can be considered the number of
mined by (1) the properties of the compounds theoretical plates N (although N varies to
that have to be separated, (2) the specific a certain extent from compound to
column properties that are compound indepen- compound, this variation is not significant
dent, (3) the requirements of the separation, and the choice of a specific molecular species
independent of analytes and mobile phase, for measuring N eliminates this variability).
and 4) the mobile phase requirements. As previously discussed, columns with
smaller and more uniform particles lead to
1) The column choice depends mainly on the higher N values. Also the columns with core-
properties of the compounds to be shell particles are highly recommended for
separated. This should be associated with their high N values although they have
the selection of the mobile phase/elution lower J and therefore lower k values.
program such that a successful separation Geometric properties of the column also
is achieved. Further details regarding the contribute to the success of a separation.
selection of a chromatographic column Longer columns lead to a higher N, but at
in function of the compound/class of the same time the analysis time is typically
compounds that are subject to analysis are increased. Also, the column backpressure is
given in this book. The main goal in higher for longer columns. The linear
a separation is to obtain good R values for variation of the retention time with the
the compounds of interest. This is achieved column length is shown in Figure 6.3.1 for
by having large N, a, and k values for the seven test compounds on a Zorbax SB-C18;
separation. Further discussions will be 3.5 mm particle diameter column.
found in this book regarding ways to obtain The change in column diameter also may
columns with large N values, good affect the separation. Relation 6.3.2 shows
separation characteristics, and high k and the requirement in volumetric flow rate
a values for the compounds of interest. change for achieving identical linear flow
Sometimes other special requirements are rates in columns of different diameter.
imposed on the analysis; for example, low tR Columns with a larger diameter offer better
(that typically imply low k values) in resilience regarding the number of samples
a separation are necessary when rapid that can be injected without affecting results
analyses are needed. Also, as the separation but in general have a slightly lower N (for
time increases, the broadening of the the same length) and require larger volumes
chromatographic peaks increases (see rel. of mobile phase.
2.1.29 and Figure 2.1.2). Since an Besides the properties of a column related
improvement in the resolution R is related to to the separation in itself, other
the increase in the k value, other procedures characteristics related to the column
are involved that attempt to reduce both t0 utilization are important. For example, the
and tR, such that the value of k (tR e t0)/t0 column should be chemically stable and
is not affected, although tR is smaller. This show no bleed. The bleed indicates column
244 6. STATIONARY PHASES AND THEIR PERFORMANCE
3
choice of the chromatographic column.
4) Mobile phase requirements such as pH,
2 water content in the mobile phase, mobile
1 phase viscosity may also influence the
column selection.
0
0 20 40 60 80 100 120 140 160
L (mm) A number of other column characteristics are
listed in Table 6.3.6 and labeled as desirable.
FIGURE 6.3.1 The variation of retention time for seven The importance of these characteristics may
test compounds with the variation of the column length vary. When some of them cannot be fulfilled
(Zorbax SB-C18; 3.5 mm particle diameter) (as shown in [71]).
but the separation is good and the results are
reproducible, the column is still preferred to
degradation but can also interfere with a similar one that offers a better property in the
detection, in particular for specific detectors desirable group but notr, for example, such
(such as MS). The resilience to a wide pH a good separation. At this point, the choice of
range is another desirable property. The the column is more related to individual prefer-
backpressure produced by the column is ences and needs.
another practical parameter to consider.
High backpressures are not desirable for The Use of Guard Columns
a number of reasons, including frequent
and Cartridges
instrument failure associated with it.
The HPLC separations are typically Some of the column qualities, especially the
performed repeatedly, and analyzing capability to maintain identical separation char-
numerous samples by the same method is acteristics for a large number of injections, can
common in many laboratories. The analysis be improved with the use of guard columns or
of numerous samples and the associated guard cartridges (e.g., SecurityGuard from
data analysis is typically performed using Phenomenex). Even if the frits that are used in
computer controlled instruments. Maintain- the path of the mobile phase eliminate solid
ing the same separation characteristics contaminants that may plug the analytical
when numerous samples are analyzed is column, when the column is used for a longer
another important property for the chromato- period of time, certain strongly retained mate-
graphic column. This will allow peak rials can accumulate on the column, may never
identification without human intervention be eluted, and may dramatically reduce column
and facilitates accurate data reporting. lifetime. By modifying the surface of the
3) The analysis requirements such as speed, stationary phase, these retained materials can
number of samples to be analyzed, desire to cause shifts in peak retention, loss of resolution
collect the separated analytes or not, and and efficiency, and degradation of peak shape.
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 245
Another factor contributing to column degrada- isopropanol or hexane. Other columns such as
tion can be the use of "harsh" mobile phases that those used for ion exclusion can be received
can destroy in time the stationary phase (e.g., filled with water. Typically, buffers are avoided
when a silica-based stationary phase is sub- as storage solvent, although some columns are
jected to pH < 2 or pH > 8 solutions). Even received filled with a specific solvent with
when common solvents are used, some dissolu- buffer, such as HILIC columns that can be
tion of silica (for silica-based columns) may received in acetonitrile/water (e.g., 90:10 v/v)
occur, leading to the formation of a void volume that contains 100 mM of HCOONH4. The
at the head of the column and subsequent columns must be properly installed, and pre-
reduced column resolution. A common proce- filtering and adequate guard columns are
dure to protect the analytical column from these always recommended. The mobile phase that
problems is to use a guard column. Guard is selected with the goal of achieving separation
columns are short (or very short) columns or must comply with specific restrictions
cartridges, packed with a stationary phase regarding the nature of the solvent and the
having an identical or a slightly less retentive pH range. When using gradients, the solvents
bonded-phase as the analytical column. The used as mobile phase must be miscible. Also,
guard column should have a very small dead switching from one solvent to another should
volume such that it should not affect the column be done considering solvent miscibility. The
performance. At the same time, the guard solvent composition must be kept within the
column retains the noneluting contaminants limits recommended by the vendor. For
and provides saturation of the mobile phase example, the water content used with an RP
with silica by bleeding silica into the mobile column should not exceed the recommended
phase instead of from the analytical column. limit in order to avoid phase collapse by dewet-
The guard column must be replaced from time ting. The addition of salts for the modification
to time, before the analytical column starts to of pH or ionic strength must be done at the
degrade. The replacement time interval is deter- lowest possible concentration, and salt precipi-
mined by the specific experimental conditions tation in the column will destroy it. The pH
and can be done either at a fixed schedule range where the column is stable should be
(such as 100 injections) or when signs such as strictly obeyed. The temperature at which the
increase in the backpressure or some loss of column is used should not exceed the vendor
resolution begin to be noticed. specifications. For RP columns this tempera-
ture is typically not higher than 60 oC. The
maximum backpressure of the column (indi-
Column Protection, Cleaning,
cated by the vendor) should never be exceeded,
Regeneration, and Storing
and the flow should go in the recommended
Commercially available chromatographic direction (indicated on the column). Sudden
columns are typically received filled with a pressure changes must be avoided. Also, the
specific solvent that is specified by the vendor, samples injected in the column should be (as
and their storage is recommended to be done in much as possible) completely eluted.
a similar solvent as received. The columns for After a period of usage, some column degra-
reversed phase are usually received filled dation may be noticed, with broader peaks,
with acetonitrile/water 65:/35 v/v; silica- lower retention times, decreased separation
based ion-exchange columns are received filled capability, and increased column backpressure.
with methanol or methanol/water; normal- When any of these problems appear, a column
phase columns are received filled with cleaning may be useful. The cleaning is typically
246 6. STATIONARY PHASES AND THEIR PERFORMANCE
done by rinsing the column (e.g., 10- to 20- compound or to a mixture of unseparated
column volume) with a specific solvent or compounds. The use of detectors that allow
sequence of solvents at 1/5 to 1/2 typical flow the measurement of a compound even if it is
rate. For example, for RP columns, a set of present in a mixture improves the capability
solvents covering a wide range of polarity is rec- of analysis even if the HPLC separation is
ommended, starting with 95:5 water/acetoni- not good. This is, for example, the case of the
trile v/v, followed by tetrahydrofuran (THF), use of UV detection for a compound with
followed by 95:5 acetonitrile/water v/v. a unique absorption wavelength; the use of
Columns used for protein separations may be fluorescence detection when the compound
washed with 0.1% trifluoroacetic acid (TFA) in has unique excitation/emission wavelengths;
water followed by 0.1% TFA in acetonitrile/ or the use of MS detection. However, in
isopropanol v/v, followed by rinsing with many cases the use of selective detection is
a common mobile phase (columns should not either not sufficient or not possible. In such
be stored in THF). Other specific cleaning proce- cases, the use of two columns with very
dures may be recommended by the column different selectivity may solve the problem. If
manufacturer. In general, one should not use the second column does not lead to the separa-
a flow in the opposite direction than recommen- tion of additional peaks, it is more likely that
ded, in order to diminish the backpressure the first separation is complete. Columns
increase. However, this restriction seems to be with very different selectivity are also desir-
less important for columns with small particles, able for two-dimensional separations, when
when back-flushing with a strong solvent are in segments from the eluate from one column
some cases used to wash materials accumulated are further separated on the second column
at the head of the column. (2-D or multidimensional separations). The
Columns that are not used for a certain period HPLC separations performed on different
of time must be stored appropriately. For this types of columns and using different solvents
purpose, the clean column must be filled with that lead to a different separation are labeled
a specific solvent. For RP-type columns, a mixture as orthogonal. The orthogonality of one sepa-
of 65% acetonitrile and 35% water is recommen- ration versus another one can be evaluated
ded. Before storing, the column must be cleaned using procedures specifically developed for
from the presence of buffers. For normal-phase this purpose. These procedures are based on
columns, isopropanol is typically recommended the use of test mixtures on the two columns
as storing solvent. Ion-exchange columns are and the comparison of separation results [83].
usually stored in methanol or methanol/water. A potential procedure for testing orthogonality
For size-exclusion columns, water with 0.05% is to make a plot of retention times for the
NaN3 (to avoid bacteria growth) or with 10% compounds from the test mixture for the sepa-
methanol is typically recommended. However, ration on the two columns. A good correlation
column manufacturers may provide specific between the retention times indicates a poor
recommendations for column storage that must orthogonality. Because the separation selec-
be followed. tivity in the HILIC mode is complementary
to that in reversed phase and other modes,
Selection of Columns for Orthogonal combinations of the HILIC, RP and other
systems are attractive for two-dimensional
Separations
applications. Other criteria are based on
In HPLC practice, it is sometimes difficult to comparing column properties such as hydro-
decide whether a peak belongs to a unique phobic character and hydrogen-bonding
6.3. PROPERTIES OF STATIONARY PHASES AND COLUMNS 247
characteristics. Column differences are dis- spectrometry (ICP-AES). For example, the Fe2
cussed further in Section 6.4. content of the silica in many packing materials
is between 5 and 400 ppm, Ni2 content is about
1e2 ppm, and Al3 is about 100 ppm. Other
Physicochemical Characterization
elements are at the ppb levels or lower. Acid
of Stationary Phases washing was proved by ICP to reduce the metal
Because the quality of stationary phases for content of silica by 70%, while washing the
HPLC is determined by the phase physical support with ethylenediaminetetraacetic acid
and chemical properties, the measurement of (EDTA) resulted in silica that is completely
some of these properties provides important free of metal impurities [87].
information regarding the quality of the phase Various adsorption studies are utilized
[84]. The investigation of physicochemical prop- to evaluate the surface area of the HPLC
erties can have the following purposes [85]: stationary phase. Surface area is one the
most important physical parameters because
1. Determination of bulk properties of stationary
HPLC retention is proportional to this param-
phase, such as specific surface area, volume,
eter. Several common experimental methods
and size distribution of the pores and of the
are available for measurement of the surface
particles.
area of porous silica, and nitrogen or argon
2. Characterization of the surface structure of
adsorption isotherms at the temperature of
the packing materials considering the
liquid nitrogen (around 77 K) are typically
concentration, conformation, and mobility
used in accordance with BET theory for
of the organic attached groups, as well
calculating the total surface area per unit of
as characterizationof the structure,
adsorbent weight [88, 89]. Inverse SEC has
chemistry, and accessibility to the phase
been utilized for the same purpose [90e92].
support.
For example, retention data of polystyrene
The procedures used to achieve these samples of narrow molecular size distribution
purposes can be destructive or nondestructive. and known average molecular mass were used
In the destructive procedures, the packing to estimate the external, internal, and total
material is subjected to chemical or physical porosities of monolithic columns (Chromolith
modifications that alter the phase. The nonde- Performance from Merck) and compared to
structive procedures involve instrumental those of the conventional packed column
analysis, such as fluorescence, infrared spectrom- (Luna C18 from Phenomenex) [93].
etry, or NMR techniques. These methods leave Other techniques for studying the
the packing material intact so that it is suitable stationary phase involve scanning electron
for direct chromatographic characterization [86]. microscopy (SEM), infra red spectroscopy
Among the destructive techniques is the mea- (IR), diffuse reflectance infrared Fourier trans-
surement of carbon load (C%) of the stationary form spectrometry (DRIFTS), nuclear magnetic
phase. For this measurement, the combustion resonance (NMR), fluorescence spectroscopy,
of the stationary phase and the measurement and thermogravimetric analysis (TGA). The
of total ash and CO2 can be performed using DRIFTS technique is used in particular for
elemental analysis instruments. Metallic impu- evaluation of silanol density and water
rities in silica (before and after acidic washing) adsorption on silica surface. Usually, in the
or modified silica materials are also determined IR spectra of silicas (silica gels) there are two
by a destructive technique, typically using intense IR absorption bands situated at 3400
inductively coupled plasmaeatomic emission and 1600 cm1, which are assigned,
248 6. STATIONARY PHASES AND THEIR PERFORMANCE
respectively, to the stretching and deformation Figure 6.3.2, on the low-coverage density of
vibrations of absorbed water molecules. After the bonded phases, the heat of immersion for
the absorbed water molecules are removed, hexane is lower than for methanol or acetoni-
the infrared spectrum of silica is characterized trile, but for high-covered bonded phases,
by a sharp band near 3750 cm1, assigned to hexane adsorbs higher heat than acetonitrile
the vibration of the isolated silanol groups on because the interactions with the C18 chains
the surface and a broad band in the range of start to have more contributions.
3900e3200 cm1 for the H-bonded silanols Hydride silica stationary phases that have the
and the residual water molecule. Also, TGA typical groups Si-H were studied using IR spec-
has found wide application in estimation of troscopy based on the characteristic stretching
silanol number. Silanol activity and, indirectly, band situated at 2250 cm-1. The decrease of the
the coverage density with the bonded phase intensity of this band can be used in evaluating
can also be characterized using microcalorim- the substitution with alkyl group during deriva-
etry, based on the heat of adsorption of tization of silica surface, with the CeH bonds
a solvent on the stationary phase material from bonded phases characterized by strong
(heat of immersion) [94]. When the silica stretching bands situated between 3000 and
surface is covered with a bonded phase (e.g., 2800 cm-1. The Si-O bonds have absorption
C18), the number of free silanol groups bands situated between 900 and 1300 cm-1.
decreases, and this will produce a decrease Alumina surfaces are characterized by the
in the heat of adsorption for polar solvents. same procedures [25].
This effect is shown in Figure 6.3.2 represent- Considerable attention has been given to
ing the decrease in the heat of immersion for the study, by spectroscopic techniques, of the
several C18 stationary phases obtained from stationary phase structure. For example, the
the same silica (7.1 mmol/m2 silanol groups) conformational state of the alkyl chains in silica
and having different coverage densities with gel modified with alkyl chains of various
C18 groups, for three different solvents (meth- lengths in the dry state was investigated over
anol, acetonitrile, and hexane). As seen from a temperature range of 123e353 K using
Hexane
10
0
0 1 2 3 4
Coverage density ( m ol/m 2)
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 249
infrared spectroscopy [95]. The conformational and unbranched alkyl groups resulting
state of the alkyl chains in their bound state during silica derivatization and functionaliza-
via various conformation-sensitive vibrational tion [97].
bands of the symmetric and antisymmetric
CH2 stretching bands was monitored. CH2
wagging type vibration bands were used to
6.4. HYDROPHOBIC STATIONARY
identify and determine the relative amounts
PHASES AND COLUMNS
of different conformers over the whole chains.
The conformational state of the chemically
Types of Hydrophobic Stationary Phases
attached alkyl chains was found to depend
strongly on temperature, actual chain length, The technique most often utilized in prac-
and chain position. Shorter alkyl chains, such tice is reversed phase (RP), and this type
as octyl and decyl chains, were reported to of HPLC requires hydrophobic stationary
possess a high degree of conformational phases. The related technique known as
disorder, and increase in alkyl chain length nonaqueous reversed-phase chromatography
was found to decrease the conformational (NARP), as well as ion-pair reversed-phase
disorder, most probably due to better chain chromatography, also use hydrophobic
packing. stationary phases. The most common hydro-
High-resolution 29Si-NMR has proved to be phobic phases are octadecyl (C18 or ODS)
a powerful tool for structural elucidation of and octyl (C8), which consist of octadecyldi-
organic and inorganic silicon compounds. methylsilane groups or octyl-dimethylsilane
Because of the sensitivity of 29Si chemical groups, respectively, usually bonded to high-
shifts, detailed information can be obtained purity, spherical silica gel. Other alkyl phases
by 29Si-NMR on the structural surroundings and phenyl phases on silica are used occasion-
of a given Si atom in a complex molecular ally, and long-chain phases such as C27 or C30
framework. This information can be used to are sometimes used for special applications.
study microstructural details of Si-O-Si link- Also considered as hydrophobic stationary
ages connected in chains or branched and phases but having some polarity are the fluo-
crosslinked structures. In solid-state samples, rinated phases, and intermediate between
due to the limited motion, strong dipolar- hydrophobic and polar phases are the cyano-
dipolar and chemical shift anisotropy interac- propyl type. Most bonded phases are made
tions occur. The line-broadening effects can be using so-called base-deactivated silicas (Type
canceled using the magic angle spinning B purity). The deactivation consists of acid
(MAS) technique. Other problems that arise washing of the silica before bonding single
in solid-state NMR spectroscopy related to or multiple reactant groups (ligands) to form
the strong heteronuclear interactions and to the desired active phase.
the existence of very long spin-lattice relaxa- Besides phases bonded on porous silica,
tion times of the order of minutes to hours core-shell particles are successfully utilized
are solved through the cross polarization as support for hydrophobic phases. Also,
(CP) technique [96]. Solid-state 13C-NMR monolithic silica-based materials with bonded
using cross polarization and magic angle hydrophobic phases are used in practice.
spinning (CP/MAS) can help characterize (Various types of silica supports were dis-
different carbon atoms from bonded phases. cussed in Section 6.1, and basic procedures
For example, this technique can be useful for silica surface derivatization were discussed
for characterization of different branched in Section 6.2.)
250 6. STATIONARY PHASES AND THEIR PERFORMANCE
Both octyl and octadecyl phases can have ethylene-bridged hybrids or BEH phases).
quite a large range of properties. These proper- Also, polar-embedded phases with a polar
ties are a result of different reagents used for group embedded between the silica surface
the silica derivatization (mono-, di-, or tri- and the hydrophobic moiety are successfully
functional), the reaction conditions (vertical or used. Among other advantages that may
horizontal polymerization), the degree of deriv- come from using the polar-embedded phases
atization (leading to different carbon loads), the include a wider pH range of stability and less
type of silica, and the treatment of free silanols effect on the separation of the free silanol
remaining on the silica surface (end-capping). groups. The covering or encapsulating of the
Silanol coverage is related to phase resistance silica surface with polymeric materials is also
to the pH of the mobile phase, as well as the used in some phases to reduce the activity of
intensity of other interactions exhibited by silanols. The results were phases showing
the phase. minimal interaction and no or little tailing
The common pH range for the stability of with strongly basic compounds (from where
silica-based stationary phases is between 2 the name base deactivated).
and 8. This range is not entirely satisfactory in Polymeric materials such as poly(styrene-
some applications. Basic pH is able to deterio- divinylbenzene) (PSeDVB), with or with no
rate the silica structure, and pH below 2 phase attached to the polymer base, can
produces hydrolysis of the fragments that also be used as reversed-phase media (these
make the active phase. Silica-based stationary being more resistant to high pH mobile
phases resistant to a wider pH range are highly phases), but they typically have lower column
desirable, particularly when more acidic mobile efficiencies than those of silica gel-based pack-
phases are necessary. Lowering the pH of the ings. Special phases such as graphitized
aqueous component in the mobile phase to carbon, silicon hydride, and zirconia based
very acidic values, close to 1, may improve are also known and have hydrophobic
some of the retention parameters (retention character.
time, peak shape, or selectivity), in particular Several hydrophobic types bonded on silica
for analytes having pKa < 2. One reason for and polymeric materials used in RP-HPLC are
this effect is that at low pH values the interac- presented in Table 6.4.1. As shown in the
tions of residual silanols with amino containing table, there are other types of interactions
solutes are suppressed, and thus peak tailing is besides the hydrophobic interactions that are
no longer observable. However, such low pH common to all columns.
values may not be acceptable for the stationary
phase stability. The end-capping of silanol
End-capping of Free Silanols
groups, with the purpose of reducing secondary
interactions with the analytes, also has some A considerable difference can be seen in
effect on extending the acceptable pH range the characteristics of stationary phases that
for the mobile phase. This effect is due to the are end-capped and those that are not. This
blocking of the access of H or OH- ions toward difference is illustrated, for example, in Table
the silica. 6.3.5 where values for log Kow are given for
Besides the phases with the hydrophobic models for the two types of phase. Besides
groups bound directly to a silica substrate, that, differences in peak shape and behavior
hydrophobic phases are also generated by toward polar compounds can be significant.
bonding groups such as C8 or C18 on Several strategies have been used to reduce
organic/inorganic silica particles (such as the undesirable effect of residual silanols.
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 251
TABLE 6.4.1 Several types of hydrophobic stationary phases.
1 n-Alkyl on silica* end-capped C2, C4, C8, C12, C14, C18, C20, Medium or strong hydrophobic
C22, C27, C30
2 n-Alkyl on silica not end-capped C8, C12, C14, C18 Strong hydrophobic and weak
polar
3 n-Alkyl on silica polar end-capped C8, C18 Strong hydrophobic and medium
polar
4 Polar embedded on silica C8, C18, etc. Strong hydrophobic and weak
polar
5 Cyclic alkyl on silica Cyclohexyl, n-C6 linked cyclohexyl Medium hydrophobic
6 Mixed alkyl on silica C18-short alkyl, C4-C18 Medium or strong hydrophobic
7 Aryl on silica Phenyl, diphenyl, C2 linked phenyl, Medium hydrophobic and strong
C6 linked phenyl aromatic
8 Mixed alkyl-aryl on silica C18-phenyl, C6-phenyl, cyclopropyl- Strong hydrophobic and medium
phenyl, C14-phenyl aromatic
9 Cyano on silica Cyanopropyl, phenylcyanopropyl Medium hydrophobic and strong
polar
10 Fluorinated phases on silica Pentafluorophenyl, perfluoroalkyl Medium hydrophobic
11 Other on silica Fulerene, cholesterol Strong hydrophobic
12 Silicon hydride C8, C18 bonded, silicon hydride Special
partially graphitized
13 Graphitized carbon Graphytized carbon Strong hydrophobic
14 Polymeric block Polystyrene/divinylbenzene, Strong hydrophobic
ethylvinylbenzene/divinylbenzene
15 Phases bonded on organic C18 bonded on polymethacrylate, Medium or strong hydrophobic
polymers phenyl bonded on polymethacrylate,
C18 bonded on divinylbenzene,
pentafluorophenyl bonded on
divinylbenzene
16 Phases bonded on zirconia C8, C18, etc. Strong hydrophobic
* Note: Phases indicated on silica include, besides silica, phases on organic/inorganic silica particles such as ethylene-bridged hybrids (BEH) or Gemini NX.
Among these strategies can be included the as providing further coverage of silanol
end-capping using trimethylsilyl groups by groups (e.g., Eclipse XDB). Another method
treating the phase with reagents such as trime- used for increasing the stability of the silica-
thylchlorosilane or hexamethyldisilazane after based stationary phase below pH 2 relies
the desired groups were already attached. on a special derivatization approach by which
This procedure was previously discussed. protecting groups are present close to the silica
Double end-capped columns are reported surface [98]. By steric hindrance, these groups
252 6. STATIONARY PHASES AND THEIR PERFORMANCE
provide a kind of shield for the area where the show the excellent resistance of these columns
active phase is attached, protecting it from an to both higher and lower pH values compared
acid attack from the mobile phase. This type to the stationary phase obtained on simple
of protection is achieved with voluminous silica.
groups, as shown schematically in the Another procedure to protect the silica
formulas in Figure 6.4.1. The figure shows surface is the use of very long hydrophobic
the chemical structures as well as the models alkyl chains (C27, C30) for derivatization.
of molecules (with R C4) picturing van der Because of the higher degree of surface shield-
Waals surfaces obtained from simplified struc- ing, long-chain stationary phases are stable
tures, with the geometry optimized using an over a wider pH range than C8 or C18
Alchemy 2000 program (Tripos, Inc., MO). bonded phases.
The larger volumes of isopropyl and isobutyl Besides end-capping with small hydro-
groups provide some protection to the bond phobic groups such as trimethylsilyl, end-
connecting the silica surface, with the group capping with small fragments containing polar
acting as stationary phase. End-capping can groups was found to protect the silica struc-
also be used to further protect from hydrolysis ture and extend the range of pH where the
stationary phases that are based on inorganic/ columns can be utilized. This procedure is
organic base structures, such as those contain- also utilized for generating phases with some
ing ethylene bridges. The reported results polar character in addition to the hydrophobic
R R
H3C R CH3
H3C Si CH3 CH Si CH CH2 Si CH2
H3C CH3
H+
..O.. H3C ..O .. CH3 CH
O .. ..
CH
H3C CH3
Si Si
Si
FIGURE 6.4.1 Schematic formulas for bonded phases having two methyl groups attached on the silicon bonded to the
active fragment, and for phases sterically protected with two steric-hindrance diisopropyl groups or diisobutyl groups (the
R chain was selected C4). Van der Waals surfaces with the geometry optimized using an Alchemy 2000 program are also
shown.
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 253
character of long alkyl groups. One such The silanol groups not connected to the silica
column (Synergi 4u Hydro-RP 80A) can be gel surface may have lower acidity and a protec-
used, for example, for the separation of amino tion effect against the attack of strong bases or
acids by ion-pair chromatography (see Section acids.
5.2). The polar-end-capping groups are usually Other procedures utilized to shield the inter-
amino or hydroxyl, bonded to a propyl chain. action of silanol groups with the analytes
These polar-end-capped stationary phases include the use of reagents having multi point
possess hydrophobicity similar to noneend- reactive functionalities for attaching the desired
capped ones, and they display enhanced group on silica surface [99]. Also, horizontal
hydrogen bond-type interactions, but the polymerization leads to better protection of sila-
acidity of the polar groups is reduced, and it nol surface interactions [50]. The introduction of
is better controlled than that of silanols. Polar a small percentage of groups displaying positive
end-capping makes the phases more compat- charges on silica surface (charge surface hybrid
ible with highly aqueous mobile phases, and or CSH) also has been used for blocking the
this type of column has useful specific applica- interaction with the silanols and reducing peak
tions. For example, use of a polar-end-capped tailing for basic compounds.
C18 phase can retain more polar water-soluble
compounds such as water-soluble vitamins, Preparation of Polar-Embedded
being a better choice for their separation.
Hydrophobic Phases
One additional procedure applied to have
polar groups shielding the silanol groups A special type of hydrophobic phase are those
from the silica surface is the use for derivatiza- phases containing a polar-embedded moiety in
tion of trifunctional reagents as previously the chain connecting the hydrophobic group
shown in reactions 6.2.5 or 6.2.6 (see Section with the silica. These phases are used in RP chro-
6.2) and the generation of silanol groups that matography and have a number of advantages
are not directly bonded to the silica gel over common RP stationary phases (such as C8
surface. In reaction 6.2.5, these silanol groups or C18), including a wider range of selectivity
are further reacted, but in special conditions versus the analytes, higher stability to phase
(with a higher proportion of water) the final collapse in eluents high in water content,
product may remain with intact OH groups. reduced silanol activity with more symmetrical
This type of reaction is schematically shown peaks, and better stability in low and high pH
in reaction 6.4.1 for a tri-functional chlorinated mobile phases (see Section 6.3).
reagent. The hydrophobic character of the columns
with RP phases containing a polar-embedded
group is close to that of a regular corresponding
Si OH phase. The calculated log Kow for the model of
Cl Cl an amide-embedded phase shows, for example,
O + Si
-2 HCl close values to that of a not end-capped C18
Si OH Cl R
phase (see Table 6.3.5.)
Polar-embedded stationary phases can be
(6.4.1) synthesized using a two-step process, as
Si O + H2O Si O
Cl OH shown in reactions 6.2.11, 6.2.12, and 6.2.13.
O Si O Si A single-step procedure can also be utilized
R - HCl R
Si O Si O for generating embedded phases. As an
example, an embedded polar stationary phase
254 6. STATIONARY PHASES AND THEIR PERFORMANCE
that contains a polar group within a long where eXe can be one of the following groups
hydrophobic chain attached to the silica shown in Figure 6.4.2.
surface can be obtained by reaction 6.4.2: Polar embedded phases having two zones of
polarity or an extended carbon linker (e.g., 5 or 6
CH3 O CH3 atoms to the silica) are also known [100]. These
C (CH2)17 phases reduce the possibility of interactions
Si OH + Cl Si
O NH between the main polar group and silanol
CH3 - HCl
from the silica surface. Bidentate-embedded
stationary phases have also been reported in
CH3 O CH3 the literature [101]. The additional end-capping
Si O Si C (CH2)17 of polar-embedded stationary phases can be
O NH done in the same manner as for other common
CH3
hydrophobic phases.
(6.4.2)
O
O O O O
O O S
C C C C N O
O S
NH NH NH O NH S NH
CH3
ether amide urea carbamate sulfone thiocarbamate N-methylsulfonyl
FIGURE 6.4.2 Bidentate groups that can be used in the synthesis of polar embedded stationary phases.
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 255
TABLE 6.4.2 Characteristics of the phase and of columns with hydrophobic stationary phases.
Property Range
* Note: Some of the phases indicated on silica include, besides silica, phases on organic/inorganic silica particles such as
ethylene-bridged hybrids.
important such as the pore size of the stationary (1) the analyte, (2) the stationary phase, and (3)
phase, which is very important when macromol- the mobile phase. For this reason, in order to
ecules are separated. For protein separation, for compare stationary phases regarding their capa-
example, where the molecules are larger than bility of retention, the other two factors, which
about 2000 Da, the pore size of the column are the mobile phase and the analyte, must be
must be large (> 300 A ) in order to allow the ana- specifically selected and kept unchanged within
lyte to penetrate the pores of the stationary phase a comparison.
and interact with the bonded moiety. Otherwise, The hydrophobic character of the stationary
the molecules are excluded from the interaction. phase is a very important property in RP-HPLC.
A detailed discussion of hydrophobic interactions
Retention and Separation Properties in general was given in Section 4.1, and the mech-
anism in RP-HPLC was described in Section 5.1.
of Hydrophobic Stationary Phases
In Section 5.1 has been shown that under the
Retention in chromatography is a complex name hydrophobic interactions are incorpo-
process whereby various interactions take place rated several types of contributions. They include
between the three participants in the separation: the energy from the ideal gas phase
256 6. STATIONARY PHASES AND THEIR PERFORMANCE
interactions, energy from van der Waals interac- where log Kow for models of different phases are
tions, and the energy from the cavity creation in listed. The values of log Kow for models for phenyl
the mobile phase. All these interactions are indi- and especially for cyanopropyl are significantly
cated as hydrophobic since the cavity creation lower, for example, than those for C18 columns.
is the largest energy contributor, but they include However, physicochemical phase properties are
polar and polarizability contributions (see rel. difficult to be directly related to a value that gives
5.1.26). As discussed in Section 4.1 (and schemat- the strength of the hydrophobic interactions, and
ically suggested in Figure 5.1.3), hydrophobicity only a higher or a lower resulting hydrophobicity
is mainly a "response" to the polar character of can be suggested. Each of the physicochemical
the mobile phase the contribution from the van phase properties also depends on other
der Waals interactions being small. This aspect stationary phase characteristics. For example,
of hydrophobic interactions points to the major the carbon load (%C, as determined by elemental
role of the solvent in RP-HPLC, which can be analysis), which is also important for phase
considered to "overrule" the role of the stationary hydrophobicity, is related to: (1) the nature and
phase. Most organic molecules have both hydro- length of the hydrocarbon chain used in the
phobic moieties and polar moieties, and for bonded phase, (2) the degree of hydroxylation
a given solvent polarity, the solvophobic interac- of silica surface subjected to the derivatization
tions depending mainly on the hydrophobic char- procedure, and (3) the derivatization yield giving
acter of the molecule and of the stationary phase the ratio between derivatized hydroxyl number
differentiate molecular retention in the HPLC to the initial number of OH groups on the silica
process. surface. In general, a higher carbon load (which
Several properties of the stationary phase are can reach up to 22e24% for RP stationary phases)
indicative of its hydrophobicity. For silica-based is an indication of a more hydrophobic character.
phases, these properties include (1) the nature of With new, more modern phases that have polar-
the bonded substituent (C8, C18, phenyl, etc.), embedded groups, bidentate attachments to the
(2) the carbon content (carbon load C%), (3) silica surface, or organic/inorganic solid support,
the density of surface coverage with the bonded it is even more difficult to predict hydrophobicity
phase, (4) the weakness of the silanol activity from the stationary phase construction.
(increased, e.g., by the impurities in silica), and To characterize the hydrophobicity of a
(5) the level of solvent molecules adsorbed on stationary phase, one parameter that is typically
the stationary phase. used is the capacity factor k (or log k) for
The nature of the bonded substituent influ- hydrophobic-tested compounds [102]. The
ences the hydrophobicity by various character- values of capacity factor k can be used to
istics. For saturated hydrocarbon substituents, compare columns with different carbon
the increase in the number of carbon atoms in contents, chain lengths, or other derivatization
the chain typically leads to an increased hydro- approaches. A higher k (for a hydrophobic ana-
phobicity, although this increase is not linear, as lyte) indicates a more hydrophobic character for
shown in Figure 5.1.6. In addition, a consider- the phase. However, the value of k must be
able overlapping in the hydrophobicity can be taken in a relative way, since it depends not
seen between columns with different numbers just on the nature of the stationary phase, but
of carbons in the bonded moiety. also on the nature of the mobile phase and the
Another factor contributing to column hydro- particular hydrophobic analyte used in the test.
phobicity is the chemical nature of the substit- As is well known, RP-HPLC is commonly used
uent, in particular the polarity of the bonded for the separation of a variety of organic mole-
ligand. This has been illustrated in Table 6.3.5, cules that have a hydrophobic moiety, it may
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 257
also contain other groups such as polar or even part, the result of the fact that hydrophobicity is
ionic ones. However, for the phase characteriza- more a response to the polar character of the
tion regarding exclusively the hydrophobic inter- mobile phase than a force in itself.
actions, the analyte must have only a hydrophobic As expected, other factors also contribute to
character. Therefore, the retention must be the value of the capacity factor. One important
measured for compounds that have very low or such factor is the surface area of the stationary
no polarity. Such compounds are characterized phase. As shown by rel. 2.1.13, k KJ, and the
by little or no tendency to adsorb water, and water value for J depends on the pore volume (and/
forms beads on their surface. Hydrophobic or the surface area) of the stationary phase;
substances lack the groups that can participate therefore, k should also depend on the surface
in the formation of hydrogen bonds with water. area of the stationary phase. For the columns
Many of the hydrophobic substances are hydro- listed in Table 6.4.3, the surface areas are shown
carbons, although several other moieties are in Figure 6.4.5, and the dependence of capacity
not hydrophilic (halogen, ester, ether, and other factor on surface area is shown in Figure 6.4.6.
less polar moieties). Specific hydrophobic Similar to the carbon load, the correlation pre-
compounds commonly used for the evaluation sented in Figure 6.4.6 is positive, but the R2 value
of hydrophobic character include toluene, ethyl- for it is not very good (R2 0.4779). Some
benzene, naphthalene, and acenaphtene. When improvement in the R2 value of a correlation is
such compounds are used, a greater k (and obtained if both carbon load and surface area
log k) will indicate a more hydrophobic stationary are considered, but the correlation coefficient is
phase. For the selected compound, the calculation still not very high, indicating that more factors
of k is performed based on retention time tR and contribute to the value of capacity factor. Even
the value t0 for the column, where k (tR t0)/ better results are obtained when a parameter is
t0 and t0 is typically measured using the retention included to account for silanol activity of the
time of uracil or of thiourea, which practically are column. The variation in silanol activity for the
not retained on hydrophobic columns but at the columns listed in Table 6.4.3 is shown in
same time are not excluded from the pores of Figure 6.4.7.
the packing material (as are, for example, certain The values of carbon load, phase surface area,
ionic compounds). and silanol activity (with arbitrary assigned
The capacity factor for toluene (expressed as ln values 1 for very low and 4 for high activity)
k) for a number of C18 columns, measured for allow an estimation of k, using a dependence
a mobile phase consisting of 80% CH3OH and equation of the form:
20% aqueous buffer 0.25 mM KH2PO4 at pH
6 and at 24 oC (in isocratic conditions), are given kcalc a b C% c Surface area
in Table 6.4.3. The capacity factor k depends on d Silanol activity (6.4.4)
several variables, the carbon load of the column
being a contributor to the value of k [104]. For A graph showing the variation of the calcu-
example, for the columns listed in Table 6.4.3, lated values kcalc for toluene as a function of
the carbon load is given in Figure 6.4.3, and the measured k values is shown in Figure 6.4.8. As
dependence of capacity factor on the carbon shown in this figure, there is a positive correla-
load is shown in Figure 6.4.4. As seen in tion between the measured and the calculated
Figure 6.4.4, a positive correlation exists between k values. However, the correlation is not very
the capacity factor and the carbon load of the good. This indicates that the retention proper-
column, although only a R2 0.3942 is obtained ties of a column are a function of a number of
for this correlation. This poor correlation is, in stationary-phase characteristics not accounted
258 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.4.3 Capacity factor ln k for toluene on C18 stationary phases (5 mm particles) [103]. Mobile phase consisted of
80% CH3OH and 20% aqueous buffer 0.25 mM KH2PO4 at pH 6.
500
400
200
100
0
0 5 10 15 20 25 30 35 40 45 50 55 60
Column No. in Table 6.4.3
FIGURE 6.4.5 Values for surface area m2/g for the columns listed in Table 6.4.3.
2.5
R2 = 0.4779
2
ln k (toluene)
1.5
0.5
0
0 100 200 300 400 500 600
Surface area m 2/g
FIGURE 6.4.6 Variation of capacity factor ln k as a function of surface area of the particles.
RP-HPLC is a technique widely used not approximation, it can be assumed that the
only for hydrophobic compounds separations. separation of polar compounds containing
A range of molecules that have only a small hydrophobic moieties is achieved on RP
hydrophobic moiety can also be successfully columns only due to the interaction of their
separated on this type of column. In a first hydrophobic moieties with the hydrophobic
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 261
High
Medium
Silanol activity
Low
Very low
0 5 10 15 20 25 30 35 40 45 50 55 60
Colum n No. in Table 6.4.3
FIGURE 6.4.7 Silanol activity (qualitative) for the stationary phase for the columns listed in Table 6.4.3.
2.5
2
R2 = 0.5322
Calculated ln k
1.5
0.5
0
0 0.5 1 1.5 2 2.5
Measured ln k
FIGURE 6.4.8 Dependence of calculated ln kcalc for toluene as a function of measured log k values for this compound,
with ln kcalc a + b C% + c (Surface area) + d (Silanol activity) (a 0.367045, b 0.049043, c 0.001141, d 0.06382).
stationary phase of the column (in equilib- phase, an excellent correlation has been
rium with the hydrophobic character of the reported between log kc1 and log kc2 for a large
mobile phase). For two different columns number of compounds that have hydrophobic
(indicated as c1 and c2) and the same mobile moieties [105]. This correlation between the
262 6. STATIONARY PHASES AND THEIR PERFORMANCE
retention on two columns can be written in retention capability. From rel. 6.4.8 it can be
the form: seen that Hc describes how much the hydro-
phobic character of the column influences the
log kcl A1 A2 log kc2 (6.4.6a) separation between ethylbenzene and another
hydrophobic compound.
where A1 and A2 are constants depending on
Similar to log k, parameter Hc is dependent on
the two compared columns. Relation 6.4.6a can
column/stationary phase properties such as n
be easily generated if it is assumed that the
the number of carbons in the alkyl chain of the
capacity factor kc1 for a compound j is expressed
active phase, the pore diameter dp, the density
by a formula of the type:
of the active phase cL, and the end-capping
log kc1 j ac1 h0 jHc1 (6.4.7) parameter Ec. For example, a formula success-
fully used for estimating Hc is the following:
where h0 (j) is a parameter characterizing the
hydrophobicity of compound j, Hc1 is the Hc a b log n c log dp d log cL e Ec
parameter for the hydrophobicity of column (6.4.10)
c1, and ac1 is a constant. For a second column
and the same evaluated compound, rel. 6.4.7 As in the case of rel. 6.4.5, the coefficients a, b,
will be log kc2 ac2 h0 (j) Hc2. By replacing c, d, and e are obtained from best regression fit
h0 (j) from one expression into the other, the for a large number of compounds. The value
following result is obtained: of Hc increases with the number of carbons in
ligand that forms the bonded phase (b > 0),
Hc1 Hc1 and it also increases for smaller pore diameters
log kc1 ac1 ac2 log kc2
Hc2 Hc2 (c < 0), for higher ligand density (d > 0) and
(6.4.6b) for end-capping (e > 0). Columns obtained by
vertical polymerization having a high carbon
which is identical to rel. 6.4.6a when replacing content (large cL) usually have a high Hc value.
the first term in rel. 6.4.6b with A1 and the coef-
ficient for log kc2 with A2 (index j is omitted).
By selecting ethylbenzene (EB) as a standard
Methylene Selectivity
reference (h0 (EB) 0), rel. 6.4.7 can be written Besides using the capacity factor and param-
for any given hydrophobic compound in the eter Hc, the separation capability for hydro-
form: phobic compounds of an RP column is
frequently described by another parameter that
log kc1 log kc1 EB h0 Hc1 (6.4.8) is also related to hydrophobicity, indicated as
methylene selectivity and noted as a(CH2). This
For a compound j, rel. 6.4.8 (with some parameter is defined as the selectivity a for two
changes in notation) gives: log kc1(j) log kc1 compounds that are different by a CH2 group
(EB) log (k/kEB) log [ac1(j,EB)], which can (one example being the pair toluene and ethyl-
be written in the form: benzene). The formula for a(CH2) is as follows:
log ac1 logk=kEB h0 Hc1 (6.4.9) kRCH2 R
aCH2 (6.4.11)
kRR
Parameter Hc has the advantage of indicating
the capability of a column to separate hydro- The value of a(CH2) depends as expected on
phobic compounds (separation from EB in the stationary phase and on the selected mobile
particular), while log k indicates only the phase. However, it has been found that for
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 263
several classes of solutes with an adjacent selectivity can be used for comparing columns
number of carbons, a(CH2) is a constant (for of the same type (e.g., C18 columns USP code
a given column and mobile phase). Even more, L1) or even for comparing RP columns of
a linear dependence for log a(CH2) on the different types such as C18 and C8, or C18 and
number of CH2 units was noticed for certain CPhenyl.
homologous series such as the one from Although the capacity factor k and the methy-
benzene to amylbenzene. This linearity was lene selectivity for hydrophobic compounds are
verified for various columns [108] and also for related, their correlation is not very strong. As
various mobile phases [109]. Based on this an example, the correlation between the values
observation, the values for log a(CH2) can be of k for butylbenzene and the values of a(CH2)
obtained from the slope of the trendline of the obtained for the homologous series from benzene
graph of log k versus the number of methylene to amylbenzene on different columns, when the
groups in the alkylbenzene: mobile phase is composed of acetonitrile and
aqueous buffer (20 mM KH2PO4-K2HPO4 at
log aCH2 log ki =kj log ki log kj pH 7) in the ratio 65/35 (v/v), is shown in
tan b Figure 6.4.10 (data from [108]). As seen from
the graph, R2 0.6645 indicates a modest posi-
(6.4.12) tive correlation. The list of the columns on which
where i and j are two consecutive homologs the values for the graph in Figure 6.4.10 were
(i j 1). measured is given in Table 6.4.4.
The graph showing a(CH2) calculation for the When a column is selected for an application
case of a Zorbax Bonus-RP column, with the requiring the separation of hydrophobic
mobile phase composed of acetonitrile and compounds, the preferred characteristic should
aqueous buffer (20 mM KH2PO4-K2HPO4 at be a high a(CH2) for obtaining a better separa-
pH 7) in the ratio 65/35 (v/v) as reported in tion [110]. However, methylene selectivity
[108], is given in Figure 6.4.9. The methylene depends on the characteristics of both the
0.8
0.7
y = 0.1472x - 0.0051
0.6
0.5
0.4
log k
0.3
0.2
0.1
0
0 1 2 3 4 5 6
-0.1
No. of CH2 units in the alkylbenzene
FIGURE 6.4.9 Calculation of a(CH2) from the k values of a homologous series of alkylbenzenes [108].
264 6. STATIONARY PHASES AND THEIR PERFORMANCE
LiChr.C18 ACN
3
LiChr.C18 MeOH
Puro.C18 ACN
2.5 Puro.C18 MeOH
(CH 2)
LiChr.C8 ACN
LiChr.C8 MeOH
2
SymmC18 ACN
SymmC18 MeOH
1
10 20 30 40 50 60 70 80
% Organic phase
FIGURE 6.4.11 Variation of a(CH2) for several columns and two mobile phase systems (acetonitrile/water and meth-
anol/water) at different concentrations.
also include p-p interactions among those silica support. For this reason, before the
acting in RP, when the stationary phase contains derivatization of the base silica gel (bare silica)
phenyl or cyano groups [113]. In general, the it is desirable to have a large number of silanol
p-p molecular interactions were not very groups. These groups are supposed to be
important, as shown by ab-initio calculations, derivatized with the bonded phase. However,
or by experimental results obtained for cyano- because of steric hindrance during the
propyl columns [114]. All interactions may, or derivatization of silica, the density of silanols
may not, play a role in the separation. The on chromatographic-grade silica (about 8
complexity of interaction process is schemati- 1 mmol/m2) is much greater than the maximum
cally shown in Figure 6.4.12 [115]. possible concentration of alkyl groups in
The silanol groups on the silica have an a bonded phase (about 4.5 mmol/m2 for C18
opposite role in separation compared to the ligands). Therefore, after the silica surface has
hydrophobic character of the bonded phase. been modified, numerous unreacted (residual)
It is generally accepted that the surface of silanol groups are left within the bonded phase
modified silica still contains active sites given (about 50%). These residual silanols are weakly
by SieOH groups (silanols), regardless of the acidic, with pKa values typically between 5 and
end-capping treatment of the material. The 7, and they can interact with polar compounds
polar or apolar activities of the siloxane groups through strong hydrogen bond and dipole-
(Si-O-Si) are so low that they are not taken dipole interactions. At higher pH values of the
into consideration for the interactions between mobile phase, the silanol groups may also
analytes and stationary phase. The silanols have an ion-exchange type of contribution. As
serve as attachment sites for the covalent silyl a result of the heterogeneous surface of the
ether bonds that anchor-bonded phases to the stationary phase, a mixed retention mechanism
266 6. STATIONARY PHASES AND THEIR PERFORMANCE
X X
H X
CH3 CH3 CH3 CH3 X CH3 CH3
Si Si Si Si Si Si
H3C O
..O H H3C
O
H ..O H3C
O O
- H3C
O
H3C
O
H3C
O
n+
M
is in place. This mechanism may influence the contain phenyl- or cyano-bonded phases. The
separation of all analytes. However, when only columns with embedded polar groups may be
a small proportion of the analyte molecules included in this category. For these columns,
participate in other interactions besides hydro- besides the hydrophobic interactions, other
phobic ones, peak tailing and loss of chromato- forces contribute to the separation. When the
graphic resolution are seen. This happens separation is intended for highly hydrophobic
especially when basic solutes are involved compounds, these polar interactions are not
[116]. For this reason, silanol activity is some- important. However, the separation of organic
times characterized based on the values of compounds with hydrophobic and polar groups
As (j) or TF (j) (see rel. 2.2.16 and 2.2.17) for a is more complex than that based on simple
specific polar compound j. The acidity of silanol hydrophobic interactions, and in this case
groups can increase in the presence of transi- more parameters must be considered for
tional metal ions. The metal ions can also describing the separation.
become adsorption sites for compounds that Due to various types of interactions that may
are able to form complexes, and the take place between an analyte and the stationary
metals from silica can interfere in the retention phase, a deviation from rel. 6.4.9 is observed for
process [117]. For this reason, high purity particular analytes. In these cases the relation
silicas are typically used for stationary phase 6.4.9 can be written in the form:
manufacturing.
Besides some polarity resulting from the sila- log ac1 h0 Hc1 D (6.4.13)
nol groups (and adsorbed solvent), various
columns are available having groups that confer In rel. 6.4.13, D 0 for hydrophobic
some intentional polarity besides hydrophobic compounds, with no steric hindrance of inter-
character. Examples are the columns that acting with the stationary phase. For other
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 267
compounds, an extension of rel. 6.4.9 can be k0 (j) can be determined by multiple regression
written for log a such that it will account in of values for log ac1 when Hc1, S*c1, Ac1, Bc1,
the value for a D, including all possible interac- and Cc1 are known.
tions. These interactions are: (1) steric interac- Parameters s0 (j) and S*c1 (this term is taken
tions, (2) hydrogen bonding between a basic with a negative sign, although in some literature
solute and an acidic column group (column reports the positive sign is utilized [118]) are
acidity), (3) hydrogen bonding between an typically related to the steric resistance to inser-
acidic solute and a basic column group (column tion of a bulky solute characterized by s0 (j)
basicity), and (4) cation exchange and/or ion- into a stationary phase characterized by S*c1.
ion interactions. When solutes with a diversity However, a different steric interaction process
of molecular structures were evaluated, it was was observed for molecules that can penetrate
possible to identify solutes for which the value the phase but have restricted orientation toward
of D is influenced (mainly) by only one type of it, and the interaction with the phase is similar to
the four listed interactions. Average values of D a size-exclusion effect. Also, the shape of the
were then determined for each solute group (1 molecule may affect the extent to which a hydro-
to 4), for a number of columns and using phobic moiety of the molecule can be pre-
a specific mobile phase. For each column it sented to the stationary phase such that it will
was possible to generate a parameter S*c1 diminish the exposure to interactions with the
accounting for steric interactions, Ac1 mobile phase. The formula 6.4.14 for describing
accounting for hydrogen bonding between the selectivity is known as the hydrophobic
a basic solute and an acidic column group subtraction model [119e121]. As further shown,
(column acidity), Bc1 accounting for hydrogen the hydrophobic subtraction model is very useful
bonding between an acidic solute and a basic for column characterization using a set of
column group (column basicity), and Cc1 selected compounds, while the true calculation
accounting for cation exchange or ion-ion inter- of log k for different analytes is not possible
actions. Since these interactions can be without knowing the specific parameters h0 (j),
assumed to be additive, for a column c1 and s0 (j), b0 (j), a0 (j), and k0 (j). Expression 6.4.14
for the compound j the expression for ac1(j) shows that by using rel. 6.4.14, it is possible to
will be: estimate how much a compound is separated
from ethylbenzene on the specific column.
log ac1 j h0 j Hc1 s0 jSc1 b0 jAc1 The hydrophobic subtraction model shows
a0 j Bc1 k0 jCc1 that when the analyte-depending parameter is
zero or extremely small, that particular interac-
(6.4.14)
tion does not contribute to the value of log ac1(j).
Since log ac1 j log kj=kEB , rel. 6.4.14 is For example, formula 6.4.9 will result from
equivalent to the expression: rel. 6.4.14 for an analyte j with b0 (j), a0 (j), k0 (j),
and s0 (j) all zero. In this case, a hydrophobic
log kj log kEB h0 jHc1 s0 jSc1 compound with no polar groups and with very
b0 jAc1 a0 jBc1 k0 jCc1 little steric interactions, such as toluene or ethyl-
benzene, will generate a log k value describing
(6.4.15) only hydrophobic interactions. This concept
where the hydrophobicity is described by log generated the idea that specific compounds in
kEB for the specific column, and the parameters a specific mobile phase may lead to a unique
h0 (j) for the solute and Hc1 for the column (see contribution to log ac1(j). For example, a mixture
rel. 6.4.9). Parameters h0 (j), s0 (j), b0 (j), a0 (j), and of uracil, toluene, ethylbenzene, quinizarin (or
268 6. STATIONARY PHASES AND THEIR PERFORMANCE
mmol/m2 of the stationary phase. The bonding literature (PQRI approach). For example, the
density BD is assumed to be related to the steric values for all the parameters from rel. 6.4.14 for
parameter S*. The program also provides a specific mobile phase were reported in the liter-
a column distance F (see rel. 6.4.17). An ature for 16 different analytes [118]. The parame-
example of such information for several ters characterizing this set of compounds in
columns compared with an Ascentis C18 a mobile phase consisting of acetonitrile/
column is given in Table 6.4.6. aqueous 60 mM phosphate buffer 50/50 v/v
Other test solutions, more elaborate than those are given in Table 6.4.7 (thiourea was used for
in SRM 870 mixture, have been suggested in the measurement of t0) [118]. The table also lists the
TABLE 6.4.6 Example of information from USP information regarding column description and comparison.
TABLE 6.4.7 The parameters for solute hydrophobicity, steric effects, hydrogen bonding, ion exchangeand log Kow
[114,118].
(Continued)
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 271
TABLE 6.4.7 The parameters for solute hydrophobicity, steric effects, hydrogen bonding, ion exchangeand log Kow
[114,118]. (contd)
No. Solute h0 s0 b0 a0 k0 log Kow
log Kow values for each compound. The struc- The parameters from Table 6.4.7 were
tures of the compounds from Table 6.4.7 are as obtained based on the values log a on a large
follows: number of columns for which the parameters
H
O
N
O CH3 CH3 CH3
C O CH3 H
O
N
O O
Acetophenone Benzonitrile Anisole Toluene Ethylbenzene 4-Nitrophenol 5-Phenylpentanol
O O O
H H
N H3C O
H3C O
N H
O N
N
H3C CH3
CH3 CH3
O
CH3
5,5-Diphenylhydantoin Chalcone (trans) N,N-Dimethylacetamide N,N-Diethylacetamide 4-Butylbenzoic acid
N O
H O H CH3 CH3
H3C CH3 N N
H CH3
Mefenamic acid Nortriptyline Amitriptyline
272 6. STATIONARY PHASES AND THEIR PERFORMANCE
Hc, S*c, Ac, Bc, Cc for pH 2.8 (C(2.8)), and Cc The parameters from Table 6.4.8 are very
for pH 7.0 (C(7.0)) were also calculated, using useful in evaluating a specific column char-
multiple regression. These values are given in acter. For example, a column such as Acclaim
Table 6.4.8, together with log kEB for various 120 C18 has a large Hc value and relatively
columns 150 mm in length, 4.6 mm i.d., and low values for the other parameters, indicating
with 5 mm particles and the same mobile phase a good separation of compounds based on
acetonitrile/aqueous 60 mM phosphate buffer their hydrophobic character, but lower effi-
50/50 v/v [118,124]. Assuming that all other ciency for other interactions. Relation 6.4.14
parameters remain unaffected when changing allows the calculation of log a values for
the buffer solution from pH 2.8 to pH 7.0, compounds with known parameters. For
it can be easily determined that the parameter example for toluene, with the values from
C(7.0) can be obtained from C(2.8) from the Table 6.4.7, the following log a value can be
following relation: obtained (using C(2.8) value) for Acclaim 120
C18 column:
k7:0
C7:0 C2:8 log (6.4.16)
k2:8 log a 0:21156 0:00171 0:00156
TABLE 6.4.8 The values of Hc, S*c, Ac, Bc, Cc(2.8), Cc(7.0), and log kEB for various columns.
1 Acclaim 120 C18 1.032 0.018 0.142 0.027 0.086 0.002 1.002
2 Acclaim 120 C8 0.857 0.004 0.274 0.012 0.086 0.016 0.780
3 Acclaim 300 C18 0.957 0.018 0.170 0.170 0.261 0.222 0.462
4 Acclaim Organic Acid 0.833 0.063 0.385 0.001 0.316 0.349
5 Acclaim Polar Advantage C16 0.855 0.068 0.116 0.023 0.270 0.357
6 Ace 5 CN 0.409 0.107 0.729 0.008 0.086 0.441 0.019
7 Ace Phenyl 0.638 0.145 0.305 0.031 0.128 0.461 0.445
8 Ace 5 AQ 0.804 0.051 0.129 0.034 0.009 0.167
(Continued)
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 273
TABLE 6.4.8 The values of Hc, S*c, Ac, Bc, Cc(2.8), Cc(7.0), and log kEB for various columns. (contd)
No. Column Hc Sc Ac Bc Cc(2.8) Cc(7.0) log kEB
24 Develosil C30-UG-5 (C30) 0.976 0.036 0.195 0.011 0.158 0.177 0.892
25 Develosil ODS-HG-5( C18) 0.980 0.015 0.171 0.008 0.187 0.221 0.911
26 Develosil ODS-MG-5 (C18) 0.963 0.036 0.164 0.003 0.012 0.051 1.051
27 Develosil ODS-UG-5 (C18) 0.997 0.025 0.145 0.004 0.150 0.154 0.926
28 Discovery BIO Wide pore C18 0.836 0.014 0.253 0.028 0.121 0.119 0.528
29 Discovery BIO Wide pore C5 0.655 0.019 0.305 0.029 0.091 0.220 0.059
30 Discovery BIO Wide pore C8 0.840 0.018 0.224 0.034 0.201 0.195 0.345
31 Discovery C18 0.984 0.027 0.128 0.004 0.176 0.153 0.683
32 Discovery C8 0.832 0.011 0.237 0.029 0.119 0.143 0.522
33 Discovery CN 0.397 0.110 0.615 0.002 0.035 0.513 0.198
34 Discovery HS F5 0.631 0.166 0.325 0.023 0.709 0.940 0.603
35 Fluophase PFP 0.675 0.129 0.311 0.065 0.817 1.375 0.653
(Continued)
274 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.4.8 The values of Hc, S*c, Ac, Bc, Cc(2.8), Cc(7.0), and log kEB for various columns. (contd)
No. Column Hc Sc Ac Bc Cc(2.8) Cc(7.0) log kEB
48 Hypersil Beta Basic-8 0.834 0.016 0.248 0.029 0.110 0.114 0.619
49 Hypersil BetamaxNeutral (C18) 1.099 0.035 0.068 0.031 0.038 0.012 1.231
50 Hypersil Bio Basic-18 0.975 0.025 0.099 0.007 0.253 0.217 0.512
51 Hypersil Bio Basic-8 0.821 0.011 0.232 0.029 0.231 0.211 0.253
52 Hypurity C18 0.981 0.020 0.090 0.002 0.192 0.168 0.744
53 Hypurity C8 0.833 0.010 0.200 0.034 0.157 0.161 0.546
60 JSphere L80 (C18) 0.762 0.036 0.216 0.001 0.400 0.345 0.764
61 JSphere M80 (C18) 0.926 0.026 0.123 0.004 0.294 0.139 0.957
62 Jupiter 300 C18 0.945 0.031 0.224 0.008 0.234 0.218 0.467
63 Jupiter 300 C4 0.698 0.008 0.426 0.019 0.153 0.142 0.126
64 Jupiter 300 C5 0.729 0.021 0.382 0.016 0.129 0.331 0.183
65 Kinetex XB-C18 0.975 0.013 0.083 0.023 0.046 0.305
(Continued)
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 275
TABLE 6.4.8 The values of Hc, S*c, Ac, Bc, Cc(2.8), Cc(7.0), and log kEB for various columns. (contd)
No. Column Hc Sc Ac Bc Cc(2.8) Cc(7.0) log kEB
(Continued)
276 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.4.8 The values of Hc, S*c, Ac, Bc, Cc(2.8), Cc(7.0), and log kEB for various columns. (contd)
No. Column Hc Sc Ac Bc Cc(2.8) Cc(7.0) log kEB
115 XTerra Phenyl 0.690 0.076 0.374 0.003 0.102 0.033 0.409
116 XterraMS C8 0.803 0.004 0.292 0.005 0.058 0.009 0.571
117 YMC Pro C18 1.015 0.013 0.117 0.006 0.154 0.005 0.939
118 YMC Pro C8 0.890 0.014 0.214 0.007 0.322 0.020 0.814
119 Zorbax Eclipse XDB-C18 1.077 0.024 0.062 0.033 0.055 0.089 0.958
120 Zorbax Eclipse XDB-C8 0.919 0.025 0.219 0.008 0.003 0.012 0.823
121 Zorbax Rx-18 1.077 0.040 0.310 0.037 0.096 0.415 0.886
122 Zorbax RX-C8 0.792 0.076 0.117 0.018 0.012 0.948 0.703
123 Zorbax StableBond 300A C18 0.906 0.050 0.045 0.043 0.253 0.700 0.344
124 Zorbax StableBond 300A C8 0.701 0.085 0.002 0.047 0.146 0.820 0.106
125 Zorbax StableBond 300A C3 0.526 0.122 0.194 0.047 0.057 0.711 0.151
126 Zorbax StableBond 80A C18 1.008 0.021 0.215 0.002 0.077 0.822 0.884
127 Zorbax StableBond 80A C3 0.601 0.124 0.080 0.038 0.084 0.810 0.450
128 Zorbax StableBond 80A C8 0.795 0.079 0.138 0.018 0.014 1.020 0.710
A negative value for log a indicates that end-capping, and columns with a higher acidity
toluene elutes ahead of ethylbenzene (as of the silica base have higher Ac values. Also,
expected). The contribution to the value for log the presence of higher content of metals in the
a (and for log k 0.79680) comes mainly from silica affects the increase in Ac. The value of Bc
the hydrophobic effects. is in general affected by the nature of the
Similar to the rules that lead to an increase bonded phase. The ion-exchange character is
in Hc, several observations can be made affected by the metal impurities in the silica
regarding the increase in the other column char- and is very different for silica-based columns
acteristics (some desirable and some not). For compared to zirconia-based columns. The steric
example, Ac decreases with the column interaction parameter S*c is typically increased
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 277
for longer chain ligands that form the bonded stationary phase. Similar parameter values and
phase and also increases with the density of results for various other types of columns are
the active phase. For phases indicated as mono- reported in the literature [114, 126, 127].
meric (obtained with a monofunctional silaniza- The values for parameters Hc, S*c, Ac, Bc,
tion reagent) or horizontal polymeric (see Cc(2.8), Cc(7.0), and log kEB for various columns
Section 6.2), the values for S*c are typically lower may vary significantly depending on column
than for the phases obtained by vertical poly- characteristics (e.g., the chemical nature of the
merization. These rules remain only directional, bonded phase, carbon load, pore size, end-
however, and no direct quantitation of a param- capping, etc.). Several typical values for these
eter can be obtained by a simple relation with parameters for different types of columns are
the physicochemical characteristic of the listed in Table 6.4.9. Comparison between
TABLE 6.4.9 Minimum, maximum or average* values for Hc, S*c, Ac, Bc, Cc(2.8), Cc(7.0), and log kEB for various
hydrophobic columns.
8 C18 wide pore min. 0.98 0.00 0.09 0.01 0.05 0.02 0.54
9 C18 wide pore max. 1.03 0.02 0.02 0.01 0.27 0.27 0.99
10 C18 monolith average 1.01 0.02 0.12 0.02 0.11 0.31 0.51
11 C18 embedded polar group min. 0.74 0.01 0.22 0.00 0.27 0.17 0.77
12 C18 embedded polar group max. 0.97 0.07 0.08 0.12 0.14 0.53 0.91
13 C30 average 0.96 0.01 0.10 0.02 0.24 0.29 0.48
* Note: The minimum, maximum, and average values were obtained from a limited set of about 150 columns. Columns not included in the set may display
other values.
278 6. STATIONARY PHASES AND THEIR PERFORMANCE
columns can be obtained based on parameters where fH, fS, fA, fB. are weighing factors
described in Table 6.4.8 by different procedures. selected based on the relative contribution of
One visual procedure is the use of a radar each parameter to the value of a. For the
graph. Figure 6.4.14 shows such a graph for columns listed in Table 6.4.8, the weighing
four synergy-type columns. From Figure 6.4.14 factors can be chosen with the following values:
it can be seen, for example, that Synergy fH 12.5, fA 30.3, fB 142.8, fC 83.3, and fS
Fusion-RP has strong hydrophobicity and 100 [118]. A selection of weighing factors all
moderate polar character, Synergy Hydro-RP equal to 1 can also be made, and rel. 6.4.17
has strong hydrophobic interactions and also will in this case become:
strong polar interactions (strong hydrogen
bonding and average cation-exchange char-
F Hc1 Hc2 2 Sc1 Sc2 2 Ac1 Ac2 2
acter), Synergy Polar-RP has lower hydrophobic
character but the strongest polar character, and Bc1 Bc2 2 Cc1 Cc2 2 1=2 (6.4.18)
Synergy Max-RP has strong hydrophobic inter-
actions and low other interaction types. (Note: A similar distance between columns
The values from Table 6.4.8 can also be used can be developed by using the parameters
to develop a formula for a distance between measured by USP test Hc, CTF, CFA, TFA, BD.)
two different columns. This distance F can be Use of the distance parameter can allow the
calculated using an expression of the form:
selection of columns to be similar in case of
F fH Hc1 Hc2 2 fS Sc1 Sc2 2 a need for a replacement with similar proper-
ties, or in case of search for an orthogonal sepa-
fA Ac1 Ac2 2 fB Bc1 Bc2 2 ration, the distance may facilitate the choice of
1=2 a very different column. The USP database
2 [123] has a calculation that also allows an
fC Cc1 Cc2
online comparison of numerous columns by
(6.4.17) displaying the F value and the characteristic
Hydrophobicity
1 Hc
1.2
Hydrogen bonding
5 3
Cation exchange acidic column A c
pH 2.8 Cc(2.8)
Hydrogen
4 bonding
basic column B c
FIGURE 6.4.14 Radar plot displaying column properties for four different Synergy columns.
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 279
Hc, S*c, Ac, Bc, Cc(2.8), and Cc(7.0) values for the 2,20 -dipyridyl and 4,40 -dipyridyl in the pres-
alternative columns. An example of such ence of metal ions on the silica surface is rather
a comparison for an Acclaim 120 C18 column different: 2,20 -dipyridyl can form complexes
selected from more than 500 columns is shown with metallic species in particular with iron,
in Table 6.4.10. whereas 4,40 -dipyridyl cannot form complexes.
Therefore, the peak of 2,20 -dipyridyl will
exhibit a post-tailing with an asymmetry
Other Tests for Comparing Hydrophobic
depending on the concentration of metallic
Columns ions from the silica surface, while the peak of
Various other tests and analytes are 4,40 -dipyridyl will remain symmetric.
reported in the literature for comparing
different column characteristics [119, 127,
128]. For example, metal activity in a stationary
N N
phase can be evaluated using other reagents
besides quinizarine. Measurement of the effect N N
of metals is important since metallic ions can 2,2'-dipyridyl 4,4'-dipyridyl
increase the acidity of adjacent silanols and
can become adsorption sites for compounds
that are able to form complexes. Overall, they Although these dipyridyl derivatives are
can interfere in the retention process. For basic compounds that can be involved in
measuring metal activity on the surface of strong interactions with dissociated silanol
stationary phase, acetylacetone and also the groups from silica surface, this inconvenience
couple of 2,20 -dipyridyl and 4,40 -dipyridyl was solved by using buffered (pH 7) mobile
were used. The retention behavior of phase. [117].
TABLE 6.4.10 Example of column comparison as obtained from U.S. Pharmacopeia web program [123] based on PQRI
test.
Another type of test for comparing columns The possibility of using ionic compounds
is related to the evaluation of stationary phase in a 100% aqueous mobile phase for the char-
polarity. The ability of a reversed-phase acterization of RP columns has also been sug-
column to retain polar compounds depends gested [130]. In this test, various naphthalene
not only on the extent of hydrophobic interac- disulfonic acids and naphthalene trisulfonic
tions between the column and the hydro- acids were employed as test compounds.
phobic moiety of the compound, but also on These compounds are practically completely
the two participants polarity. For example, ionized in aqueous solution, and when using
the selectivity difference between a conven- only water as a mobile phase, they are
tional C18 and a polar-embedded phase excluded from the pores of the hydrophobic
results from their polarity differences. In packing material, having a retention time
general, higher polarity of the embedded shorter than compounds such as uracil typi-
group in a stationary phase results in a longer cally used for measuring the column dead
retention of polar compounds. One simple test time (or volume). This effect is attributed to
to characterize the polarity of the stationary ionic repulsion between the slightly nega-
phase is based on using a mixture containing tively charged stationary phase surface (as
uracil, pyridine, phenol, N,N-dimethylaniline, a result of silanol group ionization) and the
p-butylbenzoic acid, and toluene in a mobile sulfonic acid anions. The test was performed
phase made of acetonitrile/aqueous buffer using as mobile phase the solution 0.4 M of
60/40 (v/v) (the buffer consisted of 50 mM Na2SO4. With this mobile phase, the retention
KH2PO4/KH2PO4 pH 3.2). With this times of the test compounds increases
mixture, a column polarity index (P) was compared to the pure water mobile phase, on
defined [128]: all tested hydrophobic columns. However, the
dead time t0 for the columns as measured,
2
P kp-butylbenzoic acid kphenol =ktoluene (6.4.19) for example, using uracil cannot be used for
calculating a capacity factor, and the differ-
This test shows that the polarity of polar- ences in retention must be compared versus
embedded stationary phases with the same the least retained test compound (1,5-naphtale-
hydrocarbon chain varies in the following nedisulfonic acid for disulfonic acids and
order: 1,3,5,7-naphthalene-tetrasulfonic acid for tri-
sulfonic acids). The test provides information
amide > carbamate > sulfonamide > alkyl on the hydrophobicity of the column and
peak asymmetry.
Besides the nature of the column (and
Another test for measuring polarity uses
considering the nature of the test compounds),
butyl paraben and dipropyl phthalate [129].
both the capacity factor, the methylene selec-
Retention studies on amide-embedded
tivity a(CH2), and also other interactions are
stationary phases showed that dipropyl phath-
significantly influenced by the mobile phase
late elutes before butyl paraben, whereas the
used in the test [110, 131, 132]. However, the
elution order on carbamate-embedded group
previous discussion did not explicitly include
phases is opposite. The order of polarity for
the nature of the mobile phase, except that
the polar-embedded phases using this test is
the mobile phase must be kept the same for
the following:
a pertinent comparison between columns.
Since the selection of the mobile phase
amine > amide > carbamate > ester > alkyl strongly affects the value of capacity factor, it
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 281
can be concluded that rel. 6.4.14 and 6.4.9 are TEST 1. Mobile phase: MeOH/water 55/45 (v/v),
incomplete, and a coefficient dependent on several buffers instead of water, column
solvent should be included in these expres- temperature: 40 C. [132,133]
sions. Since the nature of each interaction is Compound Test
dependent on the nature of the mobile phase
1 thiourea dead time t0(1)
that may affect a particular interaction inten-
sity (e.g., by the dissociation or suppressing 2 toluene hydrophobicity from capacity
dissociation of a given analyte), each interac- 3 ethylbenzene factor k(2), k(3)
methylene selectivity a(CH2)
tion type should have a different solvent-
k(3)/k(2)
dependent parameter. This would lead from
rel. 6.4.15 to a formula for the capacity factor 4 phenol capacity factor for polar
5 ethyl benzoate solutes k(4), k(5)
of the type:
6 aniline capacity factor for basic
log kj log kEB h0 jHc h00 jHmo 7 m-toluidine solutes k(6), k(7), k(8), k(9)
8 p-toluidine asymmetry As(6), As(7), As(8),
s0 jSc b0 jAc Amo 9 N,N-dimethylamine As(9)
a0 jBc Bmo k0 jCc
(6.4.20) TEST 2. Mobile phase: A. 80% MeOH in water, B. 30%
MeOH in phosphate buffer (0.02 M) pH 7.6,
In rel. 6.4.20, log kEB depends the column C. 30% MeOH in phosphate buffer (0.02 M)
and on the mobile phase, Hc, S*c, Ac, Bc, Cc pH 2.7, column temperature: 40 C [134]
depend only on the column, while Hmo, Amo, Compound Test
and Bmo are parameters related only to
a specific mobile phase and account for 1 alkylbenzenes methylene selectivity a(CH2)
polar/polarizability, hydrogen bonding for 2 amilbenzene a(CH2) k(2)/k(3)
a basic solute, and hydrogen bonding for an 3 butylbenzene
acidic solute, respectively. The sign of respec- 4 triphenylene steric selectivity a k(4)/k(5)
tive coefficients depends on the effect they 5 o-terphenyl
have on separation (decreasing log k when the
6 caffeine hydrogen bonding capacity
solvent interactions are stronger). Further 7 phenol a k(6)/k(7)
discussion of the solvent contribution to sepa-
8 benzylamine ion exchange capacity at pH > 7
ration can be found in Chapter 7.
9 phenol a k(8)/k(9)
The combination of the idea of choosing ion exchange capacity at pH < 3
a specific analyte and a specific mobile pha- a k(8)/k(9)
se for putting in evidence a specific character
of the stationary phase led to the develop-
ment of several tests allowing the character-
Other Properties of Reversed-Phase
ization of a hydrophobic column from
the point of view of hydrophobicity, polar
Columns Critical for Separation
interactions, hydrogen bonding, ion-exchange Resolution of an HPLC column is a function
interactions, and steric interactions. Several of the number of theoretical plates N, in addi-
such tests are further listed (see Test 1 to tion to the capacity factor k and the selectivity
Test 5). Other tests were also suggested for a. For this reason, an important parameter in
characterization of RP chromatographic column characterization is N. The number of
columns [137]. theoretical plates is less dependent on the nature
282 6. STATIONARY PHASES AND THEIR PERFORMANCE
TEST 3. Mobile phase: A. acetonitrile, B. n-heptane TEST 5. Mobile phase: A. 65% acetonitrile in 20 mM
(dry), C: acetonitrile/water 65/35 (v/v) phosphate buffer pH 7, B. 15% acetonitrile
[135] in 20 mM phosphate buffer pH 7, C. 30%
acetonitrile in 20 mM phosphate buffer pH
Compound Test 2.5, D. 30% acetonitrile in 20 mM phosphate
buffer pH 7, E. 75% acetonitrile in 20 mM
1 uracil dead time t0(1) phosphate buffer pH 2.5 [108]
2 N,N- silanol index a k(2)/k(3)
dimethyltoluamide Compound Test
3 anthracene
1 butylbenzene hydrophobicity k(1), k(2)
4 nitrobenzene silanol index k(4) 2 amylbenzene (kBB for capacity factor of
butylbenzene: see Table
5 benzene hydrophobicity a k(6)/k(5) 6.4.10)
6 anthracene
3 propylbenzene methylene selectivity a(CH2)
7 toluene theoretical plates N from 4 ethylbenzene from the series benzene to
Wb(7) 5 toluene amylbenzene
6 benzene
7 caffeine hydrogen bonding capacity
TEST 4. Mobile phase: A. 60% MeOH in water, colu- 8 phenol a k(7)/k(8) (acaffeine/phenol)
mn temperature, B. acetonitrile/water 65/35
(v/v), column temperature: 30 C. [106,136] 9 benzylamine silanol activity index at
pH 2.5, a k(9)/k(8)
Compound Test (abenzylamine/phenol pH 2.5)
100000
n/meter (toluene)
80000
60000
40000
20000
0 5 10 15 20 25 30 35 40 45 50 55 60
Column No. in Table 6.4.3
FIGURE 6.4.15 Effective plate numbers (per meter) n for the columns listed in Table 6.4.3.
occur if a column having a phase with low with solvents with a higher content of water
wettability is subject to a mobile phase too (for a similar derivatization degree) [138].
high in water. To avoid this problem, stationary Dewetting of the hydrophobic phases is
phases that have higher wettability can be possible when concentration of water in the
selected. Wettability can be estimated by mobile phase is higher than 80e90%. It is man-
measuring the maximum water concentration ifested by the loss of retention capability of the
in common organic solvents (methanol, acetoni- stationary phase, retention irreproducibility,
trile, and isopropanol) that allow the phase to increased tailing, and inability of the column
remain wetted and not float to the surface of to regenerate when returned to a high concen-
the solvent. All the brush phases were shown tration of organic component. Study of this
to tolerate higher concentrations of water in process [139] showed that two potential mecha-
the mobile phase than the bulk phases before nisms are responsible for dewetting. One mech-
they become un-wettable. It was also proved anism is related to the disturbance in the
that the more dispersive and less polar is the conformation of the alkyl chains in the presence
solvent, the more water the solvent can contain of high water content in the mobile phase and is
before any of the phases become unwettable. indicated as phase collapse. This explanation
Stationary phases that are incompletely derivat- starts with the observation that short-chain
ized can be wetted even by pure water. This can stationary phases such as C4 and very long
be explained by the high content of residual sila- stationary phases such as C30 are less prone
nol on their surfaces, which allows the polar to dewetting. The short chains on the silica
interactions with water molecules. C18 phases surface are not very well oriented, and the
are wetted by a lower content of water in lack of effect from the water is expected since
organic modifier as compared to shorter chain no specific conformation is to be disturbed.
phases such as C2, which becomes wetted The very long-chain phases are in a strongly
284 6. STATIONARY PHASES AND THEIR PERFORMANCE
bundled form, and the water is not able to easily water and air on the adsorbent surface. The
disturb the phase conformation, which may pressure necessary for the mobile phase to
explain the resilience of this type of phase to reenter the pores can be quite high (in an
higher water concentrations [140]. On the other experiment using a C8 column and 0.1%
hand, the interaction forces between the alkyl CH3COOH in water, a pressure of 270 bar
chains in C8 or C18 phases are easier to disturb was necessary to re-wet the column [142]).
(e.g., the melting point of C18H38 is 27e28 oC This proposed mechanism is in agreement
while C30H62 has a melting point of 64e67 with several experimental findings and can
o
C), and the water perturbs the aligned confor- easily explain why short-chain alkyl phases
mation of the phase, reducing its capability of are less prone to dewetting, their exclusion
retaining the analyte. However, other studies of water being much lower than that of C8 or
[141] indicated that the alkyl-bonded phases C18 phases. The explanation for C30-type
are always in the most compact conformation, phases may be related to the fact that such
regardless of the concentration of the organic phases are less densely packed, although the
modifier in the mobile phase, and the change carbon content (C%) is high. However, it is
in conformation due to the water is not the more likely that the dewetting is a complex
main cause of the dewetting. This suggests process whereby the exclusion of the highly
that dewetting is very likely caused by the aqueous mobile phase from the pores of the
exclusion of the aqueous mobile phase from stationary phase plays an important role in
the pores covered with a hydrophobic material the phases loss of separation capability, but
and the inability of the mobile phase to reenter changes in the conformation of the alkyl
the pores. Since most of the stationary phase chains from the silica surface under the influ-
surface is inside the pores, the exclusion of the ence of water is also likely to occur and to
mobile phase from the pores is the reason for play a role in the modification of phase
a reduced active phase. The process is depicted properties.
in Figure 6.4.16. Dewetting/phase collapse can be reversible
A dry sorbent requires a specific pressure for (not always 100%) by regenerating the column
the solvent to reenter the pores. This pressure with a partially organic aqueous mobile phase.
can be estimated with the formula: The process of regenerating the phase can
4g0 cosq require hours of flushing the column with
Dp (6.4.21) partially organic phase (e.g., 60% CH3CN and
d
40% H2O). To avoid the dewetting problem,
where Dp is the pressure required for the columns with special stationary phases [143]
liquid to enter the pore, g0 is the surface can be used in highly aqueous mediums and
tension of the liquid, d is the effective pore are preferred for specific applications such as
diameter, and q is the contact angle between the separation of very polar analytes. One such
No Description No Description
(Continued)
286 6. STATIONARY PHASES AND THEIR PERFORMANCE
(Continued)
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 287
TABLE 6.4.11 List of various columns used in RP-HPLC. (contd)
No Description No Description
81 Waters SymmetryShield RP8 113 Waters Xbridge C8 (Acquity UPLC BEH C8)
82 Thermo Hypersil BDS C18 114 Waters Nova-Pak Phenyl
83 Waters Xterra RP18 115 Agilent Zorbax SB- Phenyl
4.0
1 Area enlarged
2 8
3.5
3 9
10
4 7
6 12 15
5 13 14
9 11 17
22 16
18
3.0 23 19 20
24
21
28 37 25
32 27
33 41 26
39 40 42
38 29 30
46 48 4950 52
54 56 51 34 31
55 57 4759 53 35 36
2.5 62 58 43
60 61 64 45
69 70 63 65
66 67 68 72 44
71 74
73 75 77 78 79
80
81 82
83 84 76 86 87
2.0 85
89 100
Hydrophobicity as In(k) for acenaphthene
90
88 91 96 97
92 93 94 95 98 99
101
111 102 103 104 106 107
105 108
109 110 112
1.5 113
114
115
116
119
117 118
1.0
120
121
122
126 123 124
127
0.5
125
128
129
130
0.0 131 132
135
0.5
134
133
1.0 136
1.5
1.0 0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Selectivity as In() amitriptyline / acenaphthene
FIGURE 6.4.17 The values of ln k for acenaphthene and the values for ln a for the pair acenaphthene/amitriptyline for
the columns listed in Table 6.4.11 [145].
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 289
pair (a z 44) is obtained for a phenyl-type analyte molecules. In this way, the content of
stationary phase. organic modifiers in the mobile phase could be
The columns listed in Table 6.4.11 and dis- used at levels above 5%, such that the columns
played in Figure 6.4.17 include a variety of are less prone to dewetting [146]. Some polar
columns commonly used in RP chromatog- compounds are better separated on columns
raphy. Among them are more standard columns such as cyano or pentafluorophenylpropyl (e.g.,
with C8 or C18 stationary-phase and end- Ascentis Express F5) that involve larger contribu-
capped with methyl groups, along with olumns tion from van der Waals interactions compared to
showing other bonded phases and some with solvent cavity formation energy. The separation
polar-embedded functional groups and C8, of polar and nonpolar compounds together is
C18, phenyl, alkyl chains of different lengths best achieved on columns with an intermediate
(e.g., C12). The nature of the hydrophobic hydrophobic character as seen in Figure 6.4.17
bonded phase such as CN (with some polarity), for the separation of acenaphthene and amitrip-
phenyl, alkyl, and the length of the hydrocarbon tyline, which is best achieved on a phenyl or
fragment (for alkyl chains) influences the phase CN-type column (with high values for a).
hydrophobic character, as characterized, for The list of columns from Table 6.4.11 is incom-
example, by the k values of acenaphthene. The plete since more than 500 column types are
separation of very nonpolar compounds is commercially available (not considering different
not achieved well on columns with low hydro- formats). These columns offer a wide range of
phobicity (such as CN phases). However, very characteristics allowing diverse separations. For
hydrophobic stationary phases leading to very example, very fast separations can be achieved
high k values are also not appropriate for the by using shorter columns, with small particle
separation, since they lead to unacceptably size, higher flow rates, and possibly temperature
long retention times. Probably the best choices of the column higher than ambient. One example
of columns for the separation of hydrophobic of such a separation is given in Figure 6.4.18 for
compounds are those with a C8 stationary tenoxicam and piroxicam on a Zorbax Stable-
phase. Bond C18 column, 50 mm length, 4.6 mm i.d.,
For organic compounds that have a polar char- and 1.8 mm particle size [147]. The separation
acter, there are two choices. One is to use more was performed in gradient elution with mobile
hydrophobic columns that can point up small phase starting from 30% acetonitrile and 70%
differences in the hydrophobicity of the mole- aqueous solution of 0.1% H3PO4 to 100%ACN
cules, and the other is to use columns with in 1.5 min; flow rate: 2 mL/min and column
some polarity that involve additional interac- temperature: 60 C. The detection was performed
tions besides hydrophobic ones. For the choice by UV absorption at 368 nm
of using highly hydrophobic columns, stationary
phases with long alkyl chain bonded to silica Hydrophobic Stationary Phases
surface (e.g., C27 to C30) are recommended.
with Some Polarity
The separation of polar compounds on C8 or
C18 stationary phases require a very low content The common hydrophobic stationary phases
of organic modifier in the mobile phase (less than are successfully used in a wide range of applica-
5%), which is sometimes a problem due to tions, and compounds with a large range of
processes such as phase collapse and dewetting. polarities and having only some hydrophobic
The increase in the hydrophobicity of the moiety, are well separated using hydrophobic
stationary phase can increase the hydrophobic HPLC columns. However, some analytes are
interactions with the hydrophobic moiety of the more problematic to separate. Examples of
290 6. STATIONARY PHASES AND THEIR PERFORMANCE
such analytes are compounds with a basic char- Some important types of phases used in RP-
acter such as amines that give peak tailing, large HPLC that have groups with some polarity
molecules that are not soluble in organic are those containing cyano groups (e.g., cyano-
solvents such as proteins or saccharides, and propyl), phenyl groups connected on the silica
very small polar compounds that are very little surface with a hydrophobic linker, or those
retained on hydrophobic stationary phases and with an ether-linked phenyl (e.g., Synergy
require a low level of organic component in Polar-RP from Phenomenex). Several cyano
the mobile phase to be eluted from the column. and phenyl phases are listed in Table 6.4.11.
Also, some isomers that differ minimally in The separation on such phases involves, besides
hydrophobicity may be difficult to separate on hydrophobic interactions, a number of polar
conventional hydrophobic phases. For these interactions that are stronger or weaker depend-
problem analytes, either more polar ing on the analyte. Besides better separation of
stationary phases or special properties of compounds with some polarity, these phases
common-type stationary phases were devel- can be very useful for the separation of isomers
oped, such that the compounds can be success- when hydrophobic interactions alone are not
fully analyzed. Various such phases were sufficient to assure separation.
obtained using procedures such as the use of A very good solution for obtaining hydro-
a polar group in the bonded phase, sterically phobic stationary phases with the capability
protected base silicas, bidentate bonded phases, to have more contribution from polar interac-
polar-embedded stationary phases, the use of tions and withstand very high water content
hybrid organic-inorganic particles, polar end- in the mobile phase is the use of polar-
capped silica [49], or polymerization of specific embedded hydrophobic stationary phases. As
organic monomers on the silica support [148]. previously discussed, a variety of embedded
Such phases may offer, besides hydrophobic phases can be made. Columns with various
interactions, other types of interactions such as embedded polar groups such as ether, amide,
hydrogen bonding, dipole-dipole interactions, urea, carbamate, sulfone, thiocarbamate, mixed
aromatic p-p stacking, and steric selectivity, as amide and carbamate, were manufactured.
pictured in Figure 6.4.12. Common commercially available columns
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 291
contain embedded urea, amide, carbamate, or pyrimidine bases) can be achieved, even at
ether groups with C8 or C18 hydrophobic intermediate pH values [143, 149, 150]. A
chains. Some of these phases are included in comparison of conventional C18 columns with
Table 6.4.8 (Phase No. 4, 15, 28, 33, 59, 60, 66, a number of polar-embedded C18 columns
73, 81, 83, 84, 85, 88, 109). The wide distribution and a number of polar-end-capped columns
in the k values for those phases indicates the reported in the literature [108] showed that, on
versatility in the hydrophobicity that can be average, considerable differences exist in the
achieved with embedded materials. For some separation properties of these types of columns.
analytes, the phases provide the same type of The results of the study are summarized in
retention as their C8 or C18 counterpart, as Table 6.4.12 [108].
seen from Figure 6.4.17 when comparing the k Other phases with hydrophobic character
values for the embedded phases with other and some polar interaction capability are
conventional C18 or C8 phases from also available. For example, embedded phases
Table 6.4.11. However, a different selectivity containing two zones of polarity are known.
compared to alkyl phases can be seen for other One such type of phase contains one ether
analytes, in particular for those with a stronger group, one sulfonamide moiety, and a C16
polar character. Also, with the incorporation in hydrophobic moiety. Such a phase can be
the alkyl chain of a polar functional group that used in a totally aqueous mobile phase and
is placed close to the surface of the silica gel, exhibits a high selectivity toward N-contain-
these phases retain the hydrophobic character, ing compounds. Another special phase has
while the phase can remain solvated by water two anchor points to the silica support (it is
at low percentages of organic solvent, even a bidentate phase). The structures of a phase
with 100% water in the mobile phase. Under with two polar zones and that of a bidentate
these conditions, the alkyl chains maintain phase are further shown:
O CH3
R
O Si O Si
O CH2
CH3 CH3
O CH3
O Si OH H2C
CH2 (CH2)15
O Si Si CH2 CH2 N
O O CH2 S O CH2
O CH3 CH2 O O O Si O Si
R
O CH3
R = C8H17 or C18H37
their conformational freedom and can interact The propylene bridges can sterically cover the
with polar analytes. The presence of the polar silica matrix and the silanol groups, offering
functionality close to the surface also shields superior stability to low and high pH of mobile
the effects of unreacted silanol groups protect- phase, and hindering the interaction of the ana-
ing the phase from the attack of stronger acids lytes with basic character with the silanol groups
and stronger bases. Since the silanol activity is that lead to better peak shape for these
suppressed in polar-embedded phases, better compounds [151].
peak shape and decreased tailing of basic The polar groups may also result from addi-
compounds (such as amines, purines, and tional interactions besides those with the main
292 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.4.12 Comparison on several test parameters for conventional, polar end-capped, and polar embedded
columns [108]*.
Note*: The columns were evaluated using Test 5 at page 283 where parameters kBB, a(CH2), acaffeine/phenol, abenzylamine/phenol pH 2.5,
abenzylamine/phenol pH 7 are defined.
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 293
bonded phase (e.g., C8 or C18). These polar mobile phase. For this reason, this type of
groups may contribute to the separation and column may have reproducibility problems,
may also offer additional protection against depending on the water content in the mobile
uncontrolled acidic silanol groups in the phase. Also, due to ion-exchange type interac-
stationary phase. These two effects are achieved tions, the compounds with basic character may
with the use of polar end-capping. This tech- show severe tailing.
nique protects the stationary phase from
hydrolysis and offers a dual type of interaction
with the analytes. The columns with polar
Other Types of Hydrophobic Phases
end-capping can be considered to have a kind Other special hydrophilic stationary phases
of mixed-mode character. This type of column are those with very long alkyl bonded frag-
may still show tailing when a mixed-mode ments. These phases are particularly useful for
type of interaction is not desirable. Columns the separation of compounds with very little
with C8 or C18 hydrophobic phase and end- hydrophobic character that are easily eluted
capped with polar substituents (e.g., NH2 or from typical C18 columns. For such
OH on a propyl handle) are commercially avail- compounds, the use of mobile phases with
able, and they offer an alternative-type column a very high content of water is necessary,
with its characteristics between a stationary imposing on the column the need to have resil-
phase noneend-capped and with short-alkyl ience to dewetting. The long alkyl chain
chains and that of a more hydrophobic column stationary phases, such as C27 or C30, were
(such as C8 or C18). found useful for the separation of this type of
One special type of stationary phase offering compound. These phases are more retentive
additional polar interactions are those none for polar and nonpolar analytes than are most
end-capped with short-alkyl chain bonded polar-embedded and even high-coverage C18
phase. As previously discussed, the unreacted phases. Because of a higher degree of surface
silanol groups present on an alkyl-bonded silica shielding, long-chain phases also offer greater
impart a degree of polarity to the phase, and pH stability than do C8 and C18 phases. They
frequently this polarity is detrimental since it are also more resistant to phase collapse under
produces tailing on compounds with polar or high aqueous conditions than are C18 phases.
ionic moieties. However, in some cases, the alkyl This behavior may be explained by the resis-
function alone provides insufficient separation tance of long chains to conformation changes
selectivity, and the presence of silanols can in the presence of water at the column tempera-
cause polar interactions with the polar function- ture, or it may be related to a lower density of
ality on analytes, which can be useful. The C30 chains on the silica surface for similar C%
resulting mixed mechanisms can yield content with a C18 phase [79].
improved separations, for example, for small Special stationary phases also include those
polar molecules. Also, the phases with short that are based on core-shell (or fused core) tech-
alkyl chains (e.g., C4) have a low propensity nology. The main advantage of this type of
for phase collapse when high concentrations of phase is the higher number of theoretical plates
water are used in the mobile phase. The main compared to columns similar in dimensions
problem with this type of phase is the difficulty but filled with porous particles. A variety of
in having a uniform and reproducible level of core-shell columns with different brand names
silanol groups. The activity of the free silanol are available from different commercial sour-
groups on the phase surface is also dependent ces. One series of core-shell columns is the
on the amount of water adsorbed from the Kenetex (from Phenomenex), available in C8,
294 6. STATIONARY PHASES AND THEIR PERFORMANCE
C18, XB-C18 (which has butyl side chains for base support with a silicone polymer that shields
protecting against silanol access), phenylhexyl, the silanol groups, the bonded phase being con-
and pentafluoro-phenyl (PFP), with particle nected to the coated surface. Other columns
sizes of 2.6 mm and 1.7 mm [152, 153]. Another take advantage of the ethylene bridged structure
series is Ascentis Express (from Supelco), of the silica base but use it only on the surface of
which is available in C18, phenyl-hexyl, C8, the silica particles (TWIN technology), as used,
and pentafluorophenyl. Another series is Accu- for example, in Gemini NX columns.
core from Thermo Scientific that offers C18 and Silica monoliths can also be considered special
PFP columns as well as a column end-capped columns. The C18-bonded phase on monoliths is,
with polar groups (Accucore aQ). Agilent however, a relatively common type of column,
offers Poroshell series. such as Onyx Monolithic C18 or Chromolith
One additional group of stationary phases Performance RP-18 columns, which are available
consists of those with a fluorinated bonded frag- in various formats. These columns have a typical
ment. Typically, the fluorinated fragment is dual porous structure with mesopores of about
attached to the silica surface with a short 130 A and macropores of about 2 mm diameter.
handle, and the phase has the following sche- The nature of the bonded phase is similar to
matic structure: that for spherical particle C18 columns, and the
silica surface is end-capped. This type of column
allows a reduction in elution time up to nine
Si S i CH 2 R
O times due to the capability to use higher flow
CH 2
rates without having problems with the column
backpressure. Due to a rapid mass transfer of
where R can be perfluorohexyl straight chain, the solutes between the bonded phase and the
perfluorohexyl branched chain, perfluorooctyl, mobile phase, the decrease in the number of
perfluoropropyl, perfluorododecyl, pentafluoro- theoretical plates at higher flow rates, as pre-
phenyl, pentafluorophenyl alkyl chain, penta- dicted by the van Deemter equation (see rel.
fluorophenylpropyl, and other similar phases. 2.2.8), is not as intense. Shorter retention times
In Table 6.4.8, phases No. 23, 43, 51, 123, and also lead to better resolution [156]. For compar-
132 are fluorinated. These phases cover a wide ison, the variation of the theoretical plate height
range for the k values, indicating that the fluori- in various columns is shown in Figure 6.4.19.
nated materials offer a wide range of hydropho- As shown in this figure, the flow rate for which
bicity, some with very high log Kow for the the measurements were possible is higher for
corresponding model as shown in Table 6.3.4. the monolith column, and at the same time the
The fluorinated phases offer an alternative mate- increase in the plate height is smaller.
rial for separations and were proven very useful An example of a separation of a monolith-type
in particular separations [154, 155]. column is given in Figure 6.4.20 for the separa-
A variety of other phases with hydrophobic tion of norfloxacin, one of its metabolites and of
character are also available, either commercially ciprofloxacin. The column utilized for the separa-
or at an experimental stage. As an example, tion is a Chromolith Performance RP-18e (Merck
EnviroSep type phases (from Phenomenex) KGaA), 100 mm x 4.6 mm. Two overlaid chro-
contain a silica and polymer support and matograms are shown in Figure 6.4.20, corre-
a hydrophobic bonded phase. Other columns sponding to a blank sample (A) and a sample
are made using a variety of special technologies. collected from a same human volunteer using
For example, Capcell Pak type columns (Shiseido norfloxacin (B). Sample preparation was based
Co. Ltd.) involve a surface coating of the silica on deproteinization of 200 mL human plasma
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 295
35
5 m irregular
30
25
5 m spherical
Plate height ( m)
20
15
3 m spherical monolith
10
0
0 1 2 3 4 5 6 7 8 9
Flow rat e in mL/min
FIGURE 6.4.19 Variation in theoretical plate height for different types of stationary phases (irregular particles 5 mm,
spherical particles 5 mm, spherical particles 3 mm, and monolithic).
LU
HN
35 N N
NH2
30 F COOH
CH3 N N
HN O
N N
Ciprofloxacin
25
F COOH
OH
20 F COOH
Norfloxacin
O
metabolite
Norfloxacin
15
10
B
0
A
0 2 4 6 8 10 min
FIGURE 6.4.20 Separation on a Chromolith Performance RP-18e (Merck KGaA), 100 mm x 4.6 mm column of nor-
floxacin, one of its metabolites, and ciprofloxacin (added as internal standard); (A) blank plasma sample, (B) sample from
human volunteer using norfloxacin [157].
296 6. STATIONARY PHASES AND THEIR PERFORMANCE
with 50 mL acrylonitrile that also contained cipro- columns less adequate for the new develop-
floxacin as internal standard, followed by mixing ments toward UPLC. For these reasons, the
the supernatant resulting from deproteinization use of polymeric columns, particularly for RP-
with 3400 mL mobile phase. A volume of 100 mL HPLC, is limited. Some polymeric phases can
was then injected into the analytical column. operate at pH values as low as 1 and as high
The mobile phase for the separation was made as 13. Some examples of RP polymeric phases
of 1% triethylamine in aqueous solution brought are given in Table 6.4.13, where certain reported
to pH 4.0 with phosphoric acid and methanol characteristics are also shown.
as organic modifier. The elution program con- The stationary phase from these columns
sisted of: 85/15 (v/v) aqueous / methanol for may consist of the polymer in itself or may
0e9 min, a linear gradient between 9 and 10 have a specific bonded phase (such as C8, C18)
min to 40/60, followed by a linear gradient on the polymeric support. As seen from Table
between 10 and 11 minutes to the initial compo- 6.4.13, the polymeric columns display a wide
sition (85/15), and kept constant up to 11.5 min. range of acceptable pH for the mobile phase.
The flow rate was 2.5 mL/min, and temperature Also, the reported values for N/m in the range
25 C. The detection was done with a FLD setup of 80,000 are typical for many silica-based
at 268 nm excitation and 445 nm emission [157]. columns with 5 mm particles. The only param-
eter that may contribute to a lower rating of
Organic Polymer-based Hydrophobic polymeric columns is the acceptable maximum
pressure, which may bring some limitations to
Stationary Phases
the flow rates that can be used. The pore system
Although most RP-HPLC columns are silica- generated in the polymer is an important factor
based, the polymeric columns can be very regarding the rate of mass transfer effects
useful for specific applications. This is particu- during the chromatographic process, and this
larly the case when a very low or a very high is difficult to assess from data such as those
pH of the mobile phase is necessary. The main given in Table 6.4.13. Besides stationary phases
advantage of polymeric supports is that they made with particles of rigid organic polymers,
are more resilient to very low and very high polymeric monoliths are also used as chromato-
pH. Also, the polymeric substrate does not graphic media [37]. Other polymeric phases
have a potential layer of unwanted polar groups with hydrophobic bonded groups such as C18
as do the silica-based stationary phases, which or pentafluorophenyl are commercially avail-
even after derivatization still have unreacted able (e.g., Jordi phases from MicroSolv). Among
silanol groups besides the desired ones. different polymeric materials used as stationary
However, polymeric materials typically show phase in HPLC, several molecular imprinted
lower theoretical plate numbers N for the same polymers (MIPs) were also evaluated. The poly-
dimensions as the silica-based particles. meric structure has been typically based on
Another disadvantage of polymeric supports is methacrylic acid and/or styrene crosslinked
caused by the variation in the swelling of the with ethyleneglycol dimethacrylate that were
polymeric particles when they are used in polymerized in the presence of a template
various solvents. This variation in swelling molecule (e.g., nortriptyline [158]).
affects the volume occupied by the stationary
phase in the column and can lead to the forma-
Special Hydrophobic Stationary Phases
tion of void spaces and loss of efficiency.
Organic polymeric materials are also less resil- Several special hydrophobic stationary phases
ient to high pressures, and this makes polymeric have been used successfully in RP-HPLC.
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 297
TABLE 6.4.13 Examples of polymeric columns and their main characteristics.
One of these phases is porous graphitic carbon This operation eliminates certain surface
(PGC). This material is obtained by decomposing attached functional groups, produces a structural
organic matrices using silica as template. For rearrangement of C atoms, and removes the
example, the silica used as template is impreg- micropores. After cooling down to 1000 oC, the
nated with a mixture of phenol and formalde- replacement of argon by hydrogen can induce
hyde and then heated to 80e160 oC to initiate reactions between hydrogen and free radicals
the polycondensation. The characteristics of the or functional groups still present at the carbon
silica gel as template material determine the surface. By deactivating the PGC surface
size and porosity of the particles that will becomes more uniform. The result is porous
be obtained. The polymer is then pyrolyzed graphitic carbon, which is now commercialized
under inert atmosphere (N2) at 1000 oC. Thus, (trade name of Hypercarb) [159]. Unlike
highly porous amorphous carbon is produced. silica-based packing materials, carbon-based
This carbon corresponds to what is normally stationary phases have the advantages of being
called carbon black. After this step, the silica more resistant to hydrolysis, while the lack of
template is dissolved with a hot aqueous swelling or shrinking makes them more useful
NaOH solution. The graphitization is realized than the polymeric materials. The efficiency of
through a thermal treatment at high temperature columns packed with these materials is compa-
(about 2300 oC) under inert atmosphere (Ar). rable to modified silica-based columns. Some
298 6. STATIONARY PHASES AND THEIR PERFORMANCE
physical properties of PGC are given in Table to these characteristics, the PGC phases have
6.4.14 [160]. the capacity to separate very polar compounds
The PGC columns were successfully used in (carbohydrates and compounds with several
RP-HPLC practice. The retention of nonpolar hydroxyl, carboxyl, amino and other polar
compounds is similar to that of silica-based groups). Compounds with low or no affinity to
hydrophobic columns (C18 or C8), but the C18 stationary phase can have strong retention
graphitic columns show a higher a(CH2) value and can be separated in accordance with their
compared to any C18 or C8 column (using meth- polarity in an order that is not specific to the
anol as an eluent) [161]. However, the retention RP mechanism [162, 163]. Also, the PGC phases
mechanism on PGC stationary phases is more have the ability to separate structurally related
complex than for silica bonded phases. These compounds (geometric and diastereoisomers)
phases are able to interact with hydrophobic due to the flat and highly adsorptive surface
moiety from the analyte structure, but also of the graphite [164]. Several properties of
with the polar groups from their structure. The graphitic columns such as resilience to a wide
strength of interaction depends on the molecular pH range, temperature resistance (up to
area of the analyte in contact with the graphite 200 oC) [165], and the capability of their surface
surface and on the nature and the type of func- to be covered with surfactants behaving as an
tional groups at the point of interaction with ion-exchanger make the PGC columns of consid-
the flat graphite surface. Under specific elution erable utility [166].
conditions, PGC behaves as a strong retentive As an alternative to porous graphitic carbon,
stationary phase for both nonpolar analytes phases made of carbon-clad zirconia or titania
and polar compounds. Unlike silica-bonded have been suggested. This type of material can
phases when increased polarity of analyte deter- be produced either by high-temperature graph-
mines a decreased retention behavior, the reten- itization of organic polymers covering the
tion on PGC is less influenced by polarity, and in zirconia or titania support or by chemical vapor
some circumstances it may increase with the deposition of carbon on zirconia. However, in
increase of polarity of analyte. Consequently, practice, the surface of this type of stationary
the elution order on PGC is not necessarily the phase is not perfectly homogeneous, and a
hydrophobicity order of analytes, as usually significant proportion of the surface of zirconia
takes place in reversed-phase mechanism. Due still remains uncovered by carbon, thus creating
highly acidic residual zirconia groups [167].
Among other special stationary phases used
TABLE 6.4.14 Some properties of graphitic carbon
in HPLC are those based on silica hydride.
phases.
Bare silica hydride can be used for direct phase
Property Values separations (in organic-normal phase HPLC)
or for aqueous normal-phase separations.
Particle shape Spherical 3, 5, or 7 mm diameter
Commercial stationary phases with common
Porosity type Porous 70e75 % dimensions such as 4 mm particle diameter and
Specific surface area Higher than 100 m2/g pore size of 10 nm are available. Bonded phases
on silica with C18, C8, cholesterol groups, and
Pore volume ~0.7 m3/g
the like are commercially available (e.g., Cogent
Average pore diameter ~25 nm Bidentate C18, Cogent Bidentate C8, Phenyl
Carbon load 100% Hydride, Cogent UDC Cholesterol, etc.). These
types of columns can be used in reversed-
Mechanical strength >400 bar
phase-type conditions [19].
6.4. HYDROPHOBIC STATIONARY PHASES AND COLUMNS 299
Selection of a Column for RP-HPLC a particular reason, for example, related to the
availability or price of the column. In this case
Separations
a column equivalent with the one recommended
The selection process of a column useful for an can be selected. For selecting a similar but not
RP separation should be guided by the work identical column, the calculated distance using
previously performed and reported in the litera- rel. 6.4.17 or 6.4.18 [123] is very useful, and
ture. An enormous body of information is avail- columns with a small distance F to the recom-
able regarding numerous analytical procedures. mended one can be employed successfully.
Information may be available for compounds The adequacy of a column depends mainly
identical with those of interest (possibly in on achieving a good resolution R for the solutes.
a different matrix) or regarding similar Capacity factor k plays an important role in
compounds, although not exactly the same. achieving this goal. In general, for increasing k,
When such information is available, the problem for compounds with smaller hydrophobic moie-
of column selection remains open, but a much ties, columns with stronger hydrophobic char-
more limited search is necessary for finding acter (large Hc values) are necessary. More
a good fit. Starting with a recommended column, hydrophobic compounds can be separated on
improvements can be made such that the chroma- less hydrophobic columns (see Table 6.4.8).
tography will be done in a shorter time or with This recommendation is based on the assump-
better resolution. This can be achieved, for tion that larger capacity factors k for a hydro-
example, by selecting the same stationary phase phobic compound are obtained when Hc is
in a different format or with smaller particle larger. However, in order for a large Hc to
size, in a narrower column, in a shorter or in contribute to a large k, the hydrophobicity
a longer column depending on the improvement parameter h for the compound must be large.
objectives. The criteria listed in Table 6.3.3 must Since log k is well correlated with log Kow (see,
be considered when format modifications e.g., Figure 3.1.3 and [168]), a good correlation
are implemented. The type of available equip- between h values and log Kow values would
ment influences the decision regarding the be expected. Figure 6.4.21 shows this correlation
column selection. When UPLC equipment is between h and log Kow for the compounds
available, columns with small particles and nar- listed in Table 6.4.7. When nortriptyline and
rower diameter can be selected, since they amitriptyline are not included in
provide higher N for the same length, in spite of the correlation, the linear correlation gives
their higher backpressure. Some of the advan- R2 0.8277, but when the whole set of
tages regarding column quality can be opposite compounds is used, R2 0.3657, which indi-
to each other. For example, a column with small cates extremely poor correlation. Therefore, the
particle size and smaller diameter can lead to an assumption that large Hc and h values alone
increase in theoretical plate number N improving lead to high log k values is not correct for certain
resolution and in reduction of the analysis time compounds. In cases where besides hydro-
and use of solvents, which are very advanta- phobic moieties the compounds have polar
geous. On the other hand, the same parameters groups, columns with additional interactions
may lead to a reduction of the number of injec- may be more appropriate for the separation.
tions n that can be made on a column without These columns include cyano of phenyl-bonded
affecting performance and to an unacceptable phases, or are C8, C18 columns with polar-
increase in column backpressure. embedded groups, have end-capping done
Athough a specific column is recommended, using small polar fragments, or are not end-
a change is sometimes needed or desired for capped. As an example, Figure 6.4.17 displays
300 6. STATIONARY PHASES AND THEIR PERFORMANCE
5 a mi tri ptyl i ne
nortri ptyl i ne
4
log Kow
R2 = 0.3657
2
1
R2 = 0.8277
0
-1
-2
-2.5 -2 -1.5 -1 -0.5 0 0.5
'
FIGURE 6.4.21 Correlation between h values and log Kow for the compounds given in Table 6.4.7 (including nortrip-
tyline and amitriptyline R2 0.3657, or excluding them R2 0.8277).
the case of higher a for the pair amitriptyline As previously discussed, the column nature
versus acenaphthene, which is constantly together with the nature of the compounds to
higher for more polar columns. be separated also influences the peak tailing.
Even for columns leading to high values for The selection of columns with embedded polar
k for a certain type of compound, the separa- groups can solve some of the tailing problems.
tion is not necessarily good since the resolu- The resilience to a wide pH range, also desirable
tion R also depends on a. Most compounds in certain separations, is obtained using special
analyzed by RP-HPLC have both hydrophobic end-capping or polar embedded groups. The
and polar moieties and other column charac- use of pure base silica (Type B), which is less
teristics (S*c, Ac, Bc, Cc(2.8), and Cc(7.0)) acidic, is another factor contributing to a wider
contribute to the separation, as previously dis- pH range of use for silica-based columns.
cussed in this section. For this reason, the Also, dewetting problems are alleviated with
separation of compounds that have mainly special types of end-capping or with the use of
a hydrophobic character can be evaluated hydrophobic columns with embedded polar
based on column hydrophobic character. groups.
When polar groups are present (as is the case
for most analytes), not only the hydropho-
bicity of the column is important, but other 6.5. POLAR STATIONARY PHASES
characteristics such as a partial polar character. AND COLUMNS
This can be seen, for example, from the value
for ln (a) for the pair acenaphthene versus
General Aspects
amitriptylene shown in Figure 6.4.17. The
largest a values are obtained for columns Polar stationary phases are used in normal-
with some polar character (cyano and phenyl). phase chromatography (NPC), in hydrophilic
Further discussion of column selection can be interaction chromatography (HILIC), and in
found in Section 9.4. aqueous normal-phase chromatography
6.5. POLAR STATIONARY PHASES AND COLUMNS 301
(ANPC or ANP). More recently, HILIC has and by their surface that is wettable with polar
started to gain considerable popularity. Some solvents such as water. Several types of polar-
delay in its application was caused by fewer bonded moietries on silica and on polymeric
available columns as compared to RP-HPLC. substrates used in NPC and HILIC chromatog-
This lack of column availability is being raphy are presented in Table 6.5.1 [170]. The
rapidly eliminated, and various HILIC polarity of the phase is the highest for silica,
columns are now commercially offered [169]. followed by phases with weak ion-exchange
The technique can be successfully applied for character (anion, cation, zwitterions), such as
the analysis of highly polar organic molecules amine phases, and followed by diol, amide,
such as sugars, amino acids, and numerous and cyano.
pharmaceuticals.
Normal-phase chromatography is currently Specific Procedures for Polar-Phase
practiced mainly on silica, although other
Synthesis
polar stationary phases were used in the
past, such as alumina and magnesia. HILIC The main attention in this chapter was given
chromatography can also be performed on to synthesis of hydrophobic stationary phases
silica, but bonded phases containing terminal for RP chromatography. However, as previously
polar groups such as aminopropyl, diol indicated in Section 6.2, some of the procedures
bonded, amide bonded, and peptide bonded, used for synthesis of hydrophobic stationary
are now common phases for HILIC. Cyano phases can also be used for making polar phases
(e.g., cyanopropyl) phases can also be by having a polar group in the radical R of
included in the polar phases category (see chloro-R-silanes or alcoxy-R-silanes used for
log Kow in Table 6.3.4), although they are silica gel derivatization. Other procedures
used in RP chromatography with a mobile were specifically developed to generate polar
phase that is more polar than the bonded phases. One such procedure starts
phase of the column. Some cyano RP phases with preparation of a bonded polysuccinimide
were indicated in Section 6.4. The polar phases on silica by reacting this compound with amino-
are typically characterized by their polar propyl silica, as shown schematically in the
groups (such as silanol in the case of silica) reaction 6.5.1:
O O
O H3C O
O Si O Si NH2 +
[ N ]n N
O H3C N
O O
O NH O
O
O H3C O
(6.5.1)
O Si O Si NH O
O H3C N
N
O
O
302 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.5.1 Several types of polar stationary phases Parts of the poly(succinimide) rings are
used in NPC and HILIC opened to form amide bonds with the amino-
chromatography. propyl moieties. In this way a polymeric-type
Type of phase Phase phase bonded on silica is generated. Only
a fraction of the succinimide rings are
Silanol Bare silica
engaged in this bonding; the rest of the rings
Diol on silica Diol, ether embedded and diol remain intact and susceptible to reaction with
Amide on silica Amide terminal, polyamide nucleophiles, which can be used to produce
a variety of functional silicas [170]. The poly(suc-
Weak polar on silica Cyano, perfluorinated (also
cinimide)-silica material can further undergo an
used in RP mode)
alkaline hydrolysis in water to form a
Weak anionic on silica -C3H6-NH2, diethylamine, poly(aspartic acid) bonded phase, it can suffer
triazole, etc.
hydrolysis in the presence of aminoethanol to
Weak cationic on silica Sulfonylethyl, etc. form poly(2-hydroxyethyl)aspartamide bonded
Zwitterionic on silica Amino-sulfonic, amino- phase, and in the presence of aminoethansulfonic
carboxilic acid it can form poly(2-sulfonylethyl)aspart-
amide-bonded silica. The formation of different
Mixed mode on silica Both polar and hydrophobic
groups functional groups for poly(succinimide)-silica
phases is schematically shown in reaction 6.5.2.
Weak anionic polymeric Different polar groups on
porous polymers
O
HO
O N SO3H
HO
H2O
O N O SO3H
O
HN
O N NH O H2N
SO3H O N
HN
OH
N O N O
OH
NH O
O HN NH O
H2N
OH O N
HN
N O
NH O
(6.5.2)
6.5. POLAR STATIONARY PHASES AND COLUMNS 303
O CH3 O CH3
O Si Cl + HO O C CH3 O Si O O C CH3
O - HCl O
CH3 CH3
CH3 CH3
O O O-
O Si O O C CH3 + n O P N+
O O CH3 (6.5.3)
O CH3 CH3
O
polymerization and O CH3
bonding to silica O Si O [ CH2 C] O C(CH3)3
n CH3
O O O
P + CH3
O O N
O O-
CH3
O O
O C2H5O O O
CH2 CH2
O Si OH + Si O O Si O Si O
O C2H5O OC2H5 CH3 O O CH3
X
H2C O
+n O O
O [ ]
O Si O Si O n CH3
O O CH3 X
(6.5.4)
Another method for preparing polar phases Synthesis of other active phases may include
leading to monolithic columns used for HILIC macrocycles such as perhydroxyl-cucurbit[6]
separations is based on the polymerization of uril. The macrocycle can be obtained following
vinyl monomers having various X functional the reactions 6.5.5:
304 6. STATIONARY PHASES AND THEIR PERFORMANCE
O
O O O
C
H2N NH2
+ HN NH N N CH2 N N CH2
O HCHO K2S2O8
HC CH HO OH (6.5.5)
O + HN NH H2SO4 N N CH2 N N CH2
n=6 n=6
H2N NH2
C O O O
cucurbit[6]uril perhydroxyl-cucurbit[6]uril
O
TABLE 6.5.2 List of several commercially available HILIC columns, the capacity factor k for uridine, and several
a values.
1 ZIC-HILIC (5mm) 2.11 2.03 1.67 1.5 1.11 1.14 0.33 1.57
2 ZIC-HILIC (3.5 mm) 2.10 2.07 1.71 1.51 1.12 1.14 0.27 1.64
3 Nucleodure HILIC (3 mm) 2.20 1.55 1.28 1.46 1.08 1.14 0.34 0.95
10 LiChrospher Diol (5 mm) 1.50 1.36 1.15 1.32 1.06 1.17 0.63 1.16
11 Chromolith Si 0.31 1 1.12 1.16 1.11 1.31 0.09 8.21
12 HALO HILIC (2.7 mm) 0.64 1.08 1.16 1.18 1.13 1.29 0.64 29.03
13 COSMOSIL HILIC (5 mm) 1.60 1.6 1.14 1.36 1.03 1.13 0.8 0.49
14 Sugar-D (5 mm) 1.58 1.74 1.44 1.45 1.1 1.22 1.9 0.25
15 NH2-MS (5 mm) 2.44 1.88 1.3 1.36 1.07 1.2 0.82 0.28
2.2
1.8
1.6
(OH)
1.4 (CH2)
1.2
0.8
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column No. in Table 6.5.2
FIGURE 6.5.1 The values of a(CH2) and a(OH) obtained using formula 6.5.7 and 6.5.8 for the columns indicated in Table
6.5.2 (mobile phase acetonitrile:aqueous buffer 90:10, the buffer being 20 mM ammonium acetate at pH 4.7) [179].
6.5. POLAR STATIONARY PHASES AND COLUMNS 307
NH2 NH2 O O
N H3C
N N NH NH
NH N NH O NH O NH O
Adenine Cytosine Thymine Uracil
A C T U
O O O O
H3C
NH NH NH NH
HO HO HO HO
N O N O N O N O
O O O O
OH OH OH OH OH
Uridine 5-Methyluridine 2'-Deoxyuridine 2',3'-Dideoxyuridine
Uri 5MeUri 2dUri 2,3ddUri
FIGURE 6.5.2 Formulas for nucleic bases and for some nucleosides based on uracil/thymine, used for the calculation of
a(CH2) and a(OH).
NH2 NH2 O O
N N N N
N N N N
HO HO HO HO
N N N N N NH2 N N NH2
O O N O O
OH
OH OH OH OH OH
Vidarabine Adenosine 2'-Deoxyguanosine 3'-Deoxyguanosine
Vid Ade 2dGua 3dGua
FIGURE 6.5.3 The formulas of diasteroisomers vidarabine (Vid) and adenosine (Ade) and of regioisomers 2-deoxy-
guanosine (2dGua) and 3-deoxyguanosine (3dGua).
1.6
1.5
1.4
1.3
1.2
1.1
1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column No.in Table 6.5.2
FIGURE 6.5.4 The values of adia(Vid/Ade) and aregio(2dGua/3dGua) obtained using formula 6.5.9 for the
columns indicated in Table 6.5.2 (mobile phase acetonitrile: aqueous buffer 90:10, the buffer being 20 mM ammonium acetate
at pH 4.7) [179].
given in Figure 6.5.5, and the expression for aqueous buffer 90:10, the buffer being
ashape is given by expression 6.5.10. 20 mM ammonium acetate at pH 4.7)
kNPaGlu [179], the values for ashape are shown in
ashape NPaGlu=NPbGlu
kNpbGlu Figure 6.5.6.
(6.5.10) 5) Two selectivity parameters can be used for
evaluation of ion-exchange interactions. One
For the columns listed in Table 6.5.2, and selectivity is used for describing anion-
using the same mobile phase (acetonitrile: exchange properties aAX, and the other for
6.5. POLAR STATIONARY PHASES AND COLUMNS 309
HO
O
OH HO
O NO2
O
OH OH OH
O
OH OH
NO2
4-nitrophenyl- 4-nitrophenyl-
-D-glucopyranoside -D-glucopyranoside
NPGlu NPGlu
1.32
1.28
1.24
shape
1.2
1.16
1.12
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column No. in Table 6.5.2
FIGURE 6.5.6 The values of for ashape obtained using formula 6.5.10 for the columns indicated in Table 6.5.2 (mobile
phase acetonitrile:aqueous buffer 90:10, the buffer being 20 mM ammonium acetate at pH 4.7) [179].
2.5
AX
1.5
CX
0.5
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Column No. in Table 6.5.2
FIGURE 6.5.7 The values of for aAX and aCX obtained using formula 6.5.11 for the columns indicated in Table 6.5.2
(mobile phase acetonitrile:aqueous buffer 90:10, the buffer being 100 mM ammonium acetate at pH 4.7) [179]. Note: the
aAX for column No. 9 is 4.73, and aCX for column Nos. 11 and 12 are 8.21 and 29.03, respectively.
formulas for SPTS and for TMPAC are given phases. The formulas for theobromine and
in Figure 6.5.8. theophylline are given in Figure 6.5.8.
6) Another parameter used to evaluate HILIC Unlike the case of hydrophobic columns,
columns is at(theobromine/theophylline) where the parameters for column characteriza-
[184]. This parameter differentiates columns tion Hc, S*c, Ac, Bc, Cc(2.8), and Cc(7.0) were
regarding their basic, neutral, or acidic obtained using regression parameters for the
character with at < 1 for basic, at z 1 for calculation of a values, in the case of HILIC
neutral, and at > 1 for acidic stationary columns it was found more convenient to
CH3
O CH3 O
N H 3C NH
HN N
-
+ Cl O N N N
H3C N CH3 O N
O S O
CH3 CH3 CH3
ONa
Trimethylphenyl
Na p-toluenesulfonate ammonium chloride Theobromine Theophylline
SPTS TMPAC Tb Tp
FIGURE 6.5.8 Formulas for sodium p-toluenesulfonate, N,N,N-trimethylphenylammonium chloride, theobromine and
theophylline.
6.5. POLAR STATIONARY PHASES AND COLUMNS 311
simply characterize the columns based on layer of water, and a partition mechanism for
measured a parameters for specific pairs of the separation is more likely than on dry silica.
compounds. Since the HILIC mechanism is For both NPC and for HILIC applications, silica
more complex and less investigated than the of high purity (Type B purity) is commonly
mechanism for hydrophobic columns, further used. The extent of NPC utilization for analyt-
characterization of a multitude of columns is ical purposes is rather limited, and NPC is
needed for a similar approach as that used in mainly applied in other chromatographic tech-
RP-HPLC. niques, such as thin layer chromatography
HILIC column comparison can be done by (TLC) and preparative chromatography (at
different procedures based on the multitude of lower pressure). There are, however, useful
a parameters. One such procedure is the use of applications of HPLC on silica in NPC mode
a radar graph similar to the graph from (nonpolar solvents), such as separation of
Figure 6.4.14 used for hydrophobic columns very nonpolar compounds (for example, carot-
comparison. Such a graph that allows compar- enoids or lipids) that require an organic mobile
ison of four selected columns is shown in phase with no water or polar constituents,
Figure 6.5.9. separation of compounds that are extremely
polar and are not retained or are very weakly
retained on RP columns, and also separation
Bare Silica Stationary Phase
of certain isomers (achiral) since the silica in
Silica stationary phase can be used in either NPC conditions is more efficient than RP for
NPC or HILIC mode. In NPC mode, the organic such separations. As discussed in Section 5.3,
mobile phase that has no water does not imply the interactions between the stationary phase
the total absence of water from the silica surface. and the solute (analyte) in NPC and HILIC
Anhydrous silica still contains a layer of water are stronger for more polar compounds. Since
(possibly monomolecular) on its surface. In the polarity of the isomers (not chiral isomers)
HILIC mode, silica is covered with a thicker is frequently very differentdfor example,
k uridine
1
5
CX 4 (CH2)
8 2
3
1 Amide-80 3 um
Lichropher Diol 5 um
AX 0 3(H)
NH2-MS 5 um
Zic_HILIC 3.5 um
shape
6 4dia
regio
5
FIGURE 6.5.9 Radar graph for the comparison of columns using kuridine and several a parameters.
312 6. STATIONARY PHASES AND THEIR PERFORMANCE
because the polar groups are positioned in as stationary phase are diminished, but some,
different parts of the moleculedthe interac- such as peak tailing, may still be a problem.
tions with the stationary phase are different
from isomer to isomer. This explains the
efficiency of polar phases in isomer separation.
HILIC Stationary Phases with a Bonded
Besides the polar group position, other effects Surface
such as steric hindrance and additional The development of silica-based stationary
effects such as propensity to form hydrogen phases with a bonded material that has polar
bonds or to have electron donation or with- groups is the cause of a considerable expan-
drawal interactions may affect the differences sion of applications using HILIC columns.
in retention. The polarity of bonded-phase HILIC columns
Silica utilization in NPC mode has several is lower than that of bare silica. However, their
disadvantages. The main problem is related to reproducibility and rapid regeneration when
the reproducibility of such separations. Since changing solvent corrected some of the main
the silica is always covered with a layer of problems of bare silica columns. HILIC
water, the immobilized material acting as columns are widely used for the separation
stationary phase can be considered to be water. of important classes of polar organic
The variation in the amount of water retained compounds such as carbohydrates, amino
on the silica produces variations in the retention acids, and peptides [185]. Also, due to the
times. The different level of water can be caused use of solvents containing both water and an
by different levels of exposure of the stationary organic solvent, HILIC columns are frequently
phase to water from solvents with different recommended to be selected when MS or MS/
degrees of dryness or by the length of exposure MS detection is utilized, since these solvents
of the column to a dry solvent that may dissolve provide good ionization media and lead to
some of the water forming the stationary phase. good sensitivity. A variety of types of
The sensitivity of column reproducibility to the stationary phases can be used for HILIC sepa-
water exposure is very high. Also, related to the rations; some of the ones that are commercially
water layer formation is the slow equilibration available are discussed below. Most of these
of the column when changing solvents and columns are silica-based. They include mainly
frequent irreproducibility in this equilibration columns made using porous particle materials,
process. In addition, some compounds are but several are made using core-shell (fused
retained irreversibly on bare silica. Although core) support.
the structure of Type B silica is highly homoge-
neous, silica columns frequently produce tailing 1) Neutral HILIC stationary phases have the
of the peaks, indicating variability in the type of polar group amide, diol, or cyano. These
interactions on the stationary phase surface. derivatized silica phases are less polar than
Silica phases based on Type B (purity) specifi- bare silica. The columns containing cyano
cally developed for work in HILIC mode were groups have the lowest polarity (see Table
developed and are commercially available. 6.3.5 for the corresponding log Kow), and
Among these can be listed the columns indi- this type of column can also be viewed (and
cated in Table 6.5.3. When used in HILIC used) as RP column. For this reason several
mode, the layer of water on the silica surface is cyano columns were discussed in Section
much thicker than in NPC mode, and the sepa- 6.4. Some cyano columns are specifically
rations are less susceptible to mobile phase recommended for HILIC work, and several
dryness. Also, other disadvantages of bare silica examples are given in Table 6.5.4. Also
6.5. POLAR STATIONARY PHASES AND COLUMNS 313
TABLE 6.5.3 Several columns indicated as usable in columns have diol groups in their bonded
HILIC mode on underivatized silica. phase. Some diol phases may have a specific
Phase type/
structure where ether groups are also
Brand name Manufacturer pore size present in the connecting chain (handle) to
the silica surface (crosslinked diol). Polyeth-
Atlantis Waters
Silica, 100 A
ylene glycol bonded to silica is also used as
Betasil Thermo Hypersil
Silica, 100 A a polar stationary phase. The retention
ChromoLith Merck Silica monolith mechanisms for neutral HILIC stationary
phases consist of polar interactions and
Hypersil Thermo Hypersil
Silica, 120 A
hydrogen bonding between the hydroxy or
Kromasil EKA Chemicals
Silica, 100 A amide groups on the stationary media and
Spheri-5 Silica Brownlee (Alltech)
Silica, 80 A the polar groups of the analytes. HILIC
columns with amide groups have been
Promosil Silica Bonna Agela Silica
proven to be useful for sugars, amino acids,
Venusil XBP Silica Bonna Agela Silica and peptides analyses. Dihydroxypropyl
Venusil XBP-L Silica Bonna Agela Silica (diol) groups are able to form stronger
hydrogen bonds compared to the amide
Inspire Silica Dikma Silica
groups, and these columns are used when
Nucleodur Macherey-Nagel Silica stronger polar interactions are necessary for
unmodified the separation. Schematic structures of an
Nucleosil Macherey-Nagel Silica amide and a diol stationary phase are shown
unmodified in Figure 6.5.10.
COSMOSIL SL-II Nacalai Silica More complicated structures that contain
OH groups can be used as neutral HILIC
YMC-Pack SIL YMC Silica
stationary phases. Among these are phases
Zorbax Sil Agilent Silica with bonded cyclodextrin or bonded perhy-
Ascentis Express Supelco Core-shell silica droxyl-cucurbit-[6]uril groups that contain
HILIC numerous OH functionalities and can act as
HILIC stationary phases. An example of
Halo Hichrom Silica
a schematic structure of a phase containing
Gold HILIC Thermo Silica various polar groups, including a lactose
moiety, triazole, and a phenyl spacer, is
shown in Figure 6.5.11 [186].
recommended for HILIC use is a fluorinated Other materials such as those made from
stationary phase (Epic HILIC FL with silica-bonded polysuccinimide [187] or poly-
undisclosed structure), although this phase hydroxyethyl-aspatamide can be used as
can be considered a RP type. neutral phases for HILIC. Phases containing
In HILIC stationary phases, the polar a sulfur atom embedded in the chain bearing
group is typically connected to the silica on OH group, such as mercaptoethanol silica
surface by a hydrocarbon chain (such as and thioglycerol silica, have also been
propyl or longer). One common polar group reported [169]. Several neutral HILIC
in HILIC stationary phases is amide. Amide columns that are commercially available are
polarity is not very high, but it is higher than listed in Table 6.5.4. As indicated in this table,
cyano (see Table 6.3.4). An even more polar organic polymers are also used as a support
group is hydroxy, and common HILIC for phases with neutral polar groups, such
314 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.5.4 Several neutral HILIC columns that are commercially available.
YMC-Pack CN YMC
Silica, 120 A 3-Cyanopropyl
Epic HILIC FL ES Industries
Silica, 120 A Fluorinated
TSKgel Amide-80 Tosoh Bioscience
Silica, 100 A Amide
GlycoSep N ProZyme Silica Amide
XBridge amide Waters Polyetoxysilane (BEH) Amide
Unisol Amide Bonna Agela Silica Amide
HO
O OH HO OH
Si N HO
O
O N O
N O
O OH
HO
HO
FIGURE 6.5.11 Example of a schematic structure of a phase containing various polar groups and a phenyl spacer.
316 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.5.5 Several HILIC columns with weak anion exchange properties that are commercially
available.
Various other mixed-mode phases have been organic polymers with polar functionalities such
reported in the literature (see, e.g., [169]). as poly(hydroxymethacrylate) were reported in
In addition to small particle phases, monolithic the literature [189].
HILIC phases have been prepared. Besides bare
silica Chromolith (see Table 6.5.3), polymer-
coated monolithic silica was used as HILIC
Silica Hydride-based Phases
stationary phase, the coating being performed A number of silica hydride (silica Type C)
with 3-diethylamino-2-hydroxypropylmethacry- phases are commercially available (e.g., from
late, p-styrenesulfonic acid, etc. Also monolithic MicroSolv/Cogent) and can be used in HILIC
6.5. POLAR STATIONARY PHASES AND COLUMNS 317
TABLE 6.5.6 Several HILC columns with cation exchange properties that are commercially available.
TABLE 6.5.7 Several HILC columns with zwtterionic phases that are commercially available.
CH3
318 6. STATIONARY PHASES AND THEIR PERFORMANCE
mode. These columns include a bare hydride chromatography with partially aqueous mobile
column (Diamond Hydride) as well as columns phases. Application of zwitterionic ion chroma-
with a bonded phase containing polar groups tography (ZIC) as cation-exchange phases were
such as butylamino (Cogent HPS Amino). The reported for the separation of charged peptides.
types of chromatography that can be practiced Amphoteric groups can also be present in an
on bare hydride columns include ANP and HILIC. ion-exchange phase, and depending on the pH
of the mobile phase, they may act as a weak cation
or weak anion phase [190].
Based on their composition, the ion-exchange
6.6. STATIONARY PHASES AND
phases can be silica-based or based on an
COLUMNS FOR ION-EXCHANGE,
organic polymeric material (resin). Also, some
ION-MODERATED, AND LIGAND-
EXCHANGE CHROMATOGRAPHY inorganic oxides and silicates can be used as
stationary phase in ion chromatography. A sche-
matic diagram showing the types and the basic
General Aspects
composition of the stationary phases used in IC
Ion chromatography (IC) is a special type of is given in Figure 6.6.1.
HPLC used for the separation of ionic analytes. Strong ion-exchange stationary phases are
Depending on the charge of the ions, the phases completely ionized over a wide range of pH.
can be either cationic or anionic. The dissociation The degree of dissociation of weak ion
capability of the ionic groups bound to the exchangers depends on the mobile phase pH.
stationary phase leads to the classification of ion For this reason, the ion-exchange behavior of
exchangers as strong (e.g., with groups -SO 3, strong ion-exchange phases, within typical
-NR 3 ), medium (e.g., with groups -PO(OH)O-), range of pH operation, is not influenced by the
or weak (e.g., with groups -COOH or pH of the mobile phase. The selection of a strong
-NH(CH3)). Zwitterionic phases (when used in or weak ion-exchange stationary phase is deter-
totally aqueous buffers) that have both anions mined by the nature of the analytes and by the
and cations bound to the stationary phase can purpose of analysis. The use of weak ion
act either as cationic or as anionic stationary exchangers allows better modulation of the
phases, depending on the condition of the appli- retention properties depending on the mobile
cation. Such phases are also used in HILIC phase. Also, besides ionic interactions, the
FIGURE 6.6.1 Diagram showing types of ion exchange (IC) stationary phases.
6.6. ION EXCHANGE AND OTHER STATIONARY PHASES AND COLUMNS 319
weak ion-exchange phases may develop other influence the separation of the analytes by
types of interactions (e.g., polar type). undesired hydrophobic interactions. Phases
One important characteristic of the IC with low or ultra-low hydrophobicity have
columns is their ion capacity. The ion capacity been manufactured, and this property is typi-
of the ion exchanger is determined by the cally indicated for commercially available IC
number of functional groups per unit weight columns.
of the stationary phase. The most commonly The cationic resins are commonly available in
used unit is milliequivalents of charge per H or in Na form (exchangeable cation is H or
gram of dry packing or milliequivalents per Na), and the anionic resins are available in Cl
mL of wet packing. Typical ion-exchange or OH form. The exchange of the counterion
capacity in IC is 10-100 mEquiv/g. The coun- with another ion can be done by passing a (rela-
terion present in the stationary phase must be tively) concentrated solution containing the
indicated, together with the ion capacity since desired ions through the stationary phase. The
it affects the degree of swelling of the packing reconversion of an ion exchanger into its working
and hence its volume. The ion-exchange form is known as regeneration. For this purpose,
capacity of a stationary phase plays a significant different possibilities are used depending on the
role in determining the concentrations of nature of the exchanger and its acidebase prop-
competing ions used in the mobile phase for erties. For instance, strong or weak acid cation
elution. Higher capacity stationary phases exchangers are regenerated with HCl or H2SO4,
generally require the use of more concentrated and if the desirable form is Na, the regeneration
mobile phases. This is not a recommended of strong cation exchangers should be performed
feature in high-performance ion chromatog- using NaCl, while the weak cation exchangers
raphy, since common conductometric detectors are regenerated with NaOH. Anion exchangers
cannot function well with high salt concentra- are regenerated using NaOH, NaCl, NH4OH,
tions. However, the lower ion-exchange HCl, H2SO4, Na2SO4, or Na2CO3. The operation
capacity determines a low sample loading of of regeneration must be complete. Usually, the
the phase. For strong ion-exchange phases, regeneration solution has a concentration of 1N
with the ionization of the stationary phase not and is used in a volume that is more than neces-
depending on the pH of the mobile phase, the sary for stoichiometric conversion. A special
loading capacity is the same regardless the pH. precaution must be taken to avoid the formation
For weak ion exchangers, use of a pH of the of precipitates in the resin (such as CaSO4, etc.)
mobile phase where the stationary phase is not during the regeneration process.
ionized may considerably decrease the phase Besides a typical role as ion exchange, some
retention. phases can be used for the separation of neutral
Another important characteristic of the IC species between themselves and from ionic
columns is related to their hydrophobic char- species. The ionic compounds from the solution
acter, which should be as low as possible. In are rejected by the selected resin, and the neutral
many IC separations other molecules are molecules are separated through polar interac-
present besides the inorganic ions, and the tions. This type of separation is known as ion
hydrophobic character of the analyte should exclusion.
not interfere with the separation. However,
the ionic groups of the stationary phase are
Silica-based Ion Exchangers
typically bonded to an organic fragment that
is connected to either silica or an organic poly- Various groups conferring an ion-exchange
mer. This part of the ionic stationary phase may character can be bonded on silica by procedures
320 6. STATIONARY PHASES AND THEIR PERFORMANCE
similar to those used for RP, HILIC, or other 6.6.2. Various other silica-based stationary
stationary phases. The advantages of silica- phases for ion exchange chromatography were
based phases result from their large contact reported in the literature [192].
surface with the mobile phase, good mechanical
properties, and lack of swelling and shrinking Synthesis of Organic Polymeric Ion
when the mobile phase is changed. Certain
silica-derivatized phases can be used directly
Exchangers
as a cation- or anion-exchange phase. One Besides silica-based materials derivatized
such example is aminopropyl silica. Another with organic moieties containing groups such
example is a strong cation exchange phase as SO3H, COOH, and NH2, organic resins with
(SCX type) obtained by attaching to the silica specific active groups were frequently used as
surface ethylbenzene sulfonic acid ligands that ion-exchange stationary phases. These materials
lead to phase coverage with 0.4e0.7 meq/g consist of an organic polymeric (resin) backbone
capacity. However, it is more common to attach containing covalently bound groups that are able
or form ionizable groups using a second reac- to exist in ionic form. Four types of polymeric
tion step performed on a previously derivatized packings are used for making stationary phases,
silica. Reaction 6.2.10 (see Section 6.2) showed, including copolymers of styrene-divinylbenzene
for example, the formation of a SO3H group (PS-DVB), ethylvinylbenzene-divinylbenzene
from an SH group by oxidation. Similarly, (EVB-DVB), polyvinyl copolymers such as those
strong anion exchangers with N(C2H5) 3 groups obtained from polyvinyl alcohol (PVA), and
can be obtained from chloropropyl silica various polymethacrylates [193]. The presence
following the reaction 6.6.1: of the ionic groups on these resins makes these
resins act as polyelectrolytes. The main advan-
tage of resin-based ion exchangers is their toler-
O ance to eluents with extreme pH values,
Si O Si
between 0e14, in contrast to the silica-based
+ N(C2H5)3
Cl stationary phases, whose pH limits are 2e7.
O This wide range of pH values allows the use of
selectivity effects on multicharged or weakly
ionizable solutes. The use of polymeric resins
O has pressure limitations because they have lower
C2H5 mechanical resilience. Macroporous resins with
Si O Si -
+ Cl
N a high degree of crosslinking are relatively
O C2H5
H5C2 more rigid and stable, and although they have
lower ion-exchange capacity they are used in
(6.6.1)
HPLC and can be included in longer columns,
Other ionic groups can be attached to the at higher flow rates. Monolith columns are also
silica surface forming strong cation exchangers made from polymers such as polyacrylamide
(SCX), strong anion exchangers (SAX), or weak for IEC separations [194].
ones (WCX or WAX). Some information on Several procedures are used for obtaining
several common commercial silica-based polymers with ion-exchange properties. One
stationary phases used for separation of anions such procedure is the derivatization of an
(cation-exchange stationary phases) are given already polycondensed resin. This type of reac-
in Table 6.6.1 [191]. Several silica-based anion tion was previously discussed in Section 6.2.
exchange IC columns are described in Table One other procedure consists of the synthesis
6.6. ION EXCHANGE AND OTHER STATIONARY PHASES AND COLUMNS 321
TABLE 6.6.1 Some information for commercial silica-based cation exchange columns.
of the polymer already containing the desired with methylenesulfonic acid groups. Also,
ionic groups in the monomer, as shown in reac- phenols such as resorcinol, naphthol, phenoxy-
tion 6.2.22. This procedure is commonly used, acetic acid, and various aldehydes are used in
for example, for the synthesis of sulfonic resins, the condensation instead of simple phenol and
which can be obtained following the reactions formaldehyde. For example, the condensation
6.6.2 schematically shown here: of phenoxyacetic acid and formaldehyde leads
OH OH
OH OH OH
+ H 2SO4 HCHO
+ SO3H OH (6.6.2)
SO3H
mixture
SO3H
The sulfonation products of phenol can be to a weak acid resin. Other variations of the
further treated with formaldehyde without condensation reaction are utilized, such as
separation, and a highly crosslinked material condensation of a phenol, a substituted benzal-
can be obtained under appropriate conditions. dehyde, and formaldehyde. Also, condensation
Other resins can be prepared similarly to of aromatic diamines such as m-phenylenedi-
sulfonic resins. For example, starting with sali- amine with formaldehyde is used to make
cylic acid and formaldehyde, a resin with a polymer with anion-exchange properties.
carboxylic groups is obtained. Because the amino groups directly connected
The bonded ionic group also can be gener- to the aromatic ring are weak bases, further
ated on the side chain of the resin. For example, methylation can be applied to form quaternary
condensation of sodium phenolate with Na2SO3 amines that are stronger bases. This condensa-
and HCHO leads to the formation of a resin tion can be written as reaction 6.6.3:
322 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.6.2 Some information for commercial silica based anion exchange columns.
Vydac 302 IC 4.6 Separation Group strong 50 4.6 0.1 10 Spherical particles
with-NR3]
Vydac 300 IC 405 Separation Group strong 250 4.6 0.1 15
Wescan 269-001 Wescan strong 250 4.6 0.08 13
NH2
N(CH3)3+SO4H-
NH2
NH NH2
(CH3)2SO4
+ HCHO
CH2
N(CH3)2+SO4H-
NH2
CH2
NH
(6.6.3)
HC CH Anion exchangers
Cl Functional group Type
N(CH3)3
+
Quaternary -N(CH3)3] OH strong
amine
crosslinked Quaternary -N(CH3)2(CH2CH2OH)]OH medium
amine
basic than those containing dimethyl-b-hydrox- pH of precipitation varies between 6 for low-
yethylamine. The most common groups in substitution values to 1 for high substitution
commercially available ion exchangers are (D.S. of about 0.9). The material has ion-
sulfonic and carboxylic groups for cation exchange properties.
exchangers and trimethylammonium group for Ion-exchange materials also can be obtained
anion exchangers. from dextrans. Dextrans are produced by certain
Another procedure used to obtain materials bacteria growing on a sucrose substrate, and their
with ion-exchange properties starts with cellu- structure is characterized by a (1/6)-a-D-gluco-
lose. Strong alkali solutions acting on cellulose pyranosyl chain. Branches may occur at (1/3)-
(at room temperatures) produce alkali cellulose. linked points. The dextrans used for ion-exchange
Studies on the structure of alkali cellulose resins are usually crosslinked using epichlorhy-
obtained with 20e40% NaOH solutions indi- drin, followed by transformation in a cation
cated that the substance is not a true alcoholate exchange containing sulfonyl groups or an anion
but an addition complex, RCellOH:NaOH. The exchange as shown schematically in 6.6.7 for the
treatment of alkali cellulose, for example, with anion-exchange preparation:
O
O
O CH2
CH2
CH2 + HO O
HO
HO O
O (H5C2)2NH Cl- O
O
HO H2C CH CH2
ClCH2 CH CH2 OH O
OH O
O
OH O
OH
OH
CH2 (6.6.7)
H2C CH CH2Cl CH2 (C2H5)2NH
CH2 HO O
HO O
HO HO
O HO
HO
O
O
OH H2C CH CH2
H2C CH CH2
OH
OH
chloroacetic acid sodium salt, leads to the Besides the chemical structure of the ion-
formation of carboxymethylcellulose (CMC), exchange material, its physical properties are
following the reaction: also important. Many ion-exchange resins are
synthesized for different purposes rather than
RCell OH:NaOH ClCH2 COO Na to be used as a stationary phase in HPLC.
/ RCell O-CH2 COO Na NaCl H2 O The polymers are typically obtained in reac-
(6.6.6) tions involving water solutions and have the
structure of a gel with various water contents.
The degree of substitution (D.S.) that can be The elimination of this water generates a micro-
obtained for this product usually ranges porous structure of the polymer. In many
between D.S. 0.1 to D.S. 1.2. Pure CMC is applications, it is important to preserve part
commercially available. Carboxymethylcellu- of this water, and complete drying of the poly-
lose in itself is a weak acid that can be precipi- mer usually is not desired. However, the
tated from solutions with a mineral acid. The mechanical resistance of these polymers may
6.6. ION EXCHANGE AND OTHER STATIONARY PHASES AND COLUMNS 325
preclude their utilization as stationary phases have a double role: to attach the small particles
in HPLC. Besides the polymers with a micropo- to the core and to act as an ion exchanger for
rous structure, highly porous polymers are the ions in solution. The core particles are typi-
useful in some applications. Resins to be used cally PS-DVB resin of moderate crosslinking,
in solvents in which the polymer does not with a particle size in the 5e25 mm range.
swell, for example, must have a higher The outer microparticles consist of finely
porosity to allow better contact with the ground resin or monodisperse polymer (latex)
solvent. Porous polymers are, however, easily with diameters up to 0.1 mm. These particles
compressible and not always useful as have very high porosity and are functionalized
a stationary phase for HPLC. The swelling to contain an appropriate ion-exchange group.
properties of the polymer are also important This group determines the ion-exchange prop-
since on one hand the variation in volume of erties of the composite particle. For example,
the polymer is not desired, and on the other an aminated latex produces agglomerated
hand poor swelling properties do not allow anion exchangers, while sulfonated latex
a good contact of the resin with the mobile produces agglomerate cation exchangers. A
phase. Other types of physical structures for schematic view of this type of stationary phase
ion-exchange resins are also known, such as is given in Figure 6.6.2 [191].
pellicular ion-exchange resins that have an For a latex-agglomerated anion exchanger,
inert core and may be useful in HPLC applica- the core particles are typically made of polysty-
tions. Also, monolith polymeric phases can be rene-divinylbenzene with a sulfonated surface,
used for HPLC purposes [195]. which interacts electrostatically with quaternary
groups from the fully aminated latex particles.
The free quaternary amino groups that are not
Latex-Agglomerated Ion Exchangers
involved in interactions with sulfonil groups
The direct use of polymeric ion-exchange mate- from the core particle are able to participate
rials as stationary phase for HPLC encounters in the retention of anionic solutes or compo-
several problems. One problem is the relatively nents from mobile phase. This kind of anion
low mechanical stability even at moderate pres- exchanger is chemically very stable, and even
sure, and the other is the swelling and shrinkage a high concentration of NaOH is unable to
of the phase during the ion-exchange pro- disrupt the ionic bonds between substrate
cess. These problems are significantly alleviated particle and latex beads. Among other polymers
using latex-agglomerated ion-exchange station- used for latex formation are derivatized divynil-
ary phases. In addition to better mechanical benzenedvinylbenzylchloride, polystyrene, and
stability and reduced swelling and shrinkage, acrylatedbut at a lower degree of crosslinking
these phases also offer high efficiencies and an [196]. Columns with latex-agglomerated tech-
appropriate ion-exchange capacity (between 0.03 nology can be used at column backpressures as
and 0.1 mEquiv g1). high as 3000 to 4000 psi, which is close to the
Latex-agglomerated exchangers contain an use of typical silica-based columns.
internal core particle (or support) that includes Other types of latex-agglomerated stationary
ionic functionalities on its surface. On this phases can be produced. For example, Nafion
support is attached a monolayer of small (copolymer of tetrafluoroethylene with per-
diameter particles that carry functional groups fluorovinylether with terminal sulfonic groups)
consisting of bonded ions that are of the oppo- can be coated directly to an octadecyl-modified
site charge with the functionality of the silica being attached by simple hydrophobic
support. The groups of the outer particles interactions. The final material can be used as
326 6. STATIONARY PHASES AND THEIR PERFORMANCE
R
R +
+ N
N
R R R R
R
R +
- N R
SO3
Support
R
particle +
Support N R
- Latex +
particle SO3 R R N
bead
R
+
R R
N
- R R +
SO3 N R
R
R
Latex
bead
a cation exchanger, which has been proved to multiple functionalities have been manufac-
have a high selectivity. The schematic structure tured (e.g., IonPac CS5A from Dionex).
of Nafion is as follows: An important development in ion chroma-
tography is the capillary IC. A capillary system
( CF2 CF2 ) ( CF2 CF ) (e.g., ICS-5000 from Dionex) can use IonPac
n m
O capillary columns with 0.4 mm i.d. that are
CF2 packed with the same materials as the equiva-
O
F C CF2 CF2 SO3-H+ lent analytical scale version (4 mm i.d. columns).
CF3 Examples of such columns are Capillary IonPac
CS12A for cation separation, IonPac AS18-Fast
Commercial Polymeric Stationary capillary or IonSwift MAX-100 Capillary mono-
Phases Used in Ion-Exchange HPLC lithic for anion separation, and CarboPac PA20
for carbohydrate separations. These columns
A considerable number of stationary phases are used at significantly lower flow rates
for ion-exchange HPLC have been synthesized (10e20 mL/min). These capillary columns have
and are commercially available. Also, various also been developed for achieving shorter chro-
technologies have been implemented in the matographic runs.
columns used for IC (e.g., latex-agglomerated
materials extensively used by Dionex [197]).
Several materials (columns) available for
Ion-Moderated Phases
cation-exchange HPLC are listed in Table 6.6.4. Ion-moderated phases are typically made
Several anion-exchange IC columns are from a cation-exchange material in metal form
described in Table 6.6.5. Numerous other ion- (e.g., Na, K, Ca2, Pb2, and even H), which
exchange-type columns are commercially avail- is used in specific conditions for the separation of
able (see, e.g., [199]). Also, columns with neutral species based on a selective partition of
6.6. ION EXCHANGE AND OTHER STATIONARY PHASES AND COLUMNS 327
TABLE 6.6.4 Some cation exchange polymeric columns for HPLC use.
Dimensions
(length 3 Capacity
Column Manufact. Type i.d., mm)* (mEquiv gL1) Technol. Type of phase
* Note: Columns with other dimensions are also available, and the corresponding capacity (in mEquiv/g) can be different. Vendor specifications must be
reviewed (e.g. [197,198]).
the analyte between the liquid inside the resin and no higher than 1000 psi for 8% crosslinked
and the mobile phase. The retention inside the materials. Temperatures of 60e80 oC are
resin is based on weak polar interactions commonly utilized during separation.
between the analyte and the stationary phase. Besides ion-moderated polymers with ionic
Some ion-moderated phases may be considered groups or salts of ionic groups, a different type
as functioning based on a ligand-exchange mech- of phase is based on crown ether moieties. The
anism. Several columns that are commercially crown ether groups can be present in an
available for ion-moderated separations are organic-based solid support or immobilized on
listed in Table 6.6.6. Polymeric ion-moderated silica. For example, the immobilization of
columns are typically used at pressures no dibenzo-18-crown-6 ether leads to a structure
higher than 300 psi for 4% crosslinked materials, like the following:
O
O O O O
O Si O CH2 CH2 O Si O
O O O O
O
328
TABLE 6.6.5 Some anion exchange polymeric columns for HPLC use.
IonPac AS10, 11, 16, 20, etc. Dionex various PS-DVB-NR+3 and alcanol
IonPac AS18 Dionex strong Polyethylvinylbenzene-DVB-NR+3
CarboPac MA1 Dionex strong resin with tertiary amine
CarboPac SA10 Dionex strong latex nano beeds
CarboPac PA1, PA10, PA20, PA100, Dionex strong pellicular, nanoprous beeds, etc.
PA200
IC-SI-90 4E Shodex strong 250 4.6 PVA-NR+3
IC-SI-50 4E Shodex strong 250 4.6 PVA-NR+3
* Note. IonPak AS columns have different degree of hydrophobicity in addition to anionic character [197].
329
330 6. STATIONARY PHASES AND THEIR PERFORMANCE
RCU-USP Sugar Alcohols (L19 packing) Phenomenex 8 % cross-linked PS-DVB sulfonic Ca2+
IonPac ICE-AS1 Dionex PS-DVB sulfonic H+
IonPac ICE-AS1 Dionex PS-DVB sulfonic and carboxylic H+
IonPac ICE-Borate Dionex PS-DVB sulfonic H+
SUGARSH1011, SUGARSC1011, etc. Shodex
MCI CK/CA Series Mitsubishi Chem. PS-DVB sulfonic with various ions Ca2+ Ag+ etc.
6.7. STATIONARY PHASES AND functionality. The general reaction of this type
COLUMNS FOR CHIRAL is shown in 6.7.1:
CHROMATOGRAPHY
OCH3
General Comments Si OH
O + H3CO Si
Since most bioorganic molecules are chiral, the X
separation of chiral compounds is of considerable Si OH OCH3
importance, for both analytical and preparative
purposes. Chiral chromatography is therefore a
Si O
field with numerous books (see, e.g., [200e204])
and journals, including the dedicated journal O Si
X
Chirality (Wiley). Chiral stationary phases and Si O OCH3
columns are also commercially available from
a number of suppliers. This section presents only X = NH2, N=C=O, SH, N3 , etc.
a summary of this rich volume of information. (6.7.1)
The reactive group can be amino, isocyanate,
Procedures for the Synthesis of Phases
thio, or azide, and is further reacted with the
with Chiral Properties desired compound that contains a chiral selector
A number of syntheses of chiral stationary group for achieving the desired stationary
phases are reported in the literature [200]. One phase. One such procedure starts with amino-
important type of such a synthesis starts with propyl silica, which is further derivatized
a silica support on which specific chiral selectors with N-(3,5-dinitrobenzoyl)-phenylglycine,
are attached. Such phases can be obtained, for with N-(3,5-dinitrobenzoyl)-isobutylglycine, or
example, by a two-step silica derivatization with N-(3,5-dinitrobenzoyl)-a-naphtyl-a-alanine.
similar to the procedure used for the synthesis These reagents contain a chiral carbon and
of phases with embedded polar groups. A first generate phases indicated as Pirkle type [205,
step in these syntheses is the preparation of 206]). The reaction with N-(3,5-dinitroben-
a silica-based material containing a reactive zoyl)-phenylglycine is shown in 6.7.2:
O C2H5O O O
O Si OH + Si NH2 O Si O Si NH2
O C2H5O OC2H5 O O
NO2
O
O
(6.7.2)
H H
O C* NO2 O O
+ N * N
O Si O Si NH C NO2
H
OH H
O O O
NO2
332 6. STATIONARY PHASES AND THEIR PERFORMANCE
Another example of reactions used for attach- Another modified silica used as starting
ing chiral groups on a silica surface starts with material contains azidopropyl groups.
silica gel derivatized with triethoxy-isocyanato- These groups can react with an alkyne
propylsilane, followed by reaction with the group following a Huisgen cycloaddition
desired amine, as shown in the set of reactions ("click" chemistry). As an example,
6.7.3: a quinine-type chiral stationary phase was
Si OH OC2H5
Si O Si (CH2)3 NCO
O + H5C2O Si (CH2)3 NCO
O
Si - xC2H5OH
OC2H5 Si
O (6.7.3)
Si O Si (CH2)3 NCO R1* Si O Si (CH2)3 NH C R1*
O + HN N
O
Si R2 Si R2
In reaction 6.7.3, the R1* fragment contains obtained by this type of reaction [207]. The
a chiral carbon atom that imparts chiral proper- schematics of this synthesis are shown in
ties to the stationary phase. reaction 6.7.4.
O
O O CH3 Si Si
Si Si
+ H3CO Si O O +
OH OH N3 Si
O CH3 H3CO
N3
NO2 O
Si Si
HC NO2
O NH O O
Si
O CH3 (6.7.4)
O O NH
N NO2
N
O CH3 O NO2
N N
+ N
O CH3
N
chiral
selector N
molecule
6.7. STATIONARY PHASES AND COLUMNS FOR CHIRAL CHROMATOGRAPHY 333
The preparation of chiral selector molecule Polymers containing chiral selectors that are
in reaction 6.7.4 starts with quinine that is trans- bonded to silica can also be obtained by synthe-
formed into 10,11-didehydroquinine. This sizing a monomer containing two ethoxysilane
molecule is reacted with 3,5-dinitrobenzeneiso- groups, as shown in reaction 6.7.6.
cyanate to introduce one additional function- The resulting monomer can react with itself
ality necessary for chiral recognition. The or with a silica gel surface, generating a silica-
resulting molecule reacts with azidopropyl based material covered with a silica-based poly-
silica. mer with chiral properties.
A multistep synthesis is used, for example, A different type of chiral selector is offered by
to obtain a chiral stationary phase containing macrocycles. The macrocycles are rather volumi-
a polymer bonded to silica with diaminocyclo- nous, and the direct connection to the silanol
hexylacrylamide chiral selectors. This is groups is not possible. In these cases, a reactive
done by attaching N-[2-(prop-2-enamido) handle or spacer is attached first to the silica
cyclohexyl]prop-2-enamide (obtained from surface, this handle further reacting with the
acryloyl chloride and cyclohexane-1,2- desired bonded phase. For example, starting
diamine) to a pre-derivatized silica, following with amino silica a phase can be prepared having
the set of reactions shown in 6.7.5. Due to the a crown ether moiety with attached tartaric acid
active double bonds on the chiral monomer, functionalities that add asymmetry allowing the
the process leads to the formation of a poly- chiral separations. The reactions for this synthesis
meric active phase bonded to silica. are schematically shown in reaction 6.7.7 [208].
O
Si Si
Cl Cl
O O O O H3C CN
C C O
Si Si
+ Si O Si NH
C N
O +
H3C CH3 Si O Si C N
NH
O H3C CN
NC CN
N N
H2N H2N O
H2C NH
aminopropyl silica (6.7.5)
O
O O NH
CH2 O
C
NH Si O Si NH
H3C CN
+ O NH
H3C CN
NH Si O Si NH
C
HN
O
O CH2
H2C
N-[2-(prop-2-enamido)- O
cyclohexyl]prop-2-enamide
334 6. STATIONARY PHASES AND THEIR PERFORMANCE
CH2
R O O
H OC2H5
NH C catal.
C NH + H Si OC2H5
H O
O O R OC2H5
OC2H5
H2C (6.7.6)
R O O Si OC2H5
O H
NH C OC2H5
OC2H5 C NH
H
H5C2O Si O O R
OC2H5
O
O O
O
H3C O O
O
2 Si NH2 + O O
O Si O
O H3C O
O
O O O
O
HOOC O O
H3C COOH
O * * CH3
O
Si NH * NH Si
O Si O * O Si O
O
O H3C O CH3 O
O O O
(6.7.7)
Besides tartaric acid, which has asymmetric necessary asymmetry. One synthesis with the
carbons (indicated in reaction 6.7.7 as *), other goal of preparing a crown ether including
functionalities such as binaphthyl, biphe- a binaphthyl group and capable of being attached
nanthryl, and carbohydrate, can be attached to to the active silanols of silica surface follows the
the crown ether group for providing the steps schematically shown in reactions 6.7.8 [209].
6.7. STATIONARY PHASES AND COLUMNS FOR CHIRAL CHROMATOGRAPHY 335
CH3 O O
OH OH
O O
O
S O O
O O
+ KOH
+
(6.7.8)
O O O 4
O O
S
O
O O
O O
H3C O
CH3
several reactions H C O Si O O
including with 5 2 O bonding to
O
dimethylethoxysilane silicagel
H3C
O
H5C2O Si O O O
CH3 O
The bonded phase of the type shown in reaction cyclodextrins and calixarenes. For example,
6.7.8 has chiral selectivity to numerous a chiral phase containing cyclodextrin active
compounds, allowing the separation of their phase can be obtained using the following
enantiomers. set of reactions, where glycidyl groups are
Among other macrocycles that are bound to present in a pre-derivatized silica as shown
silica to be used for chiral separations are in 6.7.9:
336 6. STATIONARY PHASES AND THEIR PERFORMANCE
O
O CH CH2
Si OH OCH3 CH CH2
Si O Si (CH2)3 O CH2
O + H3CO Si (CH2)3 O CH2 cyclodextrin
O
Si - CH3OH
OCH3 Si OH O
O CH CH2
(6.7.9)
CH CH2
Si O Si (CH2)3 O CH2
Si O Si (CH2)3 O CH2 + cyclodextrin
O
O NaO Si
Si
The same procedure of a reaction with gly- a stationary phase schematically shown in
cidyl groups present in a pre-derivatized silica Figure 6.7.1. The groups attached to the calyx-
can be used to attach a preformed polymer [4]arene macrocycles may provide enough
such as derivatized cellulose. This procedure asymmetry to use such molecules for chiral sepa-
makes a bonded polymer coating on silica rations. However, this type of phase can also be
surface and is used, for example, to prepare used for nonchiral separations.
chiral phases with cellulose derivatized with
3,5-dimethylphenylcarbamate. The derivatiza-
tion of cellulose with 3,5-dimethylphenylcarba- Main Types of Stationary Phases Used
mate adds a moiety able to provide p-bond in Chiral HPLC
interactions. Section 5.5 described several types of interac-
The bonding of calixarenes to silica [210] starts tions that allow the separation of the enantiomers
with the attachment on the silica surface of of chiral molecules. Based on these different inter-
a handle having glycidyl groups, for example, action types, a considerable number of chiral
by treating active silica with g-glicidoxypropyl- stationary phases were developed. The main
trimethoxysilane. The presynthesized calyxar- types of chiral stationary phases can be classified
ene can be attached to these groups, leading to in specific groups, as follows [211].
CH3
O
Si O
O CH3
OH R
O R O
CH3
O
O Si O
CH3 OH
NO2
O
O
NH NO2
O Si NH NO2
H
O CH2 O O
O
NH
H3C CH3 O Si NH NO2
H
L-Leucine O O
O
(CH2)11 O CH NH O
O Si NO2 NO2
O H
O O O NH
O H
(S,S)-Gem 1 O Si N
O H
Pirkle 1-J
H3CO
O NO2
P O
H3CO
O CH NO2
Si NH
H3C CH3 O
CH3 CH3 O2N
HN
(R)--Burke 2 NO2
H
O
Si O N
H
CH3 CH3 O NO2
X O
NO2 DACH-DBN
X = H, dinitrobenzene
O NO2
H
(CH2)10 NH
O Si NH NO2
H3C CH3 H
O
ULMO
FIGURE 6.7.2 Commercially available chiral phases containing groups capable of donor and acceptor hydrogen bonding
and p-acceptor type bonds.
6.7. STATIONARY PHASES AND COLUMNS FOR CHIRAL CHROMATOGRAPHY 339
CH3
O O
O CH
N NH
O Si NH
O
CH3
FIGURE 6.7.3 Commercially available chiral phases containing groups capable of donor and acceptor hydrogen bondin,
and p-donor type bonds.
O NH CH3
O
H
NH CH3
H O
O NO2 O
Si n
O
CH3 CH3
O O O
CH3 CH3 O
NO2 NH HN
(S,S)-Whelk-O 1
H3C CH3
FIGURE 6.7.4 Commercially available chiral phase
containing groups capable of donor and acceptor hydrogen FIGURE 6.7.5 Schematic structure of tris(3,5-dimethyl-
bonding and both p-donor and p-acceptor type bonds. phenylcarbamate)-cellulose.
or tris(3,5-dimethylphenylcarbamate)-
possibility to anchor cellulose is through gly-
cellulose. The schematic structure of
cidyl groups already attached to the silica
tris(3,5-dimethylphenylcarbamate)-cellulose
surface (see Section 6.2). Coating of macropo-
is shown in Figure 6.7.5.
rous polymers such as poly(2-aminoethyl
The derivatized cellulose material is typi- methacrylate-co-ethylenedimethacrylate) with
cally coated on a silica support. The coating of derivatized cellulose has also been reported in
silica with these materials can be achieved the literature [217]. Several commercially avail-
either by simple adsorption on silica surface able chiral columns based on cellulose deriva-
using a proper solvent or by simultaneous tives are listed in Table 6.7.1. Utilization of
forming of ether-bridges between cellulose cellulose-based columns requires a normal
and silica layer [216]. The process must mobile phase, such as hexane-ethanol and
preserve the porous structure of silica. Another hexane-isopropyl alcohol. The use of these
340 6. STATIONARY PHASES AND THEIR PERFORMANCE
TABLE 6.7.1 Chiral columns based on derivatized cleavage of starch polymeric chain and
cellulose. formation of cyclic oligomers. The separation
Name Structure Applications
of a, b, and g oligomeric products is based
mainly on their different solubility in water,
Chiralcel OA -OCH3 small aliphatic although other separation procedures can be
compounds utilized. Larger cyclodextrins are also
Chiralcel CA-1 -OCH3 alcohols known. Cyclodextrins offer a series of
Chiralcel OB -OPh small aliphatic and
advantages to be used as chiral stationary
aromatic compounds phases. They can be anchored to silica (e.g.,
through reactions with glycidyl groups
Chiralcel OC -ONH-Ph cyclopentenones
bonded to silica, as shown in Section 6.2),
Chiralcel OK -OCCPh aromatic compounds they offer a large number of chiral centers
Chiralcel OD -ONH-3,5di- alkaloids, tropines, (30 for a, 35 for b, and 40 for g-cyclodextrin),
MePh amines, beta blockers and they also provide the possibility to be
Chiralcel OF -ONH-para- beta lactams,
derivatized at their free OH groups with less
ClPh dihydroxypyridines, polar substituents. As discussed in Section
alkaloids 5.5, the separation on cyclodextrin type of
Chiralcel OG -ONH-para- beta lactams, alkaloids
phases is based on inclusion properties
MePh provided by their conical cavity of one part
of the chiral molecule, and differences in the
Chiralcel OJ - O-para-MePh aryl methyl esters, aryl
methoxy esters
interaction with the chiral OH or OR groups
of cyclodextrin with the enantiomer
Cellulose DMP -ONH-3,5-di- beta blockers remaining substituents. The difference in
(Astec) MePh
the size of enantiomers is important for the
Lux Cellulose-1 -ONH-3,5-di- choice of a, b, or g cyclodextrin, and the
MePh molecular fit determines the range of
Lux Cellulose-2 -ONH-3-Cl-4- analytes that can be separated. For the
Me-Ph inclusion, a-cyclodextrins will host single
Note: Chiralcel (Daicel Chem. Ind.)
phenyl groups or napthyl end-groups;
b-cyclodextrins will accept naphthyl groups
and heavily substituted phenyl groups; and
columns with a mobile phase containing some
g-cyclodextrins are useful for bulky steroid-
water can also be done, but addition of a salt
type molecules. Other interactions such as
in the mobile phase, such as a perchlorate, is
those involving the polar regions of an
necessary to prevent column degradation by
analyte and the surface hydroxyls of the
the dissolution of the stationary active coating.
stationary phase, and potential hydrophobic
3) Cyclodextrins and chemically modified interactions in the cavity, provide the other
cyclodextrins represent another type of two or more points of interaction required
chiral stationary phase [218]. Cyclodextrins for chiral recognition.
are produced by the action of enzymes
cyclodextrin glycosyltransferase and amylase Besides unmodified cyclodextrins, different
on starch. The main types of cyclodextrins modified cyclodextrins have been developed,
produced are a, b, and g (with, respectively, and they expand the nature of compounds
six, seven, and eight glucose residues), and that can be separated. The derivatives are
they are generated by the enzymatic formed by bonding various groups onto the
6.7. STATIONARY PHASES AND COLUMNS FOR CHIRAL CHROMATOGRAPHY 341
surface hydroxyls of the cyclodextrin cavity. both cellulose and cyclodextrins, it also offers
The derivatives include acetyl (analogous the capability of hydrogen bonding and some
to acetylated cellulose), (S)-hydroxypropyl hydrophobic interactions. Amylose can also be
ether, (S) or (R)-naphthylethylcarbamate (analo- derivatized to generate chiral stationary phases
gous to naphthyl Pirkle type columns), with tris(3,5-dimethylphenyl-carbamate), with
3,5-dimethyl-phenylcarbamate (analogous to tris[(S)-a-methylbenzyl carbamate], and with
Chiralcell OD type column), and p-toluoylester. tris(chloro-2-methylphanylcarbamate) (e.g.,
These modified cyclodextrin phases have Lux Amylose-2 from Phenomenex).
several advantages. They offer different polari-
ties from simple cyclodextrins, and they are 4) Crown ether-based chiral stationary phases
more stable to different mobile phases, allowing are also used for enantiomer separations
the use of a wider range of solvents. Cyclodex- [219]. In order to provide asymmetry to the
trins are used mainly in normal-phase-type crown macrocyclic group consisting of
separations, with no or little water/buffers. It a polyether with the formula [e(CH2)n-O-]m
is possible to operate cyclodextrin columns in (n 2 or 3 and m 5 e 10), groups such as
an alternative polar organic mode, with the binaphthyl, biphenanthryl, tartaric acid, and
mobile phase consisting of acetonitrile with up carbohydrate are incorporated (see Section
to 10% methanol plus up to 0.5% acetic acid 6.2). Among the commercially available
and/or 0.5% triethylamine, and even in crown ethers used for chiral separations are
reversed-phase conditions using acetonitrile/ Daicel Chem. Ind. (Crownpack CR 2 types),
water mobile phase. Various suppliers offer Regis Technol. (Chirosil RCA() and SCA(-)),
chiral chromatographic columns based on and Phenomenex (Sumichiral O-8000).
derivatized cyclodextrins and resulting from 5) Another type of chiral stationary phase is
a, b, or g cyclodextrins. Among these suppliers based on macrocyclic-type antibiotics
are Advanced Separation Technologies (Astec) immobilized on silica [220, 221]. The
(Cyclobond 12 types), Diacel Chem. Ind. (Chir- macrocyclic antibiotics contain numerous
alpack AD derivatized with tris(3,5-dimethyl- chiral centers, functionalities that allow
phenylcarbamate) and Chiralpack AS), bonding to silica (e.g., using prederivatized
Machery-Nagel (4 types), Merck (2 types), Phe- silica with reactive groups) and capability to
nomenex (cyclodextrin with carboxymethyl offer p-p interactions, hydrogen bonding,
functionalities bonded to methacrylate poly- inclusion/complexation, and ionic
mer), Showa Denko (4 types), Serva (3 types), interactions. Several glycopeptide type
Thermo Hypersil (2 types), and YMC (3 types). antibiotics have been used for making
Besides cyclodextrins or modified cyclodextrins stationary phases, including: Rifamycin(s),
bonded to silica (typically with the help of Vancomycin (18 chiral centers), Avoparcin,
a spacer), derivatized cyclodextrin (e.g., carbox- Ristocetin, glycopeptide A-40,926 (MDL
ymethyl cyclodextrin) can also be bonded to 62,476), and Teicoplanin (23 chiral centers).
organic polymers. Macrocyclic antibiotics used as a bonded
To the class with chiral stationary phases stationary phase also include a family of
similar with cyclodextrins can be added that thiopeptides, with the parent compound
based on amylose and that based on cyclofruc- being thiostrepton (17 chiral centers).
tans. Although amylose is a linear polymer, it The antibiotics that are glycopeptides
can assume a helical form and can form inclu- have dissociable groups (eOH of phenolic
sion compounds, such as with iodine, fatty type; eNH2; eCOOH, and eOH from
acids, and aromatic compounds. Similar to carbohydrate). Some of the groups are able to
342 6. STATIONARY PHASES AND THEIR PERFORMANCE
exist in zwitterionic form and likely play selectivity. Several types of proteins have
a major role in the association with analytes been used so far for separation of
during the chiral recognition. The chemical enantiomers. One common protein used as
formula of vancomycin where the functional chiral selector after immobilization on silica
groups as well as three potential inclusion is bovine serum albumin (BSA) with
regions are seen (A, B, and C) and the Resolvosil BSA 7 and BSA-7PX (from
formula of thiostrepton are given in Machery-Nagel), Ultron ES-BSA
Figure 6.7.6. The connection to silica can be (from Shinwa Chem. Ind.), and Chiral BSA
made using the amino groups of the chiral (from Shandon). Other proteins include
antibiotic. The columns made with human serum albumin (HSA) with Chiral-
macrocyclic antibiotics, such as the one made HSA (from Shandon and from Regis), a1-
with Vancomycin, can be used in normal acid glycoprotein with Chiral AGP (from
phase as well as in reversed-phase mode. For Regis), glycoproteins with Chirobiotic V, V2,
normal-phase use, the typical solvents are T, T2, TAG and R (from Astec), ovomucoid
hexane-ethanol mixtures, and for reversed- with TSKgel Enantio-OVM (from Tosoh),
phase use, tetrahydrofuran-water mixtures. ovoglycoprotein with Ultron ES-OVM (from
One company offering macrocyclic Shinwa Chem. Ind.), avidin with Bioptic
glycopeptide phases is Advanced Separation AV-1 (GL Science), cellobiohydrolase (Chiral
Technologies (Astec) (4 types of columns). CHB from Regis), and others (e.g. from
6) Proteins bonded to silica were also used as ChromTech Ltd.). Some of these phases can
stationary phases for chiral separations. be used in either normal-phase or reversed-
Proteins contain a large number of chiral phase mode with solvents such as
centers and are known to interact with isopropanol, ethanol, or acetonitrile in
enantiomers to produce acceptable chiral mixture with aqueous buffers.
HO OH
OH
O O CH2 O
COOH
CH3 NH
A * NH NH2
CH3 HN O NH2 O NH S
NH NH NH N O CH2
H3C * * NH * * * * O
NH
C O O CH2 O
O O B N CH3
HO * * OH O
* NH NH
* * NH *
S
O O NH N *
O N CH3CH3 O O CH3 OH *
CH3
Cl O O Cl CH3
* * * N * NH
* * * NH S
HO O *
HO * * HN
*
HO O HN O O
* H3C
OH N
O N *
* H3C OH
H3C NH *
* S S
* * CH3
H2N O HC * CH3
3 *
OH
HO
OH
FIGURE 6.7.6 Formula of vancomycin with three potential inclusion areas (A, B, and C) and that of thiostrepton.
6.7. STATIONARY PHASES AND COLUMNS FOR CHIRAL CHROMATOGRAPHY 343
O in Figure 6.7.7, with (S)-proline attached to
_ a polystyrene type polymeric structure.
N
O
Cu
2+ The mobile phase for this type of separation
O _
H2N must contain a constant concentration of metal
O ions (e.g., Cu2). This type of polymer was first
obtained by bonding amino acids such as
FIGURE 6.7.7 L-Proline bonded into a polystyrene type proline into polystyrene-divinylbenzene matrix,
polymer and forming a ternary complex with Cu2+ and with
but other polymeric matrixes, such as cross-
L-leucine.
linked polyacrylamide and poly(glycidylmetha-
crylate), have been useful for immobilizing
Besides bonding directly to silica, proteins chiral amino acid selectors. The bonded amino
were also bonded to organic polymers such as acid can be (S)-phenylalanine or (S)-proline as
acrylates and further coated on silica or silica typical chiral selectors, since they are able to
functionalized with amino groups that provide form complexes with Cu2. Separation of
a stronger bonding [222]. Another possibility a-amino acids as analytes has been performed
for bonding proteins to silica-based supports successfully on such columns [224].
is to functionalize the silica surface to become Another possibility of binding the chiral
an anion exchanger (e.g., quaternized polyvi- selector based on ligand exchange is to use silica
nylimidazole coated silica) and then to bond as support. An example is the linking (2S,4R)-
electrostatically the proteins to its surface. hydroxyproline to the silica matrix via a short
[223]. spacer as shown in Figure 6.7.8 [225].
7) Ligand-exchange chromatographic columns 8) Chiral synthetic polymers are another type of
contain chiral molecular units bonded on an material used as stationary phase for
organic polymer or on silica, the chiral units enantiomer separation. Some such phases
being capable of forming complexes with can have the polymer bonded to silica in
transitional metal cations such as Cu2, order to assure a high contact surface and
Ni2, or Zn2. When the metal ions can also mechanical rigidity. The polymer must
participate in a ternary complex with an contain chiral moieties to act as chiral
analyte from the mobile phase, the selectors. One example is a polymer having
enantioseparation results from the diaminocyclohexyl-acrylamide chiral
difference between the stability constants of selectors with polymeric structure bonded to
the ternary complexes formed by the two silica (P-CAP from Astec). The synthesis of
enantiomers present in solution. An this polymer was schematically shown in
example of such a stationary phase is given reaction 6.7.5. A similar phase, described as
O O COOH
O Si O Si O N
O O OH
OH
FIGURE 6.7.8 (2S,4R)-Hydroxyproline bonded to silica matrix following a reaction with silica derivatized with glycidyl
groups.
344 6. STATIONARY PHASES AND THEIR PERFORMANCE
distribution) can be used only within a narrower increase the column backpressure. In SEC, the
range of molecular weights for the analytes. columns very frequently have a limited value
For the separation of mixtures of molecules for the backpressure in order to avoid collapse
within a wide range of MW values (hydrody- of the pores that damage the column. For this
namic volume), more than one SEC column in reason, when columns with small particles are
series can be used. In such cases, the column used, a decrease in the flow rate of the mobile
with the highest porosity is used first. For phase is typically recommended (below 1 mL/
example, Phenogel columns that cover a wide min, e.g., 0.2 mL/min). The increase in the
range of porosities can be used up to four column length, which also enhances resolution,
columns in series for achieving some separa- is associated with an increase in column
tions. Since high backpressure can damage gel backpressure.
porosities, the maximum acceptable backpres- Two other characteristics are inertness and,
sure of the column should never be exceeded. a related one, recovery. The inertness of the
A reduced flow rate of the mobile phase is SEC column is important in particular when
sometimes necessary for maintaining the correct the MW of polymers are estimated using SEC.
backpressure, in particular when more than one When the column is not inert, other interactions
column in series is utilized for the separation. besides size exclusion will influence the reten-
Particle-size diameter of the columns is also tion process (see rel. 3.4.9 where an enthalpic
important for resolution of the separation. For component affects separation), and the result
high column efficiency, packing materials in for the MW evaluation may be incorrect. For
SEC should be fine, uniform, and spherical preparative reasons, the recovery of the analytes
particles (3e20 mm), as in the common interac- is another important characteristic of the SEC
tive HPLC. However, smaller particles (e.g., columns. Proteins or other biologically active
3e5 mm), which lead to better resolution, also materials are often separated (e.g., for
346 6. STATIONARY PHASES AND THEIR PERFORMANCE
purification) on SEC columns. It is important packing material for SEC columns [232]. The
that the material injected in the column be surface of the porous glass can be derivatized
recovered and that its biological activity not be with diol groups, similar to that of porous
altered. When the biological activity of the ana- silica [233].
lyte must be preserved, the nature of the mobile
phase also plays an important role. For such
Polymeric-based SEC Phases
GFC separations, the mobile phase is either
water or a dilute buffer. Polymeric-based SEC phases are more
common than silica-based ones. Some phases
are designed for GPC, a very common material
Silica-based SEC Phases and Glass
used for this purpose being polystyrene-
Phases divinylbenzene (PS-DVB). This polymer is typi-
Silica-based stationary phases are common for cally prepared by suspension polymerization
size-exclusion separations, their advantages (see Section 6.1), and depending on the propor-
being mainly related to the good mechanical tion of cross-linking reagent (DVB), either soft
properties and the lack of swelling, such that their gels are obtained (2-12% DVB) or rigid polymers
pore size is independent of the nature of mobile are generated (more than 20% DVB) [234]. Other
phase. This typically is associated with good phases are used for GFC (aqueous SEC). Exam-
column efficiency. However, polymer-based ples include several soft gels based on dextran
phases are more frequently utilized than silica- or agarose, more rigid polymers such as hydrox-
based ones for SEC. This is due to the larger range ylated poly(methyl methacrylate) (HPMMA),
of pore sizes offered by the polymeric materials and polyvinylalcohol (PVA) copolymers.
(between 60 A and 4000 A ) and the progress The porosity of the polymeric materials is
obtained regarding their good mechanical controlled by the conditions in which the poly-
strength. Silica-based phases have been used in merization is carried out. When 20% or more
gel permeation-GPC (with organic-type mobile of a nonpolymerizable compound is present,
phase) but in particular in gel filtration-GFC this compound dissolving the monomer but
(with aqueous-type mobile phase) modes. not the polymer, a product with a macroporous
Silica-based phases include bare silica as structure is obtained. This macroporous struc-
well as derivatized silicas, similar to the ture remains in the dry state of material.
construction of other bonded phases on silica. Porosity of the stationary phase is critical for
Bonded-phase silica-based materials are made the separation of compounds within a specific
with groups such as diol, diol on Zr-clad silica, range of MW. However, the pore size of the
glycol ether, polyether, amide, and frequently polymer is difficult to measure when the poly-
with a propyl anchor group to the silica mate- mer is swollen, and it is usually assessed with
rial. The purity of the silica material used for calibrants of known MW. Examples of calibrants
making the packing is very important in order are sets of polystyrenes with different known
to avoid other types of interactions with the MW for GPC separations and sets of polyeth-
analytes. The silica-based phases should be ylene oxide, dextrans, and proteins with
operated within the recommended range of different known MW. Polymeric packings with
pH, which is typically from 2.5 to 7.5. The a narrow range of pore size have a separation
silica-based phases are not stable outside this capacity concentrated in a limited molecular
range. Several commercially available silica- weight range. Although the resolution of such
based packings are listed in Table 6.8.2. columns is high, their use for SEC analyses is
Besides silica, porous glass has been used as possible only for a narrow molecular weight
6.8. STATIONARY PHASES AND COLUMNS FOR SIZE-EXCLUSION CHROMATOGRAPHY 347
TABLE 6.8.2 Silica based SEC packings (for aqueous SEC or GFC)
Chemistry
Phase name (Supplier) Pore size designation Range of use Particle size on silica
Phase name (Supplier) Pore size designation Range of use* Particle size
Phase name (Supplier) Pore size designation Range of use, Da Particle size Chemistry
PL aquagel-OH (Polymer AOH 30, 40, 50, 60 100,000, 1,000,000, 5 mm, 8 mm, polyhydroxyl
Lab. Ltd./ Agilent 20,000,000 15 mm surface
PL aquagel-OH mixed bed AOH Mixed H, Mixed M 20,000,000, up to 8 mm, polymer
600,000 respectively
Shodex OHpak (Showa various indicated as various from 4,000 to various HPMMA
Denko), Protein KW KB-802, to KB-806, 20,000,000
KB-80M, SB-401, etc.
TSK-GEL PW, PWXL, various G1000 to G6000, various from 1,000 to various HPMMA
PWXL-CP (Toyo Soda) GM, PWXL G5000PW, 8,000,000
GMPW, etc.
TSK-GEL HHR and HXL G1000H to G7000H, various from 1,500 to 7 mm HPMMA
(Aldrich) GMH-H, GMH-L, GMH-M 1,000,000
Toyopearl HW 40S, 40F, 40C, 50S, 50F, 55S, 100 to 50,000,000 20e40, 30e60,
55F, 65S, 65F, 75F (five pore 50e100
sizes)
TSK-GEL Alpha Alpha-3000, Alpha-5000, various up to 10,000,000 various HPMMA
and Super AW, Super AW2500 to Super
AW6000
of the synthesized particles is still necessary for 6.9. STATIONARY PHASES AND
obtaining particles with a narrow size distribu- COLUMNS IN IMMUNOAFFINITY
tion. This is achieved by using sedimentation or CHROMATOGRAPHY
centrifugation [236]. Information regarding the
particle shape and size can be obtained by micro- General Aspects
scopic methods. Particle diameters in the range
3e70 mm are commercially available. Smaller Separation in immunoaffinity can be done as
particles offer improved resolution but result in an HPLC technique [240, 241] and also at low
higher operating pressures and can prove more pressure [242]. A large number of stationary
difficult to pack. phases, some of which are custom made for
Mechanical stability of the polymer is impor- this technique, and only a few examples are pre-
tant for establishing the limit of flow-rate opera- sented in this section (for details regarding
tion. The maximum operating pressure of the affinity chromatography phases, see, e.g., [243]).
packing should be below the compression point
of the packing material. Since mobile phase Synthesis of Stationary Phases Used
viscosity is commonly increased in both GPC in Affinity and Immunoaffinity
and GFC, particular attention must be given to
Chromatography
this parameter.
The surface chemistry of the packing material The two typical components of the stationary
is very important and must be selected so that phases in immunoaffinity are the support and
any enthalpic contribution (interactions with the active phase. Support for immunoaffinity
the polymer surface) is minimal. For GFC, the chromatography should be chemically and phys-
packing materials are typically highly hydro- ically stable, with a very good mechanical
philic and should not possess charges. These strength; should allow high flow rates of samples;
effects are difficult to completely eliminate, and should have minimal nonspecific adsorption
and salts (buffer) as well as organic modifiers capability. The immobilization of ligands of
are typically added to the mobile phase to interest should be such that high ligand accessi-
diminish such interactions. bility is achieved, while the phase properties are
The chemical and temperature stability of the not affected. For LC applications, particle-size
packing is related to its inertness to specific and pore-size distribution of the support are
solvents, and typically the crosslinked polymers very important parameters. A wide variety of
are capable of being used with a wide range of materials, including organic and inorganic poly-
solvents. Temperature stability of the packing mers, have been used for the design of these solid
is also necessary since some polymeric analytes phase supports. One of the first materials used as
are soluble in the mobile phase only at elevated support in immunoaffinity chromatography
temperature. Packing materials capable of being were natural polysaccharides such as agarose
used at temperatures in the 50e120 oC range are (the neutral gelling fraction of the complex
common. Several phases used in GPC are listed natural polysaccharide agar), cellulose, and cross-
in Table 6.8.3, and some used in GFC are listed linked dextran (sepharose, sephacryl, etc.). These
in Table 6.8.4. A variety of column formats are materials are stable over a wide interval of pH
available for specific applications [237]. Partic- (3e13) and are characterized by a high content
ular advantages and limitations as well as of hydroxyl groups available for activation and
typical applications for each type of column derivatization. Their hydrophilic surface gener-
are reported by the suppliers (see, e.g., [144, ally does not interact with proteins and exhibits
238, 239]). only a low nonspecific adsorption. A major
6.9. STATIONARY PHASES AND COLUMNS IN IMMUNOAFFINITY CHROMATOGRAPHY 351
drawback of these materials is their poor mechan- tunately, silica is unstable at mild alkaline pH
ical strength related to their swelling ability. Other values and dissolves significantly above pH of
supports for the stationary phase may include 8. In addition, nonspecific interactions can occur
silica as well as organic polymers such as methac- between silanol groups and the basic parts of
rylates, polyacrylamide, and copolymers of poly- the biomolecules. However, surface modification
acrylamide with other polyvinyl polymer. can be performed either by chemical modification
Although more resistant to pressure than or physical adsorption of ligands in order to mini-
polysaccharides, in comparison to inorganic mize this nonspecific adsorption and to introduce
materials, these organic supports exhibit a lower a high density of functional groups [246].
pressure tolerance. In addition, some of these The active phase in immunoaffinity can be
materials show swelling differences in the pres- very diverse, depending on the nature of the
ence of organic solvents, a broader pore-size material immobilized on the stationary phase.
distribution, decreased ligand-binding efficiency, The development of affinity chromatography
and nonspecific interaction due to their hydro- depends on the construction of new stationary
phobic character. Monolithic supports based on phases and affinity ligands, as well as new
glycidyl methacrylate-co-ethylene dimethacry- approaches to immobilization chemistry [247].
late (GMA-EDMA) have been shown to be useful Stationary phases containing an immobilized
in modern affinity chromatography. Such enzyme or other compounds such as heparin
columns are commercially available from BIA [248], lectins, and nucleotides were successfully
Separations (Slovenia) under the trade name of utilized in various chromatographic procedures,
CIM disk monolithic columns. The main advan- mainly low-pressure liquid chromatography,
tage of the GMA-EDMA support is the significant but also in HPLC. For silica-based stationary
concentration of chemically reactive epoxy phases, a common material utilized as a starting
groups, which can be used for immobilization of material is silica pretreated with an aminosilane
ligands, for example, containing amino groups. (e.g., g-aminopropyltriethoxysilane, g-aminopro-
[244]. The stationary phase can also be porous, pylmethyldiethoxysilane, g-aminopropyldime-
nonporous [245], or monolithic [241]. thylethoxysilane). The reaction of the
Silica is one of the most widely used inorganic propylamino silica surface with glutaraladehyde
supports for polymers in immunoaffinity chro- generates a reactive material on which an amino
matography. This support is very stable under group of a protein (e.g., with lysine units in its
pressure and can be easily derivatized in order structure) can be readily attached. This is sche-
to introduce different functional groups. Unfor- matically shown in reaction 6.9.1:
CH3 CH3
NH2 NH2
Si OH + X Si Si O Si
CH3 - HX CH3
(6.9.1)
Protein
+ Protein
OHC (CH2)3 CHO NH2
+ Sil N CH (CH2)3 CHO Sil N CH (CH2)3 CH N
Sil NH2
352 6. STATIONARY PHASES AND THEIR PERFORMANCE
Other similar procedures use succinic anhy- For an organic support, the linking process is
dride instead of glutaraldehyde as an interme- usually done using an activating reagent such as
diate step of derivatization. In this case the cyanogen bromide when the molecule to bind
reactions follow the path shown in 6.9.2: contains a free primary amine, sulfhydryl, or
hydroxyl groups for attachment. On the activated
support, proteins (or other molecules) with
specific binding capability are further immobi-
protein lized. These can be selected by the user or can be
O O O OH + general-purpose immobilized compounds.
O NH2
+ Sil NH C (CH2)2 C Immobilized heparin, for example, acts with
Sil NH2 O a specific binding site to retain certain proteins,
O lectin resins can be used for the separation of
NH protein
glycoproteins from other glycoconjugate mole-
Sil NH C (CH2)2 C
cules, and nucleotide resins are used for the sepa-
O
ration of specific proteins. Heparin resins, lectin
(6.9.2) resins, and others are commercially available.
Specific immunoproteins also can be bound, for
example, on agarose activated with cyanogen
bromide or with other activation reagents such
Some proteins that contain phenolic groups
as 6-aminohexanoic acid, carbonyldiimidazole,
(e.g., with tyrosine units) can be bound to the
and thiol [249]. These types of materials have
aminosilica using azo-coupling, as shown sche-
a very high specificity for the specific antigen
matically in reaction 6.9.3:
that generates the immunoprotein. Affinity resins
containing immobilized sugars and sugar deriva-
tives and resins with immobilized biotin or avidin
are also available. Several activation reagents for
O
agarose and crosslinked dextrans are shown in
Sil NH2 + C NO2
- HCl Table 6.9.1. Other resins are used having immobi-
Cl
lized ligands such as p-amino benzamidine
NO2 NH2
(ABA-5PW), m-aminophenyl boronic acid
[H] + NaNO 2 (Boronate-5PW), and iminodiacetic acid (CHelate
LiAlH4 5PW), all from Sigma. Numerous other immobili-
+ HCl
zation procedures are reported in the literature
C C
HN O HN O [249e251]. The affinity and immunoaffinity-type
Sil Sil phases have excellent selectivity and work
protein
(6.9.3) well in aqueous solutions, but each material
+ must be developed for a specific analyte; the
H2C OH
Sil NH phases can be unstable toward organic solvents
C N2+ Cl -
and may be stable only in a narrow pH range.
O
protein
Some bioaffinity phases are used for HPLC sepa-
CH2
rations [252e254], while others must be used at
Sil NH
lower pressures (1e2 bar) since some materials
C N=N impose considerable limitations regarding the
O maximum pressure that can be applied to the
HO phase.
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