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SPECTROSCOPIC & PHYSICOCHEMICAL METHODS IN CHEMICALS &

MATERIALS STRUCTURE & PROPERTIES DETERMINATION

Liquid Chromatography/Mass Spectrometry


LC/MS
Chromatography

Chromatography it is a broad range of physical methods used to separate and or to analyze


complex mixtures. The components to be separated are distributed between two phases: a
stationary phase bed and a mobile phase which percolates through the stationary bed.
A mixture of various components enters a chromatography process, and the different components
are flushed through the system at different rates. These differential rates of migration as the
mixture moves over adsorptive materials provide separation. The smaller the affinity a molecule
has for the stationary phase, the shorter the time spent in a column.
In any chemical or bioprocessing industry, the need to separate and purify a product from a
complex mixture is a necessary and important step in the production line. Today, there exists a
wide market of methods in which industries can accomplish these goals. Chromatography is a
very special separation process for a multitude of reasons!

First of all, it can separate complex mixtures with great precision. Even very similar components,
such as proteins that may only vary by a single amino acid, can be separated with
chromatography. In fact, chromatography can purify basically any soluble or volatile substance if
the right adsorbent material, carrier fluid, and operating conditions are employed.

Second, chromatography can be used to separate delicate products since the conditions under
which it is performed are not typically severe.

It was the Russian botanist Mikhail Tsvet (Mikhail Semyonovich Tsvet) who invented the first
chromatography technique in 1901 during his research on chlorophyll. He used liquid-adsorption
columns to separate plant pigments.

Processes which take place in chromatographic process:

Partition is a concentrational changes in the system due to the distribution of the components
between two (or more) phases.

Adsorption is the concentrational changes in the system in presence of interface with another
phase and due to the surface forces.
In chromatography we have two main phases:

Mobile phase (eluent) is a liquid solvent or mixture of solvents which is moving through the
chromatographic column and carrying analytes.

Stationary phase (adsorbent) is solid porous media which consists of the porous particles, usually
silica based, with the specific surface properties (surface chemistry).

Types of Chromatography

Adsorption chromatography

Adsorption chromatography is probably one of the oldest types of chromatography around. It


utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid
phase. The equilibriation between the mobile and stationary phase accounts for the separation of
different solutes.

Adsorption chromatography Partition Chromatography Ion Exchange


Chromatography
Partition Chromatography

This form of chromatography is based on a thin film formed on the surface of a solid support by a
liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid.

Ion Exchange Chromatography

In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently
attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are
attracted to the resin by electrostatic forces.

Size exclusion chromatography

In size exclusion chromatography molecules are separated based on their molecular size in a
sieving effect. The bigger molecules (higher molecular weight) elute earlier.

Affinity chromatography

This is a highly specific mode of chromatography in which molecular recognition process takes
place between the agents bonded to the stationary phase and the solutes. The principle of lock-
key recognition similar to enzymes takes place. For example, the immobilized molecule may be
an antibody to some specific protein. When solute containing a mixture of proteins are passed by
this molecule, only the specific protein is reacted to this antibody, binding it to the stationary
phase. This protein is later extracted by changing the ionic strength or pH.
History

Prior to the 1970's, few reliable chromatographic methods were commercially available to the
laboratory scientist. During 1970's, most chemical separations were carried out using a variety of
techniques including: open-column chromatography, paper chromatography, and thin-layer
chromatography.

However, these chromatographic techniques were inadequate for quantification of compounds


and resolution between similar compounds. During this time, pressure liquid chromatography
began to be used to decrease flowthrough time, thus reducing purification times of compounds
being isolated by column chromatography.

High pressure liquid chromatography was developed in the mid-1970's. In the late 1970's, new
methods including reverse phase liquid chromatography allowed for improved separation
between very similar compounds.

By the 1980's HPLC was commonly used for the separation of chemical compounds. New
techniques improved separation, identification, purification and quantification far above the
previous techniques. Computers and automation added to the convenience of HPLC.

Currently, one has the option of considering over many types of columns for the separation of
compounds, as well as a variety of detectors to interface with the HPLC in order to get optimal
analysis of the compound.

Although HPLC is widely considered to be a technique mainly for biotechnological, biomedical,


and biochemical research as well as for the pharmaceutical industry, these fields comprise only
about 50% of HPLC users. Currently HPLC is used by a variety of fields including cosmetics,
energy, food, and environmental industries.
Specific chromatographic methods:
Liquid chromatography (LC)
Used to separate analytes in solution including metal ions and organic compounds. The
mobile phase is a solvent and the stationary phase is a liquid on a solid support, a solid, or
an ion-exchange resin.
Gas chromatography (GC)
Applied to volatile organic compounds. The mobile phase is a gas and the stationary
phase is usually a liquid on a solid support or sometimes a solid adsorbent.
High-performance liquid chromatography (HPLC)
A variation of liquid chromatography that utilizes high-pressure pumps to increase the
efficiency of the separation.
Size-exclusion chromatography (SEC)

Also called gel-permeation chromatography (GPC), the mobile phase is a solvent and the
stationary phase is a packing of porous particles.

Theory

Retention time tR is the time between the injection point and maximum of the detector response
for correspondent compound. Retention time is inversely proportional to the eluent flow rate.

The product of retention time and eluent flow rate, so called "retention volume", is more of a
global retention parameter. Retention volume, VR represents the volume of the eluent passed
through the column while eluting a particular component. Component retention volume VR could
be split into two parts:

Reduced retention volume is the volume of the eluent that passed through the column while the
component was sitting on the surface.

Dead volume is the volume of the eluent that passed through the column while the component
was moving with the liquid phase.

Historically, a slightly different retention parameter, called "capacity factor" (k') was introduced
by the analogy with the liquid partitioning theory and widely accepted in chromatographic
practice.

Capacity factor is independent on any geometrical parameters of the column or HPLC system. It
could be considered to be a thermodynamic characteristic of the adsorbent-compound-eluent
system.

Selectivity and Resolution

Selectivity is the ratio of the capacity factors of both peaks, or the ratio of its adjusted retention
times. Selectivity represents the separation power of particular adsorbent to the mixture of this
particular components.

This parameter is independent of the column efficiency, it only depends on the nature of the
components, eluent type, eluent composition, and adsorbent surface chemistry. In general, if the
selectivity of two components is equal to 1, then there is no way to separate them by improving
the column efficiency.
Resolution is the parameter describing the separation power of the complete chromatographic
system relative to the particular components of the mixture.

By convention, resolution (R) is expressed as the ratio of the distance between two peak maxima
to the mean value of the peak width at the base line:

If we approximate peaks by symmetric triangles, then, if R is equal to or more than 1 then


components are completely separated. If R is less than 1, then components are overlapped.

High-performance liquid chromatography (HPLC)

Separation can be achieved by controlling and manipulating these interactions, which effect the relative
retention times of the various sample components.
In Reversed Phase separations organic molecules are separated based on their degree of hydrophobicity.

There is a correlation between the degree of lipophylicity and retention in the column. This is the list of

mobile phase parameters effecting retention and separation in Reversed Phase.

Elution order in Normal Phase HPLC shows that the polar solutes elute later than non-polar lypophilic

ones.
The graphic below shows basic system components:

• Solvent Delivery System - pushes the solvent stream through the instrument at constant flow

rate

• Autosampler - introduces the sample into the liquid stream of the instrument

• Column - a stainless steel tube packed with silicon beads that separates what I'm looking for

from other compounds

• Detector - An optical sensor (usually) that detects changes in the characteristics of the solvent

stream

• Data System - A means of controlling the system components and storing, processing and

displaying data

A high pressure pump is required to force the mobile phase through the column at typical flow rates of

0.1-2 ml/min. The sample to be separated is introduced into the mobile phase by injection device, manual

or automatic, prior to the column.


Detectors

Ultra-Violet (UV) detectors measure the ability of a sample to absorb light. This can be
accomplished at one or several wavelengths:

A) Fixed Wavelength measures at one wavelength, usually 254 nm

B) Variable Wavelength measures at one wavelength at a time, but can detect over a wide
range of wavelenths

C) Diode Array measures a spectrum of wavelengths simulateneously

Refractive Index (RI) detectors measure the ability of sample molecules to bend or refract light.
This property for each molecule or compound is called its refractive index. For most RI detectors,
light proceeds through a bi-modular flow-cell to a photodetector. One channel of the flow-cell
directs the mobile phase passing through the column while the other directs only the mobile
phase. Detection occurs when the light is bent due to samples eluting from the column, and this is
read as a disparity between the two channels.

Fluorescent detectors measure the ability of a compound to absorb then re-emit light at given
wavelengths. Each compound has a characteristic fluorescence. The excitation source passes
through the flow-cell to a photodetector while a monochromator measures the emission
wavelengths.

Mass Spectroscopy (MS) Detectors - the sample compound or molecule is ionized, it is passed
through a mass analyzer, and the ion current is detected. There are various methods for
ionization:

A) Electron Impact (EI)- An electron current or beam created under high electric potential
is used to ionize the sample migrating off the column.

B) Chemical Ionization- A less aggressive method which utilizes ionized gas to remove
electrons from the compounds eluting from the column.
HPLC/MS systems

Interfacing a HPLC system with a mass spectrometer is not trivial.

The difficulty is to transform a solute into a gas phase ion. The challenge is to get rid of the
solvent while maintaining adequate vacuum level in the mass spectrometer, and to generate the
gas phase ions.

Since the early seventies, a number of approaches have been used. LC/MS became really popular
with the introduction of of the thermospray interface and the particle beam interface. The next big
improvement was the introduction of the electrospray and APCI techniques. The thermospray
interface is no longer available on the market, but the particle beam is still available from Waters
because it it the only method to provide electron impact spectra.

Actually, the large majority of applications are done with electrospray and APCI ionisation. New
techniques like APPI (atmospheric photo ionisation) are appearing, but are not yet largely used.

Electrospray and APCI are both API (atmospheric pressure ionisation) techniques. Ionisation
takes place at atmospheric pressure and both are considered to be soft ionisation method. The
mass spectrum provides mainly the molecular weight information, unless fragmentation
techniques are used. The possible fragmentation techniques are in source CID (collision induced
dissociation), CID in the collision cell of a tandem type instrument, fragmentation in an ion trap.
This is very different from the spectra obtained with EI (electron impact ionisation).

Niggard spectra (polymer additive), obtained by negative APCI (A), negative APCI
with in source fragmentation (B), electron impact ionisation (C).
Principle of Mass Spectrometry
The mass spectrometer is an instrument designed to separate gas phase ions according to their
m/z (mass to charge ratio) value.

The "heart" of the mass spectrometer is the analyzer. This element separates the gas phase ions.

The analyzer uses electrical or magnetic fields, or combination of both, to move the ions from the
region where they are produced, to a detector, where they produce a signal which is amplified.
Since the motion and separation of ions is based on electrical or magnetic fields, it is the mass to
charge ratio, and not only the mass, which is of importance. The analyzer is operated under high
vacuum, so that the ions can travel to the detector with a sufficient yield.

In addition to the analyzer, the mass spectrometer also includes

• A vacuum system

• Tools to introduce the sample (LC, GC …)

• Tools to produce the gas phase ions from the sample molecules

• Tools to fragment the ions, in order to obtain structural information, or to get more selective

detection

• A detection system

• Software and computing


Principles of LC/MS
LC/MS is a hyphenated technique, combining the separation power of HPLC, with the detection
power of mass spectrometry. Even with a very sophisticated MS instrument, HPLC is still useful
to remove the interferences from the sample that would impact the ionization.

Closely related to LC/MS are some other techniques, like flow injection/MS, CE or CEC/MS,
capillary LC or nano LC/MS

In all cases, there is the need for an interface that will eliminate the solvent and generate gas
phase ions, then transferred to the optics of the mass spectrometer.

Most instruments now atmospheric pressure ionization (API) technique where solvent
elimination and ionization steps are combined in the source and take place at atmospheric
pressure.

When electron impact ionization (EI) is the choice, the solvent elimination and ionization steps
are separate.

The interface is a particle beam type, which separates the sample from the solvent, and allows the
introduction of the sample in the form of dry particles into the high vacuum region.

Electron impact is of interest for molecules which do not ionize with API technique, or when an
electron impact spectrum is necessary, since it provides spectral information independent of the
sample introduction technique (GC or LC, or direct introduction) and instrument supplier.
Electrospray Ionization (ESI)

The HPLC line is connected to the electrospray probe, which consists of a metallic capillary
surrounded with a nitrogen flow. A voltage is applied between the probe tip and the sampling
cone. In most instruments, the voltage is applied on the capillary, while the sampling cone is held
at low voltage. First step is to create a spray. At very low flow rate ( a few µl/mn), the difference
in potential is sufficient to create the spray. At higher flow rate, a nitrogen flow is necessary to
maintain a stable spray.

The API sources include a heating device, in order to speed up solvent evaporation.

A mandatory condition to work with electrospray is that the compound of interest must be
ionized in solution.

If it is not compatible with the HPLC conditions (i.e. in case of normal phase chromatography), it
is possible to use post column addition to get appropriate conditions.

In the electrical field, at the tip of the capillary, the surface of the droplets containing the ionized
compound will get charged, either positively or negatively, depending on the voltage polarity .
Due to the solvent evaporation, the size of the droplet reduces, and, consequently, the density of
charges at the droplet surface increases. The repulsion forces between the charges increase until
there is an explosion of the droplet. This process repeats until analyte ions evaporate from the
droplet.
Multiply charged ions can be obtained depending on the chemical structure of the analyte. This is
why ESI is the technique of choice for analyzing proteins and other biopolymers on quadrupole
or ion trap analyzers.

Atmospheric Pressure Chemical Ionization (APCI)

Solvent molecules (S) being protonated by the corona (SH+), then reacting with the analyte molecule (M)
to give the protonated form MH+.

The HPLC line is is connected to the APCI probe which consists normally of a glass capillary
surrounded with a nitrogen flow used for mobilization. Part of the APCI probe, or close to the
probe tip are a heating device and an additional gas flow, to instantaneously volatilize the solvent
and sample.

Close to the probe, there is a metallic needle, which is at potential of a few kilovolts. This is the
"corona discharge electrode". The "corona effect" term describes the partial discharge around a
conductor placed at a high potential. This leads to ionisation and electrical breakdown of the
atmosphere immediately surrounding the conductor. This effect is known as corona discharge. St.
Elmo's fire is an example of a naturally occurring corona. In the case of an APCI source, the
atmosphere surrounding the corona electrode consists mainly in the vapour generated from the
HPLC eluent, nitrogen, and the analyte molecules.

The eluent vapours are ionised by the corona effect, and react chemically with the analyte
molecules in the gas phase.
The application field of LC/MS is extremely large and is covered by a wide range of instruments
and techniques.

Looking globally at the users, it is possible distinguish three groups, depending on how they use
LC/MS

• Users for which the main useful information from the mass spectrometer is the mass
information (molecular weight or fragments). The quantitative aspect is of no or little
importance.
Typically, these users wish to:

o monitor or confirm an organic chemistry synthesis

o or to trigger a fraction collector when the expected compound elute from the
column or to check if a peak on a chromatogram is a metabolite or degradation
product of a known parent compound

o or to get molecular weight and structure information from their compound

• Users for which the main interest is getting a very selective and sensitive detection. These
users are targeting specific molecules. The quantitative aspect is important, but the mass
information is of secondary importance.

• Users targeting specific molecules, wanting the quantification and the confirmation of the
identity. The molecular weight, and the presence of a few specific fragments which the
expected abundance are as important as the sensitivity and selectivity.
Literature:

1. Uwe D. Neue “HPLC Columns. Theory, Technology and Practice”, John Wiley & Sons,
New York 1997.
2. J. J. Kirkland “Współczesna chromatografia cieczowa”, PWN, Warszawa 1976.
3. R. J. Hamilton, P. A. Swell “Wysokosprawna chromatografia cieczowa”, PWN,
Warszawa 1982.
4. http://www.pharm.uky.edu/ASRG/HPLC/hplcmytry.html

5. http://hplc.chem.shu.edu/NEW/HPLC_Book/index.html

6. http://www.forumsci.co.il/HPLC/topics.html

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