High Performance Liquid Chromatography

High performance liquid
chromatography
Daniel Zahn
Idstein | Kln | Hamburg | Dsseldorf | Mnchen | Frankfurt am Main | Berlin | Zwickau | New York
Agenda
Principles of Chromatography
Terms and definitions
HPLC techniques
Instrumentation
Practical applications
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Origin of chromatographic separation techniques

Mikhail Tswett (early 19th century):
Investigation of plant extracts with calcium carbonate filled
columns and petrol ether/ethanol
Zones with different colors were observed
Further investigation and application as separation
technique
Technique was termed chromatography, derived from the
greek word chroma (color) and graphein (to write)
Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

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Principle of separation
A chromatographic system consists of a mobile phase and a stationary phase
Chromatographic separation is based on the interaction of sample molecules with the
stationary phase
The mobile phase can interact with the sample molecules as well or influence their
interactions with the stationary phase
sample molecules travel with the mobile phase but are retained by the stationary phase
Retention by the stationary phase can in most cases be attributed to at least one of the
basic mechanisms:
Adsorption
Adsorption
Distribution
Distribution

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Adsorption chromatography
Stationary phase has active centers of any kind that allow adsorption of sample
molecules
Sample molecules adsorb to and desorb from the stationary phase repeatedly
Properties of the sample molecules (affinity to stationary phase) determine the
frequency of adsorption
Molecules with a higher affinity to the stationary phase are adsorbed more
frequently and therefore more strongly retained
Molecules with a low affinity to the stationary phase spend little time adsorbed and
thus are quickly transported by the mobile phase

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Distribution chromatography
Stationary phase is a liquid attached to a solid support
The stationary and the mobile phase have to be immiscible
Continuous distribution of sample molecules between the mobile phase and the
stationary phase takes place
Molecules with an distribution equilibrium shifted towards the mobile phase spend
the majority of the time in the mobile phase and are therefore transported rapidly
Molecules with an distribution equilibrium shifted towards the stationary phase
spend the majority of the time in the stationary phase and are therefore strongly
retained

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Classification of chromatographic techniques

Classification by mobile phase state of matter:
gaseous (Gas chromatography, GC)
liquid (Liquid chromatography, LC)
supercritical fluid (Supercritical fluid chromatography, SFC)
Classification by phase pair (mobile phase/stationary phase):
Liquid/solid (Liquid-solid-chromatography, LSC)
Liquid/liquid (Liquid-liquid-chromatography, LLC)
Gas/solid (Gas-solid-chromatography, GSC)
Gas/liquid (Gas-liquid-chromatography. GLC)
Classification by application technique:
Planar chromatography (e.g. thin layer chromatography, TLC)
Column chromatography (e.g. HPLC, GC)
High Performance Liquid Chromatography (HPLC) is column
chromatography with a liquid mobile phase and solid stationary phase or
liquid stationary phase attached to a solid support
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The chromatogram
Intensity
A chromatogram is a plot of the intensity against the time. It can be described by a

variety of basic parameters:
t0
dead time
tR retention time
t`R reduced retention time
A
peak area
h
peak height
h1/2 half peak height
w
peak width
w1/2 peak width at half height
k` capacity factor
time
The capacity factor is often more suitable

than the reduced retention time to describe
the retention behavior of a substance
because it is independent of the column
length and flow rate. The capacity factor
should be kept between 1 (better 2) and 10
during method development

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Peak types

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Fronting and tailing

Fronting (AS < 1): Overload of mobile phase,
instrumental problems
Tailing (AS > 1): Overload of stationary phase,
secondary interactions, instrumental problems
Reasons for fronting and tailing are diverse and have to
be investigated separately for any case. Tailing is much
more common than fronting.
Fronting and tailing may be expressed as asymmetry factor

(IUPAC) or tailing factor (USP). Both calculations are valid but a
specific calculation may be requested by regulatory authorities.
Strong fronting and tailing has adverse effects on peak high and
resolution and thus severely compromises quantification.

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Number and height of theoretical plates

Chromatography is based on continuous interactions of the
analytes with mobile and stationary phase
Theory of plates is deployed to simplify the description of the
continuous process
H
The chromatographic column is viewed as consecutive sections

in which an equilibrium is reached. The height of these sections is
the height of a theoretical plate (H) and their quantity is the plate
number (N)
The plate number

significantly influences
the broadness of a peak
l
column length
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Van-Deemter-equation
High plate numbers result in narrower peaks. The plate number can be optimized by
increasing the column length or decreasing the plate height.
height of a
theoretical
plate H
Van-Deemter-eqiation:
H = A + B/u + C*u
minimal plate
height
optimal
flowrate
eddy diffusion
longitudinal diffusion
mass transfer
mobile phase
flowrate u

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Eddy diffusion:
Different path lengths due to variety of possible ways
through the column
Independent on flow rate
Longitudinal diffusion:
Diffusion of sample molecules in or against the mobile
phase flow rate
Effect is significantly increased at low flow rates
Mass transfer:
Mass transfer in and out of stationary phase/stagnating
mobile phase in stationary phase pores
Effect is increased at high flow rates

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Chromatographic resolution
The resolution describes the separation of two adjacted peaks
A minimal resolution of 1.5 is required for baseline separation of Gaussian peaks
Baseline separation is a requirement for accurate area and height measurement
Baseline separation is
mandatory if quantification
is performed with nonselective detectors!

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Optimization of resolution
The three important factors for the

resolution are:
the efficiency
the retention factors
and the selectivity

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Optimization of efficiency
A high efficiency results in narrow peaks and therefore a better

resolution.
The efficiency can be improved by:
increasing the column length (also increases analysis time!)
deploying smaller particles (also increases backpressure!)
optimization of the flow rate
other factors

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Optimization of the retention factor
A very low retention factor is a consequence of (almost) no interactions with the stationary
phase and therefore no separation. Once retention is sufficient (k > 5) a further increase has
almost no influence on the resolution
The retention factor can be improved by:
adjusting the solvent strength (effective and simple!)
changing the stationary phase

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Optimization of the selectivity
The selectivity is the ability of a chromatographic system to distinguish between two

substances. It is the most efficient but most complex way to improve the resolution
The selectivity may be improved by:
changing the organic solvent
changing the pH value
changing the stationary phase
changing the column temperature
Every influence on the selectivity is substance

specific, therefore any of the listed changes
may or may not influence the selectivity of a
specific substance pair.
other factors
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Reversed Phase HPLC
H3C
H2O
H2O
H3C
H2O
H3C
H2O
H3C
H2O
N
H3C
H3C
H2O
H2O
H2O
H2O
H2O
H3C
H3C
H3C
Stationary phase: non-polar (e.g. C18, C8)

Mobile phase: polar to semi polar (mixtures of H2O, ACN, MeOH, additives)
Analyte: Substances of intermediate to low polarity
Mechanism:
Non-polar stationary phase acts as immobilized liquid
Liquid-liquid-partition between stationary and mobile phase takes place
Secondary interactions (e.g. with underivatized silanol) are possible
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Normal Phase-HPLC
H3C
H3C
CH3
CH3
H3C
H3C
CH3
H3C
H3C
CH3
CH3
H3C
H3C
CH3
CH3
Stationary phase: polar (e.g. SiO2,Al2O3)

Mobile phase: non-polar (e.g. hexane)
Analyte: polar (has to be soluble in mobile phase)
Mechanism:
Polar molecules tend to attach to one another in a non-polar environment
The polar analytes adsorb to the polar surface of the stationary phase

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Ion chromatography
Stationary phase: charged polymers

Mobile phase: aquatic buffer solutions (e.g. NaHCO3)
Analyte: ionic
Mechanism:
Surface of the stationary phase is charged
Analytes of the opposite charge are attracted to the stationary phase

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Hydrophilic interaction liquid chromatograph
Stationary phase: polar (e.g. SiO2, polar modifications)

Mobile phase: polar to semi polar (mixtures of H2O/ACN/MeOH)
Analyte: polar
Mechanism:
Water is strongly retained on the surface of the stationary phase
partition equilibrium between the mobile phase and the immobilized water
layer on the stationary phase
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Gel permeation chromatography
Stationary phase: No interactions with analyte.

Mobile phase: No interactions with analyte. Analyte must be soluble.
Analyte: Analytes must significantly differ in size
Mechanism:
No interactions between analyte, stationary phase and mobile phase
Small analytes can diffuse into pores and therefore take more time to reach
the detector
Separation by size
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Elution strength of the mobile phase

IC
GPC
ionic strength
HILIC
polarity
NP-HPLC
polarity
RP-HPLC
polarity
high elution
strength
low elution
strength

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Overview important HPLC techniques

HPLC technique
Stationary
phase*
Mobile phase*
analytes
Main interaction
Reversed phase
(RP)
C18, C8
modified
SiO2
H2O, methanol,
acetonitrile, buffer
salts
Semi polar and

non polar
substances
Distribution
Normal phase
(NP)
SiO2, Al2O3
Hexane, pentane,
benzene
Polar substances
Adsorption
Ion chromatography
(IC)
Polymers
with charged
moieties
Acidic or alkaline
aqueous buffers
Organic and
inorganic (an)ions
Electrostatic
interactions
Hydrophilic interaction
chromatography
(HILIC)
SiO2 and
various polar
modifications
H2O, methanol,
acetonitrile, buffer
salts
Polar substances
Distribution
(adsorption)
Gel permeation
chromatography
(GPC)
Polymers
H2O, organic
solvents
Macromolecular
substances
*most commonly used stationary and mobile phases. There are of cause more.
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Instrumentation

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Pump and injector
HPLC pumps should be able to generate a

continuous, pulsation free flow at a back
pressure of up to 400 bar (1000 for UPLC)

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UV/Vis detector, Diodenarray detector

UV/Vis detector
Very similar to UV/Vis spectrometers

but different cell geometry. Revisit the
UV/Vis spectrometry lecture for the
principles of optical spectrometry
Diodenarray detector
The UV/Vis or diodenarray
detector is the standard HPLC
detector in routine analysis. It
is suitable to detect the
majority of organic substances
with an acceptable sensitivity

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Fluorescence detector
Excitation: absorption of light
Fluorescence: emission of light
Non-radiative relaxation: emission of energy (heat)
Since a portion of the energy is lost as heat the
emitted light has a longer wavelength than the
absorbed light
The fluorescence detector is highly

selective and sensitive but is limited to
fluorescent substances (derivatisation)

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Refractive index detector
Detection is based on a different

refractive index of the pure solvent
and the solvent during elution of a
substance.
The refractive index detector is a

universal detector capable of detecting
virtually any substance but suffers form
poor sensitivity, temperature sensitivity
and is incapable of gradient elution

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Evaporative light scattering detector
The column effluent gets nebulized and

the mobile phase evaporated, resulting in
the formation of analyte particles.
These analyte particles drift through the
path of a laser and scatter the laser light.
The intensity of the scattered light is
measured.
The evaporative light scattering detector

is universal for all non-volatile
substances. It is capable of gradient
elution but limited to volatile mobile
phase compositions
All components of the mobile phase must

be volatile. Volatile analytes can not be
detected!

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Mass spectrometer
The analyte has to be ionized and

desolvatized. Detection of
substances is achieved m/z
selective.
A mass spectrometer is capable of

analyzing all ionizable substances with a
high selectivity and sensitivity. It
properties are highly dependent on the
instrumental setup.
Various ion sources, mass analyzers

and detectors are commercially
available and heavily influence the
properties and performance of the
instrument.
The details of mass spectrometry will
be covered in a separate lecture.

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Electrical conductivity detector
Measures the conductivity of the

column effluent. Changes in the
conductivity indicate the presence
of eluting ions.
A electrical conductivity detector is most commonly

deployed in combination with a suppressor to reduce the
base conductivity of the mobile phase and thus increase
the sensitivity
The electrochemical conductivity detector is the

standard detector in ion chromatography. It is
selective for charged compounds.

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Detectors overview
Detector
principle
analytes
sensitivity
selectivity
UV/Vis detector,
Diodenarray detector (DAD)
UV/Vis
absorption
UV/Vis absorbing
substances
Fluorescence detector
(FLD)
fluorescence
fluorescent
substances
++
++
Refractive index detector

(IRD)
refraction
universal
--
--
Evaporative light scattering

detector (ELSD)
light
scattering
non-volatile
substances
Mass spectrometer (MS)*
mass
spectrometry
ionizable substances
++
++
Electrical conductivity
detector (ELCD)
electrical
conductivity
ionic substances
*content of a separate lecture

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Method development LC technique selection

Selection of a suitable chromatographic technique based on the analyte properties
Semi to non-polar
organic substances
Semi-polar to polar
organic substances
Ionic
substances
RP-HPLC
NP-HPLC/HILIC
IC/HILIC
While NP-HPLC offers good

retention for very polar substances
they are often not well soluble in the
deployed apolar organic solvents
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Method development basic screening gradient

Once a chromatographic technique is selected a basic screening gradient should be run
(e.g. 95/5 water/methanol to 5/95 water/methanol on a C18 column in case of RP-HPLC)
Dont start at 0%
water unless the
column is
compatible with it

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Method development adjustment of slope

The slope of a gradient may
heavily influence resolution, peak
intensity and analysis time.
A shallow gradient results in an
improved resolution at the cost of
peak height and time.
A steep gradient increases the
peak height and shortens the
analysis time but resolution may
suffer.
In most cases the aim is to
achieve a sufficient resolution
while maintaining a short analysis
time and good peak heights

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Mobile phase adjustments methanol vs. acetonitrile

Viscosity: mixtures of acetonitrile and water have a
significantly lower viscosity than mixtures of
methanol and water, resulting in a reduced back
pressure.
UV-Absorption: methanol (205nm) and acetonitrile
(190 nm) have a different UV-cutoff wavelength,
resulting in increased sensitivity when using
acetonitrile with a UV detector at low wavelengths
Selectivity: methanol is a protic and acetonitrile a
aprotic solvent, resulting in a different selectivity
for some analytes (especially analytes with a polar
moiety)
Elution strength: methanol and acetonitrile have a
similar elution strength in RP-HPLC
Relative elution strength
Price: methanol is cheaper than acetonitrile

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Mobile phase adjustments pH value

The pH value may influence the charge state of a compound.
Ionic compounds are more polar and their non-ionized forms and thus less retained
under RP-HPLC conditions. Ionic compounds may interact strongly with silanol groups.
When analyzing ionizable compounds the pH of the mobile phase has to be adjusted
Buffers are effective within a range of
1 pH around the pKa of their compounds
Common HPLC buffers are:
Phosphate: pKa 2.1/7.2/12.3
non-volatile
Formate: pKa 3.8
volatile (NH4+ salt)
Acetate: pKa 4.8
volatile (NH4+ salt)
Always consider your detector

when selecting a buffer
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Dealing with ionic substances

Ionic substances are not well retained in RP-HPLC. There are three ways to deal with
them:
1.) Adjustment of pH (not possible for all substances)
2.) Selection of a more suitable chromatographic technique (e.g. IC or HILIC)
3.) Use of ion pair reagents
Ion pair reagents consist of an
ionic and a hydrophobic part.
They pair with the analyte and
are retained together with it.
Always consider your
detector when selecting an
ion pair reagent

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Sample solvent and injection volume

Injecting a sample dissolved in a solvent with
a higher elution strength than the mobile
phase (starting conditions in case of gradient
elution) can result in broad peaks with poor
peak shapes and reduced retention times.
ACN
H2O/ACN
70:30
This effect is more significant for early eluting

peaks and with increased injection volumes.
Always choose a sample solvent that

hast a lower elution strength than your
mobile phase

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Trends in chromatography U(H)PLC
U(H)PLC (ultra (high) performance liquid

chromatography) is a variant of HPLC with
shorter columns (50mm), smaller particles
(<2m), and higher linear velocities.
It allows for faster analysis with reduced
solvent consumption at the cost of significantly
increased backpressure (instrumentation!)

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Trends in chromatography solid core particles
Particles are composed of a solid core and a

porous shell.
Reduced diffusion path lengths (compared to
fully porous particles of the same size) result
in an decreased influence of mass transfer.
Results are increased in a similar was as in
UHPLC but without the increase in
backpressure.

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Task 1: method development
Phenol
Nitrophenol
Anthracene
Suggest a suitable chromatographic method for the quantification of phenol,

nitrophenol and anthracene from aqueous samples.
Consider the LC technique, the detector and method parameters.

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Task 2: method development
Diquat
Diclofenac
Anthracene
Suggest a suitable chromatographic method for the quantification of diquat,

diclofenac and anthracene from aqueous samples.
Consider the LC technique, the detector and method parameters.

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Task 3: method optimization
Column: C18 150*4.6mm; 5m

Mobile phase: H2O/ACN/H2SO4
75:25:0.03
Flow: 1ml/min
Detector 1: UV (205 nm)
Detector 2: RID
Your laboratory routinely performs the above mentioned analysis and you are tasked
to reduce the cost per analysis without compromising the results. Discuss the
usefulness of
a) An exchange acetonitrile for methanol
b) Gradient elution to reduce the analysis time
c) Reduction of the column dimensions (diameter lengths and particle size) and
adjustment the flow rate
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Additional information
Book:
Douglas A. Skoog, F. James Holler, Stanley R. Crouch: Principles of Instrumental
Analysis, 6th Edition, 2007
Internet:
http://www.chemguide.co.uk/analysis/chromatogrmenu.html#top
http://www.sepscience.com/Techniques/LC
http://www.studyhplc.com/novice.php
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High Performance Liquid Chromatography

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High performance liquid

Origin of chromatographic separation techniques

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Classification of chromatographic techniques

A chromatogram is a plot of the intensity against the time. It can be described by a

The capacity factor is often more suitable

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Fronting and tailing

Fronting and tailing may be expressed as asymmetry factor

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Number and height of theoretical plates

The chromatographic column is viewed as consecutive sections

The plate number

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

The three important factors for the

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

A high efficiency results in narrow peaks and therefore a better

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Optimization of the retention factor

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Optimization of the selectivity

The selectivity is the ability of a chromatographic system to distinguish between two

Every influence on the selectivity is substance

Reversed Phase HPLC

Stationary phase: non-polar (e.g. C18, C8)

Stationary phase: polar (e.g. SiO2,Al2O3)

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Stationary phase: charged polymers

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Hydrophilic interaction liquid chromatograph

Stationary phase: polar (e.g. SiO2, polar modifications)

Gel permeation chromatography

Stationary phase: No interactions with analyte.

Elution strength of the mobile phase

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Overview important HPLC techniques

Semi polar and

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Pump and injector

HPLC pumps should be able to generate a

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

UV/Vis detector, Diodenarray detector

Very similar to UV/Vis spectrometers

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

The fluorescence detector is highly

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Refractive index detector

Detection is based on a different

The refractive index detector is a

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

Evaporative light scattering detector

The column effluent gets nebulized and

The evaporative light scattering detector

All components of the mobile phase must

Principles of chromatography I Terms and definitions I HPLC techniques I Instrumentation I Applications

The analyte has to be ionized and

A mass spectrometer is capable of