INTRODUCTION TO

CHROMATOGRAPHY
Topics to be discussed
 An introduction to chromatographic methods
 General description of chromatography
 Classification of chromatographic methods
 Elution chromatography
 Chromatogram
 Migration rate of solutes
 Band broadening and column efficiency
 Theory of band broadening
 Column resolution
 Optimization techniques
 General elution problems
 Application of chromatography
Introduction
 Chromatography is
 an analytical method,
 which is widely used for separation,

 identification and

 determination of the chemical components in the

complex mixture.
 Colored band formation (Gr.Chroma- color ; graphein-
writing)
Introduction
 Invented by Russian botanist Mikhail Tswett
 He employed the technique to separate various plant
pigment such as chlorophyll and xanthophyll by passing
solution through glass column packed with finely divided
calcium carbonate.
Milestones in Chromatography
 1903 Tswett - plant pigments separated on
chalk columns
 1931 Lederer & Kuhn - LC of carotenoids
 1938 TLC and ion exchange
 1950 reverse phase LC
 1952 Nobel Prize awarded to Martin & Synge
 1959 Gel permeation
 1965 instrumental LC (Waters)
Application of chromatography –growing
explosively in the last century.

Forensic

Pharmaceutical industries Research

General description of Chromatography

 Chromatography consists of two phases: mobile

phase and stationary phase.
 Sample is dissolved in the mobile phase and then
forced through the column from injector to detector
as a flowing media.
 Mobile phase may be a gas or a liquid or a
supercritical fluid.
 Stationary phase is fixed in place in a column or on
a solid surface.
General description of Chromatography

 The injected sample is carried down by the mobile

phase along the column.
 The two phases are chosen so that the components of
the sample distribute themselves between the mobile
phase and stationary phases to varying degrees.
 All Chromatographic techniques are based on
differential mobility of components of the mixture in
stationary phase when they are carried by the flow
of mobile phase.
General description of Chromatography

 Those components strongly retained by the

stationary phase move only slowly with the flow of
mobile phase.
 Components that are weakly held by the stationary
phase travel rapidly.
 As a consequence of the differences in migration
rates, a sample component separate into discrete
band or zones that can be analyzed.
Mixture Stationary
phase

Mobile
(moving)
phase

t1 t2 t3
 Classification

Chromatography

Based on the physical means by Based on the type of mobile and stationary
which the stationary and mobile phase and the kinds of equilibria involved in
phase are brought into contact the transfer of solute between phases

Column Planer GC LC SFC

Classification
 Column Chromatography: The stationary phase is
held in a narrow tube through which the mobile
phase is forced under pressure or gravity.
 Planar Chromatography: The stationary phase is
supported on a flat surface or in the interstices of
paper. Mobile phase moves through stationary
phase by capillary action or under the influence of
gravity.
Types of Chromatography…

14

Thin layer
Paper

© RGR
HPLC Gas Column
Supercritical fluid

 A supercritical fluid is any substance at a temperature and

pressure above its critical point, where distinct liquid and gas
phases do not exist. It can effuse through solids like a gas, and
dissolve materials like a liquid.
 In addition, close to the critical point, small changes in pressure or
temperature result in large changes in density, allowing many
properties of a supercritical fluid to be "fine-tuned".
 Supercritical fluids are suitable as a substitute for organic solvents
in a range of industrial and laboratory processes.
 Carbon dioxide and water are the most commonly used
supercritical fluids, being used for decaffeination and power
generation, respectively.
Elution in Column Chromatography
 Elution involves washing a species through a column
by continuous addition of fresh solvent.
 The sample is introduced at the head of a column,
whereupon the components of the sample distribute
themselves between the two phases.
 Introduction of additional mobile phase (the eluent)
forces the solvent containing a part of the sample
down the column, where further partition between
the mobile phase and fresh portions of the
stationary phase occurs.
Fig: Concentration profiles of solute bands A and B at
two different times in their migration down the column
Elution in Column Chromatography
 The average rate at which solute moves with the
mobile phase depends upon the fraction of the time it
spends in mobile phase.
 Due to this difference in the rate of migration
components are separated into bands or zones along
the length of column.
 By passing sufficient quantity of the eluent different
components are separated or isolated .
Chromatogram
 If a detector that responds to solute concentration is
placed at the end of the column and its signal is
plotted as function of time (or of volume of the
added mobile phase), a series of peaks is obtained.
 Such a plot is called a chromatogram
 useful for both qualitative and quantitative analysis.
 The positions of peaks on the time axis may serve to
identify the components of the sample
 The areas under the peaks provide a quantitative
measure of the amount of each component.
Fig: Two component chromatogram
Migration rate of solute
❖Distribution constant (K)
❖Retention time (tR )

❖Retention factor (k)

❖Selectivity factor(α)
Migration rate of solute
 The effectiveness of separation depends upon the
relative rate at which two species are eluted .
 Rate of elution depends upon distribution constant.
 Due to different extent to which solutes are
distributed between two phases, solutes are migrated
down the column at different rate.
 Distribution constant (K)
 Retention time (tR )
 Retention factor (k)
 Selectivity factor(α)
Distribution constant/Partition
coefficient/partition ratio (K)
 The distribution equilibria involved in chromatography
involve the transfer of an analyte between the mobile
and stationary phases.
Amobile Astationary
 The equilibrium constant K for this reaction is called the
distribution constant, the partition ratio, or the partition
coefficient,
Kc = cS/cM
where cs is the molar concentration of the solute in the stationary phase
cM is its molar concentration in the mobile phase.
( Kc is constant over a wide range of solute concentrations.)
Retention time (tR )

▪ The time required by the sample component to

reach the detector after sample injection is called
retention time tR.
▪ The time tM for the unretained species to reach the
detector is called the dead time

tR =ts+tm

= L/tR
u = L/tM
Retention time (tR )

▪ The rate of migration of the unretained species is the

same as the average rate of motion of the mobile
phase molecules.
• The average linear rate of solute migration ‾ is
‾ = L/tR
where, L is the length of the column packing.
• The average linear velocity u of the molecules of the
mobile phase is
u = L/tM
 Relationship between Distribution constant
& retention time
L
Average linear rate of migration of mobile phase (u) 
tM
L
Average linear rate of solute migration ( ) 
tR

v u X fraction of time the solute spend in mobile phase.

Average numbers of moles of solutes in mobile phase at any instant
vuX
Total number of moles of solute in the column
C V
vuX M M {V and V are volume of mobile and stationary phase}
C V C V M S
M M S S
1
v  u X
1 C V / C V
S S M M This explains the rate of solute migration in
1 terms of partition ratio K and the volume of
v  u X
1  KV / V the stationary and the mobile phase.
S M
The Rate of Solute Migration:
The Retention Factor (k)
▪ The retention factor, or capacity factor, is an
important parameter that is widely used to describe
the migration rates of solutes on columns. For a solute
A, the retention factor kA is defined as
kA = KAVS/VM
where KA is the distribution constant for the species A.
kA = (tR - tM )/tM

When k is<<1.0, separation is poor

When k = 20 --30, separation is slow
When k is = 1-10, separation is optimum
Relative Migration Rates:
The selectivity Factor
 The selectivity factor  of a column for the two species A
and B is defined as
 = KB/KA
 where KB is the distribution constant for species B and
 KA is the distribution constant for species A.
  is always greater than unity.
 A relationship between the selectivity factor and retention
factors:
 = kB/kA
Where kB and kA are the retention factors.
Relative Migration Rates:
The selectivity Factor
 An expression for the determination of  from an
experimental chromatogram:

(tR ) B  tM
 
(tR ) A  tM
 Band broadening and the
column efficiency
❖Quantitative description of the column efficiency
❖Kinetic Variables Affecting Column Efficiency
Band broadening and the column
efficiency
 Efficiency is related experimentally to a solute’s
peak width.
 an efficient system will produce narrow peaks
 narrow peaks  smaller difference in interactions in
order to separate two solutes

 Efficiency is related theoretically to the various

kinetic processes that are involved in solute retention
and transport in the column
 determine the width or standard deviation (s) of
peaks
Rate theory of Chromatography

 This theory describes the variables that

influence the time for appearance of elution
band as well as the width of the band, in
quantitative terms based on a random walk
mechanism for the migration of the solute
through the column.
Distorted peak shapes
Asymmetric peaks give false area count.
The Theoretical Plate Model of
Chromatography
 The plate model supposes that the chromatographic
column contains a large number of separate layers
hypothetical, called theoretical plates.
 They also serve as a way of measuring column
efficiency: number of theoretical plates in a column, N
& plate height, H.
Quantitative description of the
column efficiency
(1) plate height H
(2) plate count plates N.
The two are related by the equation
N = L/H
where , L is the length (usually in centimeters) of the
column packing.
 The efficiency of chromatographic columns increases
as
 the plate count becomes greater and
 the plate height becomes smaller.
 Plate Height

 The plate height H is

given by
H = 2/L
 L carries units of
centimeters and
 2 units of centimeters
squared;
 thus H represents linear
distance in centimeters.
Plate Height

 The plate height can be thought of as the length of

column that contains a fraction of analyte that lies
between L-  and L.
 Because the area under a normal error curve
bounded by ±1 is about 68% of the total area, the
plate height, as defined, contains approximately 34
% of the analyte.
  is the standard deviation of the breadth of a
Gaussian curve, and 2 is its variance.
The Experimental Evaluation H and N

The time based variance in the chromatogram is denoted by τ 2

and the distance based variance by σ2. These two are related
by the relation, 

L
tR
 The Experimental Evaluation H and N
The area of the triangle is about 96% of the total area under the
peak ( that is the solute are within ±2 σ). Similarly the intercept in the
figure occurs within approximately at ±2 τ from the maximum.
Thus W = 4τ.
W is magnitude of the
width of base of the triangle LW

4t R
2
From the relation, H
L
we can obtain, LW 2
H
16t R2
2
 tR 
and using N  L/H N  16 
W
The Experimental Evaluation H and N

 Another method for approximating N, is to determine

W1/2, the width of peak at half its maximum height.
 The plate count is then given by
N = 5.54(tR/ W1/2)2

 The plate count N and the plate height H are

widely used in the literature and by instrument
manufactures as measures of column performance.
Kinetic Variables Affecting Column
Efficiency
 Band broadening reflects a loss of column efficiency.
 The slower the rate of mass transfer processes
occurring while a solute migrates through a column ,
the broader the band at the column exit.
 Some of the variables that affect mass transfer rates
are controllable and can be exploited to improve
separations.
The Effect of Mobile-Phase Flow Rate

 The magnitude of kinetic effects on column

efficiency depends upon
 the length of time the mobile phase is in contact with
the stationary phase,
 which in turn depends upon the flow rate of the mobile
phase.
 Efficiency studies have generally been carried out
by determining H as a function of mobile-phase
velocity u.
Effect of mobile phase flow rate on plate height for LC and GC
Theory of Band Broadening

Why Do Bands Spread?

a. Eddy diffusion( Multiple flow path)
b. Longitudinal diffusion
c. Mobile phase mass transfer
d. Stationary phase mass transfer
 Theory of Band Broadening
van Deemter equation can be written in the form:
 The efficiency of chromatographic columns can be approximated
by the expression
H = A + B/u + Cu
= A + B/u + (Cs + CM)u
 Where, H is the plate height in centimeters,
 u is the linear velocity of the mobile phase in centimeters per sec
 quantities A, B, and C are coefficients related to the phenomena of
multiple flow paths (eddy diffusion), longitudinal diffusion, and mass
transfer between phases, respectively.
 The C coefficient can be broken into two coefficients, one related to the
stationary phase (Cs) and one related to the mobile phase (CM).
 The van Deemter equation contains terms linearly and inversely
proportional to, as well as independent of, the mobile phase velocity
Eddy Diffusion (The Multipath term A)

 Zone broadening arises in part

from the multitude of
pathways by which a molecule
(or ion) can find its way
through a packed column.
 The length of these pathways
may differ significantly;
 thus, the residence time in the
column for molecules of the same
species is also variable.
Eddy Diffusion (The Multipath term A)

 Solute molecules then reach the end of the column

over a time interval, which leads to a broadened
band.
 This effect which is called eddy diffusion, is directly
proportional to the diameter of the particles making
up the column packing. i.e. A= 2 λ dp
Eddy Diffusion (The Multipath term A)

 A= 2 λ dp
Where, λ-Constant that depend on quality of the
packing
dp - diameter of the packing particles
 As solute molecules travel through the column, some
arrive at the end sooner then others simply due to the
different path traveled around the support particles
in the column that result in different travel distances
The Longitudinal Diffusion Term (B/u)

 Longitudinal diffusion in column chromatography is

a band broadening process in which solutes diffuse
from the concentrated center of a zone to the more
dilute regions ahead of and behind the zone center.
 The longitudinal diffusion term is directly
proportional to the mobile-phase diffusion
coefficient DM.
 The contribution of longitudinal diffusion is seen to
be inversely proportional to the mobile phase
velocity.
The Longitudinal Diffusion Term (B/u)

▪Band-broadening due to the diffusion of the solute along the

length of the column in the flowing mobile phase
Mass-transfer Coefficients (Cs and CM)

 The need for the two mass-transfer coefficients Cs and

CM arises because the equilibrium between the mobile
and the stationary phase is established so slowly that
a chromatographic column always operates under non
equilibrium conditions.
Stationary phase Mass transfer term Csu

 When the stationary phase is the immobilized

liquid , the mass transfer coefficient is
 directly proportional to the square of the thickness of the
film on the support particles df2 and
 inversely proportional to the diffusion coefficient Ds of the
solute in the film.
Stationary phase Mass transfer term Csu

 With thick films: molecules must on the average travel

farther to reach the surface, and with smaller
diffusion coefficient, they travel slower.
 Consequence is slower rate of mass transfer and
increase in H.
 Since different solute molecules spend different
lengths of time in the stationary phase, they also
spend different amounts of time on the column, giving
rise to band-broadening.
Stationary phase Mass transfer term Csu

 When stationary phase is a solid surface, Cs is

directly proportional to the time required for a
species to be adsorbed or desorbed, which in turn is
inversely proportional to the first order rate constant
for the process.
Mobile phase Mass transfer term CMu

 A process of peak broadening caused by the

presence of different flow profile within channels or
between particles of the support in the column.
 A solute in the center of the channel moves more
quickly than solute at the edges, it will tend to reach
the end of the channel first leading to band-
broadening
 H
Contribution to H, cm

CS u

C Mu

B/u

Mobile phase linear velocity, u, cm/s

Stagnant mobile phase mass transfer – band-broadening due to
differences in the rate of diffusion of the solute molecules between the mobile phase
outside the pores of the support (flowing mobile phase) to the mobile phase within the
pores of the support (stagnant mobile phase).

Since a solute does not travel down

the column when it is in the stagnant
mobile phase, it spends a longer time
in the column than solute that remains
in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase mass

transfer depends on:

1) the size, shape and pore structure of the packing material

2) the diffusion and retention of the solute
3) the flow-rate of the solute through column
Stationary phase mass transfer – band-broadening due to the movement
of solute between the stagnant phase and the stationary phase.

Since different solute molecules

spend different lengths of time in the
stationary phase, they also spend
different amounts of time on the
column, giving rise to band-
broadening.

The degree of band-broadening due to stationary phase mass transfer

depends on:
1) the retention and diffusion of the solute
2) the flow-rate of the solute through the column
3) the kinetics of interaction between the solute and the
stationary phase
Methods for Reducing Zone Broadening
 Optimization of Column
Performance
❖Column resolution
❖The Effect of Retention and Selectivity Factors on Resolution

❖The Effect of Resolution on Retention time

❖Variables that affect column performance

❖General Elution Problem

Optimization of Column Performance

 A chromatographic separation is optimized by

varying experimental conditions until the components
of a mixture are separated cleanly in a minimum
amount of time.
 Optimization experiments are aimed at either

(1) Reducing band broadening or

(2) Altering relative migration rates of the components.

Column Resolution (Rs)
 Resolution of a column tells us how far apart
two bands are relative to their width.
 The resolution Rs of a column provides a

quantitative measure of its ability to separate

two analytes .
 Column resolution is defined as

Z 2 Z 2[(tR ) B  (tR ) A]
Rs   
WA / 2  WB / 2 WA  WB WA  WB
Column Resolution (Rs)
 A resolution of 1.5 gives an essentially complete
separation of the two components, whereas a
resolution of 0.75 does not.
 At a resolution of 1.0, zone A contains about 4% B
and zone B contains a similar amount of A.
 At a resolution for 1.5, the overlap is about 0.3% .
❖ The resolution for a given stationary phase can be
improved by lengthening the column, thus increasing
the number of plates.
The Effect of Retention and Selectivity
Factors on Resolution
 Relationship between the resolution of a column and
the retention factors kA and kB for two solutes, the
selectivity factor , and the number of plates

N k'
Rs  4
(  1)(
1 k '
)

2 1 2 1 k' 2
N  16 Rs( )( )
 1 k'
Where k is the average of kA and kB
Effect of resolution on retention time
Improving Column Performance
 The separation is enhanced by increasing column
length but this will increase elution time, and band
broadening takes place, which lowers the efficiency
of the column as a separating device.
 Efficient rate of separation is that where rate of
separation is high but the rate of band broadening is
less.
 Band broadening is influenced by different physical
and chemical variables, that can be controlled.
Ways how to improve the resolution

Increase the efficiency

•Longer columns
•Smaller diameter of column or smaller diameter of particles
•Thinner stationary phase
•Optimated flow of mobile phase
Increase the retention factor(1-10)
•Bigger amount of stationary phase
•Weaker mobile phase (HPLC)
•Lower analysis temperature (GC)
•Change in solvent composition(for liq.mobile phase)
Increased selectivity
•More selective phases
•Lower analysis temperature
•Derivatization
 Gradient elution n
Isocratic elution initial, 2:3(Me and H2O). Me conc
1:1 vol of methanol and water increeased at rate of 8%/min
1
1 3 5
3 5
2 4 6
2 4

retention time
Fig: separation of different chlorinated benzenes by HPLC
Fig: General Elution Problem
APPLICATIONS OF CHROMATOGRAPHY

Chromatography has grown to be the opening

method for separating closely related chemical
species. In addition, it can be employed for
qualitative identification and quantitative
determination of separated species.
 Qualitative Analysis
 A chromatogram provides only a single piece of
qualitative information about each species in a
sample, namely, its retention time or its position on the
stationary phase after a certain elution period.
 It is a widely used tool for recognizing the presence
or absence of components of mixtures containing a
limited number of possible species whose identities
are known.
 Positive spectroscopic identification would be
impossible without a preliminary chromatographic
separation on a complex sample.
 Quantitative Analysis
 Chromatography can provide useful quantitative
information about the separated species.
 Quantitative column chromatography is based upon a
comparison of either the height or the area of the
analyte peak with that of one or more standards.
 For planar chromatography, the area covered by the
separated species serves as the analytical parameter.
 If conditions are properly controlled, these
parameters vary linearly with concentration.
 Analyses Based on Peak Height
 The height of a chromatographic peak is obtained by
connecting the base lines on either side of the peak
by a straight line and measuring the perpendicular
distance from this line to the peak.
 This measurement can ordinarily be made with
reasonably high precision.
 Accurate results are obtained with peak heights only
if variations in column conditions do not alter the peak
widths during the period required to obtain
chromatograms for sample and standards.
 The variables that must be controlled closely are
column temperature, eluent flow rate, and rate of
sample injection.
 Analyses Based on Peak Areas
 Peak areas are a more satisfactory analytical variable than
peak heights.
 On the other hand, peak heights are more easily measured and,
for narrow peaks, more accurately determined.
 Most modern chromatographic instruments are equipped with
digital electronic integrators that permit precise estimation of
peak areas.
 If such equipment is not available, manual estimate must be
made.
 A simple method, which works well for symmetric peaks of
reasonable widths, is to multiply the height of the peak by its
width at one half the peak height.
 Calibration and Standards
The most straightforward method for quantitative
chromatographic analyses involves the
preparation of a series of standard solutions that
approximate the composition of the unknown.
Chromatograms for the standards are then
obtained and peak heights or areas are plotted
as a function of concentration. A plot of the data
should yield a straight line passing through the
origin.
 The Internal Standard Method
The highest precision for quantitative chromatography
is obtained by use of internal standards because the
uncertainties introduced by sample injection are
avoided. In this procedure, a carefully measured
quantity of an internal standard substance is introduced
into each standard and sample, and the ratio of
analyte to internal standard peak areas (or heights)
serves as the analytical parameter. For this method to
be successful, it is necessary that the internal standard
peak be well separated from the peaks of all other
components of the sample.