Introduction to chromatography

Derived from two words;

chroma –color
Graphy- write
Operate in the same principle as extraction but one
phase is held constant ( stationary) while the other
one moves over it (mobile phase)

Analytical chemist use chromatographic techniques

to separate complex mixture and for analyzing very
complex samples
Mobile phase –solvent moving through the column;
could be a liquid or a gas
It carries the sample components through the
stationary phase

Stationary phase
The phase that stays in place inside the column.

It is mostly a viscous liquid chemically bonded to the

capillary tube or on to the surface of solid particles
packed in the column
Partitioning of solutes between the mobile and the
stationary phase gives rise to separation

Fluid entering the column is called eluent

Fluid emerging from the column is called eluate
Elution process of passing liquid or gas through a
chromatographic column
 Chromatographic techniques may also be
classified based on the type of support material
used in the system:
 Packed bed (column) chromatography
 Open tubular (capillary) chromatography
 Open bed (planar) chromatography
Types of chromatography
Chromatography is classified based on the
mechanism of interaction of the solute with the
stationary phase

Adsorption Chromatography
Has a solid stationary phase and a liquid or gaseous
mobile phase
Solute is adsorbed on the surface of the of the solid
particles .
The more strongly a solute adsorb the slower it
travels through the column
Solute adsorbed on
the surface of
stationary phase
Partition chromatography
Most common in gas chromatography. Stationary
phase is a liquid bonded on a solid surface
Solute equilibrates between the stationary liquid and
mobile phase

Solute dissolved in liquid

phase coated on the
surface of solid support
Ion exchange chromatography
Solute ions of opposite charge are attracted to the
stationary phase by electrostatic force

Mobile ions held near cations

that are covalently attached
to stationary phase

Anion exchange resin

attracts only cations
Size exclusion chromatography
Separates molecules based on size.

Larger ones passing out quickly no attractive

interactions between the stationary phase and the
solute

Mobile phase pass through the porous gel

 Large molecules
are excluded

Small molecules
penetrate pores
of particles
Affinity chromatography
Selective kind of chromatography.
Involves specific interaction between solute
molecules and stationary phase i.e antibody antigen
reactions
One kind of molecule in
complex mixture
becomes attached to
the molecule that is
Covalently Bound to
stationary phase

All other molecules

wash through
Planar chromatography

Stationary phase is a flat strip

of paper or solid coated onto
a glass plate.

Mobile phase moves through

the stationary phase by
capillary wetting
or combination of wetting
and gravity
Capillary gel electrophoresis

Separates solutes based on size and charge.

Smaller molecules will migrate further.

The capillary tubing is filled with a polymeric gel

that is porous.

Solutes migrate through the gel with velocity

determined by both their size and electrophoretic
mobility
A chromatogram
Solutes eluted from a chromatography column are
observed with various detectors and plotted
as chromatograms

Chromatogram a graph of detector response as a

function of elution time or volume and consists of a
peak for each of the separated solute bands.
Retention time tR- time needed for each component
after injection of mixture into column until the
component reaches the detector

Retention volume VR volume of mobile phase required

to elute a particular solute from the column

Un retained mobile phase travels through the column

in the minimum possible time tM
Compounds should have different retention volume
or retention time in order to be separated
Column Efficiency

 At the beginning of a chromatographic separation

the solute occupies a narrow band of finite width.
 As the solute passes through the column, the
width of the band continually increases in a
process called band broadening.
 Column efficiency provides a quantitative
measure of the extent of band broadening.
 Band broadening is the increase in a solute’s
baseline width as it moves from the point of
injection to the detector.
Theoretical plates
 A quantitative means of evaluating column
efficiency it treats the column as though it consists
of a series of small zones, or plates, in which
partitioning between the mobile and stationary
phases occurs.
 Column efficiency is the number of theoretical
plates, N, or the height of a theoretical plate, H
and is characterized by thinness or height of
theoretical plate H
It is calculated by dividing length of column by total
number of theoretical plates N H=
 Retention time tR or
 retention volume VR and the
 extrapolated base width of the peak

or
N=16() 2
N=16 ()2

Or alternatively

N=5.55()2

N=5.55()2
The larger the value of N is for a column,
 the better the column will be able to separate two
compounds that have small differences in
retention time

N is independent on solute retention time and the

length of the column
Plate height or height equivalent of a theoretical
plate (H or HETP):

Compare efficiencies of columns with different

lengths:
H = L/N
H is the length of the column that corresponds to one
theoretical plate

H can be also used to relate various chromatographic

parameters (e.g., flow rate, particle size, etc.) to
the kinetic processes that give rise to peak
broadening.
Why Do Bands Spread?
 Eddy diffusion
 Mobile phase mass transfer
 Stagnant mobile phase mass transfer
 Stationary phase mass transfer
 Longitudinal diffusion
Eddy diffusion – peak/band broadening due to the
presence of multiple flow paths through a packed
column.

As solutes travel through the column, some arrive at

the end sooner than others due to the different path
traveled around the support particles in the column
thus different travel distances.
Longer path arrives at end of column after 1
Mobile phase mass transfer –peak broadening due to
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 faster

than solute at the edges,
band-broadening due to eddy diffusion and mobile
phase mass transfer depends on:
 the size of the packing material
 the diffusion rate of the solute

Stagnant mobile phase mass transfer

band-broadening due to differences in the rate of
diffusion of the solutes between the mobile phase
outside the pores of the support (flowing mobile
phase) and 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:
 the size, shape and pore structure of the packing
material
 the diffusion and retention of the solute
 the flow-rate of the solute through the column
Stationary phase mass transfer – band-broadening
due to the movement of solute between the stagnant
phase and the stationary phase.

different solute molecules spend different lengths of

time in the stationary phase and on the column,
giving rise to band-broadening.
The degree of band-broadening due to stationary
phase mass transfer depends on:
 the retention and diffusion of the solute
 the flow-rate of the solute through the column
 the kinetics of interaction between the solute and
the stationary phase

Longitudinal diffusion – band-broadening due to

the diffusion of the solute along the length of the
column in the flowing mobile phase.
Band-broadening due to longitudinal diffusion
depends on:
 the diffusion of the solute
 the flow-rate of the solute through the column
Van Deemter equation: relates flow-rate or linear
velocity to H:

H = A + B/m + Cm
where:
m = linear velocity (flow-rate x V m/L)
H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer

One use of plate height (H) is to relate these kinetic

processes of band broadening to a parameter of
the chromatographic system (e.g., flow-rate).
Used to predict what the resulting effect would be of
varying this parameter on the overall efficiency of
the chromatographic system.
Number of theoretical plates (N) = 5.54 (t R/Wh)2
peak width (Wh)
H = L/N
 Plot of van Deemter equation shows how H
changes with the linear velocity (flow-rate) of the
mobile phase
 Optimum linear velocity (mopt) - where H has a
minimum value and the point of maximum
column efficiency:
Resolution
In chromatography resolution is a term used to
describe how well two elution bands are separated
from one another
The resolution Rs is taken as the distance between
two peaks divided by the average extrapolated base
width of the bands

RS = 2

Where tR is retention time for A and B respectively

W is the width of the extrapolated base width
N/B
A large value of RS means that the bands are well
separated

Example

For a column with 2.00 ×104 theoretical plates,

a) calculate the extrapolated base width of the elution

peak for a solute whose retention time is 3.50 min
a) for a solute whose retention is 3.0 min
Selectivity factor ability to distinguish between 2
species A and B

α ==

A always has smaller retention time

Capacity Factor
A measure of how strongly a solute is retained by
the stationary phase (k’).

Where tr’ is the adjusted retention time.

The adjusted retention time is the difference
between a solute’s retention time and column’s void
time (t‫׳‬r).

Example
In a chromatographic analysis of low-molecular-
weight acids, butyric acid elutes with a retention
time of 7.63 min. The column’s void time is 0.31
min. Calculate the capacity factor for butyric acid.
SOLUTION
In the same chromatographic analysis for low-molecular-
weight acids considered in the Example above, the retention
time for isobutyric acid is 5.98 min. What is the selectivity
factor for isobutyric acid and butyric acid?

SOLUTION
First we must calculate the capacity factor for isobutyric
acid. Using the void time from the example above, this is

The selectivity factor, therefore, is

Example: Given the following data and a 24.7 cm
column
Compound Retention Time (min) Peak Width(WB, min)

Nonretained 3.1 -

A 5.4 0.41
B 13.3 1.07
C 14.1 1.16
D 21.6 1.72

calculate the resolution between species C and D.

What column length is required for a resolution of
1.5?
Rs is preferred over α since both retention (tr) and
column efficiency (Wb) are considered in defining
peak separation.

Rs of 1.5 represents baseline resolution, or

complete separation of two neighboring solutes an
ideal case.

Rs of 1.0 considered adequate for most separations.