INTRODUCTION TO

CHROMATOGRAPHIC
SEPARATIONS

What is
chromatography?
Chromatography is a powerful separation
method that is usually composed of mobile
phase and a stationary phase.
This method is used to separate and identify
the components of complex mixtures.
Works by allowing the molecules present in
the mixture to distribute themselves between
a stationary and a mobile phase to varying
degrees.

 Those components that are strongly

retained by the stationary phase move
slowly with the flow of mobile phase.
In contrast, components that are weakly
held by the stationary phase travel rapidly.
As a consequence of these differences in
mobility, sample components separate into
discrete bands that can be analyzed
qualitatively and/or quantitatively.

Classification of Chromatographic
Methods
Can be categorized based on the
followings:
1. Based on physical means
2. Based on the types of mobile phase
3. Based on the kinds of equilibria involved
in the in solute transfer between the
phases

Classification of Chromatographic
Methods
Column chromatography
stationary phase is held in
narrow tube;
mobile phase moves by
pressure or gravity
E.g. - gas chromatography (GC)
- supercritical-fluid
chromatography (SFC)

Planar chromatography
stationary phase is supported
on a flat plate or in the
interstices of a paper;
mobile phase moves through
capillary action or gravity
E.g. - thin-layer chromatography (TLC)
- paper chromatography (PC)

* based upon physical means

Column chromatography can be further

differentiated based on the types of mobile phases
and the kinds of equilibria involved in solute transfer
between the phases
Mobile Phase
i) Gas

iii) Supercritical fluid

Gas Chromatography

Supercritical-fluid
Chromatography
ii) Liquid
Liquid Chromatography

Classification based on the kinds of

equilibria involved in solute transfer
between the phases
Adsorption - for polar non-ionic compounds
Ion Exchange - for ionic compounds

Anion - analyte is anion; bonded phase has positive charge

Cation analyte is cation; bonded phase has negative
charge

Partition - based on the relative solubility of analyte

in mobile and stationary phases

Normal stationary phase polar, the mobile phase nonpolar

Reverse stationary phase nonpolar, the mobile phase polar

Size Exclusion - stationary phase is a porous matrix;

sieving

Classification of Chromatographic
Methods
Chromatography

Partition

Liquid-liquid

Adsorption

Gas-liquid

Liquid-solid

Gas-solid

Ion-exchange

Liquid-solid

Size-exclusion

Liquid-solid

Partition Chromatography

 Partition chromatography
accomplished by selective & continuous
transfer of the components of the mixture
back & forth btw a liquid stationary phase and
a liquid mobile phase as the mobile phase liquid
passes through the stationary phase liquid
Stationary phase: liquid
Mobile phase: liquid or gas

 Partitioning

distribution (by dissolving) of the components

btw 2 immiscible phases:

relative solubilities of the components in the mobile

and stationary phase

e.g. stationary phase polar

Polar components will retain longer than the non-polar

components,
Non-polar components will move quickly through
stationary phase & will elute first before the polar
components, and vice-versa

 Partition chromatography
The stationary phase actually consists of a thin
film adsorbed (stuck) on or chemically bonded to
the surface of a finely divided solid particles

 Partition chromatography
If the mobile phase is gas, the volatility (vapor
pressure) and solubility in stationary phase
plays an important role.

Adsorption Chromatography

 Adsorption (Affinity) chromatography

Components of the mixture selectively adsorb
(stick) on the surface of a finely divided solid
stationary phase
As mobile phase (gas/liquid) carries the mixture
through the stationary phase, the components
of the mixture stick to the surface of it with
varying degrees of strength & thus separate
Stationary phase: solid
Mobile phase: gas or liquid

Ion-exchange Chromatography

 Ion-exchange chromatography
Method for separating mixture of ions
Sample: aqueous solution of inorganic ions /
organic ions
Stationary phase small polymer resin beads
usually packed in a glass tube

These beads have ionic bonding sites on their

surfaces which selectively exchange ions with certain
mobile phase compositions as the mobile phase
penetrates through it

 Ion-exchange chromatography
Ions that bond to the charged site on the resin
bead are separated from ions that do not
Repeated changing of the mobile phase
composition
The usual procedure is to initially use a mobile
phase with all the ions in the mixture bond &
then to change the mobile phase in a stepwise
fashion so that one kind of ion at a time is
removed
Done until complete separation achieved

Size-exclusion
Chromatography

 Size-exclusion chromatography
Also called gel permeation chromatography
Technique for separating dissolved species on
the basis of their size
Stationary phase: porous polymer resin
particles (molecular sieves)
The components to be separated enter the
pores of these particles & are slowed from
progressing through this stationary phase

 Size-exclusion chromatography
Separation depends on the sizes of the pores
relative to the sizes of the molecules to be
separated
Small particles are retarded to a greater extent
than large particles (some of which may not
enter the pores at all) & separation occurs

Terminologies in chromatography

 Terminologies in chromatography
Elution: a process in which species are washed
through a chromatographic column by addition of fresh
solvent
Mobile phase: is one that moves over or through an
immobilized phase that is fixed in place
in a column or
on the surface of flat plate
Stationary phase: a solid or liquid that is fixed in place. A
mobile phase then passes over or
through the stat.
phase
Retention time: is the time interval btw its injection onto
a column and the appearance of its peak
at the other
end of the column

Migration Rates of Solutes

Distribution constant, K
Retention time, tR
Capacity factor, k
Selectivity factor,

Distribution constant, K
In chromatography, the distribution equilibrium of analytes
between the mobile and stationary phases can often be
described quite simple.
Let say, we have analyte A. The distribution equilibrium is
written as:

Therefore, the equilibrium constant K is called distribution

constant and is defined as:
K is also called
partition coefficient
or partition ratio
C - Molar concentration of solutes

Retention Time, tR
Time required for the sample to travel from
the injection port through the column to the
detector.
A

A typical chromatogram for a two-component mixture.

The small peak on the left represents a species that is
not retained on the column & so reaches the detector
almost immediately after elution is started.

tM

- time taken for the unretained species to

reach the detector.
sometimes called dead time
Rate of migration of the unretained
species is SAME as the average rate of
motion of mobile phase molecules.
So, tM can be expressed as the time
required for an average molecule of the
mobile phase to pass through the column.

Retention Factor (Capacity

factor), k
term used to measure the migration rates of
analytes on columns.

Selectivity factor,
is defined as:

distribution constants

A measure of the relative migration rates of

species A and B with a stationary phase material
in chromatography.

Column Efficiency
Two related terms widely used as quantitative
measures of chromatographic column efficiency:
i) Plate height, H
ii) Number of theoretical plates, N

 The relationship btw H and N is:

Column length

Number of
theoretical plates
Plate height

The efficiency of chromatographic columns increases as

the number of plates becomes greater and plate height
become smaller.

 Experimentally, H and N can be approximated

from the width of the base of the
chromatographic peak.
The equation:

N can be calculated using tR and W.

To obtain H, the length of the
column must be known.

 Another method for approximating N is to

determine W1/2, the width of the peak at half its
maximum height.

N = 5.54

tR
W1/2

Resolution, Rs
a measure of the separation of two
chromatographic peaks.
Baseline resolution is achieved when Rs = 1.5

Rs values of less than 1.0 are considered unresolved peaks.

Effect of Capacity Factor &

Selectivity Factor on Resolution
Relationship btw the resolution of a column and
the capacity factor k, selectivity factor and the
number of plates N is given by this equation:

Rs = N
4

k
1 + k

Effect of Resolution on
Retention Time
Relationship btw the resolution of a column and
retention time:
tR = 16Rs H
u
2

1 + k
(k)2

Example
17.63 min

16.40 min

1.30 min

Length of column: 30 cm
Peak widths (at base) for A & B were 1.11 & 1.21 min respectively.
Calculate:
i) column resolution, Rs
ii) the average number of plates, N
iii) the plate height, H
iv) length of column to achieve Rs 1.5

i)

Rs = 2 (17.63 min 16.40 min)

(1.11 min + 1.21 min)
= 1.06

ii)

N = 16

16.40 min

= 3.49 x 103

1.11 min

N = 16

Therefore, calculate
the N average

17.63 min
1.21 min

Nave = 3.44 x 103

= 3.40 x 103

iii) H = L / N
= 30 cm
3.44 x 103
iv) (Rs)1

(Rs)2
1.06
1.5

= 8.7 x 10-3 cm

3.44 x 103
N 2
N 2 = 6.9 x 103

1 refer to original column

2 refer to new column

L=N/H
= 6.9 x 103 x 8.7 x 10-3
= 60 cm

Applications of
Chromatography
Qualitative analysis
Quantitative analysis

Qualitative analysis
Based on retention time
Provided the sample produce the peak at the
same retention time as a standard under
identical conditions

Quantitative analysis
Analysis based on Peak Height
The height of 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.

Analysis based on Peak Area

Peak areas are usually the preferred method of
quantitation since peak areas are independent of
broadening effects.
Most modern chromatographic instruments are equipped
with computer or digital electronic integrator that permit
precise estimation of peak areas.

 Calibration method
(also known as external method)
Involve preparation of series of standard solutions
that approximate the composition of the unknown.
The peak heights or areas are plotted as a
function of concentration.
The concentration of the component(s) to be
analysed is determined by comparing the
response(s) (peak(s)) obtained with the standard
solutions.

 Internal Standard Method

Equal amounts of an internal standard substance is
introduced into each standard and sample.
The internal standard should not react with the
substance to be examined; it must be stable and must not
contain impurities with a retention time similar to that of
the substance to be examined.
The concentration of the substance to be examined is
determined by comparing the ratio of the peak areas (or
heights) due to the substance to be examined and the
internal standard in the test solution with the ratio of
the peak areas (or heights) due to the substance to be
examined and the internal standard in the standard
solution.

 Area Normalization Method

In the normalization method, the areas of all
eluted peaks are computed.
The percentage content of one or more
components of the substance to be examined is
calculated by determining the area of the peak(s)
as a percentage of the total area of all the peaks,
excluding those due to solvents or any added
reagents and those below the disregard limit.
Refer example 26-2(page 784)