CHROMATOGRAPHY :

Principles and Practice

Chromatography is a technique for

separating mixtures of compounds
identifying unknown compounds
establishing the purity or concentration of
compounds
monitoring product formation in the
pharmaceutical and biotechnology industries
Selective

distribution of the component of a

mixture between two immiscible phases in
intimate contact with each other forms the basis of
separation in any chromatographic technique
.These phases are
(a) Stationary phase - Column packing material
(b) Mobile phase Solvent

Principle :
A

mixture
of
various
chromatographic process

component

enters

Different

components are flushed through the system at

different rates.
These

differential rates of migration as the mixtures

moves over adsorptive materials provide separation.
Repeated

sorption/desorption acts that take place during

the movement of sample over the stationary phase
determine the rates.
The

smaller the affinity a molecule has for the stationary

phase , the shorter the time spend in a column.

Basic operation :

1. Feed Injection
A small volume of the sample of a mixture of
components is applied to the column followed by
elution with a chosen mobile phase.

2. Separation in the Column

As the sample flows through the column, its
different
components will adsorb to the
stationary phase to varying degrees.
Those with strong attraction to the support move
more slowly than those with weak attraction. This
is how the components are separated.

3. Elution from the Column

After the sample is flushed in the stationary phase, the
different components will elute from the column at
different times.
The components with the least affinity for the stationary
phase (the most weakly adsorbed) will elute first, while
those with the greatest affinity for the stationary phase
(the most strongly adsorbed) will elute last.

4. Detection
The different components are collected as they emerge
from the column.
A detector examine the emerging stream by measuring
a property which is related to concentration and
characteristic of chemical composition. For example, the
refractive index or ultra-violet absorbance is measured.

Basic

operation :

Classification

BPC-bonded phase chromatography

GLC-gas liquid chromatography
GSC-gas solid chromatography
LSC-liquid solid chromatography
LLC-liquid liquid chromatography
PC-paper chromatography
TLC-thin layer chromatography

Chromatographic terms and parameters :

Partition coefficient :
It describes the way in which a compound or solute, which
enters a chromatographic system, immediately distributes
itself between the stationary and the mobile phase.
It is denoted by K.
K=Cs/Cm
Where Cs & Cm are the molar conc. of the solute in the
stationary and mobile phase.

Chromatographic terms and parameters :

Retention time :
It is defined as the time taken by the solute to reach the
detector from the moment of its injection into the column .
Retention time depends on the flow rate of the mobile phase
F= V*(p/tm)
where V is the product of cross-sectional area of the
cylindrical column and length, L of the column, p porosity of
the stationary phase and tm refers to the time required by a
molecule of the mobile phase to pass through the column.
Thus tm= L/u where u is the mobile phase velocity
Corrected retention time tr= tR -tm

Retention volume :
It, VR may be defined as the volume of the mobile phase
required to transport a solute from the point of its injection
into the column and its passage through the column to the
detector.
VR = tR * F

Capacity factor :
It is a measure of the retention of a solute component. It is
defined as the ratio of the total amount of the solute present
in the stationary phase to that in mobile phase.
k` = Cs Vs / Cm Vm

Column chromatography :
The stationary phase is held in a narrow tube through
which the mobile phase is forced either by pressure or by
gravity.
It is further differentiated based
On the mobile phase.
1- mobile phase (gas)
gas liquid chromatography
gas solid chromatography
2- mobile phase(liquid)
Liquid gas chromatography
liquid liquid chromatography
Bonded phase chromatography

Gas liquid chromatography

It

is based on a partition equilibrium of analyte

between a solid stationary phase and a mobile
gas . The stationary phase is adhered to the inside
of a small-diameter glass tube
(a capillary
column) or a solid matrix inside a larger metal tube
(a packed column).
Note: Though the high temperatures used in GC
make it unsuitable for high molecular weight
biopolymers or proteins.
It

is widely used in analytical chemistry.

Gas solid chromatography

Mobile phase is a gas (usually helium or

nitrogen) and the stationary phase is a suitable
adsorbent such as silica gel, alumina or carbon .
The technique is mostly used for the separation

of the permanent gases or the low molecular

weight hydrocarbons.

Liquid solid chromatography

Mobile phase is liquid and the stationary phase

is solid such as silica or alumina.

The analytes that are in the mobile phase and
affinity for the stationary phase will be adsorbed
onto it and those that do not will pass through
having shorter retention times.

liquid liquid chromatography

mobile phase is a liquid (usually a solvent or a

simple binary solvent mixture) and the stationary
phase is also a liquid (which must be immiscible
and insoluble in the liquid mobile phase).
The

liquid stationary phase is supported on some

suitable material such as a diatomaceous earth or
under certain circumstances silica gel.

Bonded phase chromatography

In liquid chromatography, liquid-liquid systems
are unstable as, however small the solubility of the
stationary phase may be in the mobile phase, the
stationary liquid phase will be eventually stripped
from the column.
It is therefore found necessary to chemically
attach the stationary phase to the support to
ensure a stable system and these materials are
called bonded phases.

type of high-pressure liquid chromatography

which employs a stable, chemically bonded
stationary phase.

Planer chromatography

Planar chromatography is a separation technique in

which the stationary phase is present as or on a plane.
The plane can be a paper, serving as such or
impregnated by a substance as the stationary bed or
a layer of solid particles spread on a support such as a
glass plate .
Different compounds in the sample mixture travel
different distances according to how strongly they
interact with the stationary phase as compared to the
mobile phase.
It is divided in two typePaper chromatography
Thin layer chromatography

Paper chromatography
In

paper chromatography, substances are distributed

between a stationary phase and a mobile phase.
The stationary phase is usually a piece of high
quality filter paper.
The mobile phase is a developing solution that
travels up the stationary phase, carrying the samples
with it.
Components of the sample will separate readily
according to how strongly they adsorb on the
stationary phase versus how readily they dissolve in
the mobile phase.
Paper chromatography is a useful technique because
it is relatively quick and requires small quantities of
material.

Thin layer chromatography

It

is a widely employed laboratory technique and

is similar to paper chromatography.
Instead of using a stationary phase of paper, it
involves a stationary phase of a thin layer of
adsorbent like silica gel, alumina, or cellulose on a
flat, inert substrate.
Application of TLC
analysing of fatty acids (chemical or biosource)
detection of pesticides or insecticides in food and
water
analysing the dye composition of fibers in
forensics

Practice of chromatography
stationary phase
It should be water insoluble, physically
rigid to withstand the operation stress,
permeable and microporous , chemically
stable.
It should have minimum non-specific
adsorption characteristics.
The material should be reusable and
preferably be of low cost.
Ex.- ion exchange resins , size exclusion gels ,
reverse phase

Mobile phase
It includes pure solvent, binary and ternary mixtures of

solvent , buffer and salt solution.

It should be compatible with the chosen stationary
phase and chemically stable with it.
The sample should be readily soluble with the mobile
phase.

Column
It

should be made of glass, acrylic plastic and

polypropylene and of steel in the case of larger scale
operation.
The column material should be chemically inert ,
pressure and solvent resistant and preferably transparent.
Most laboratory columns have 5-25 mm inner dia. and
length of 32-75 cm.

Case study
1-Determination of pharmaceuticals in biosolids
using accelerated solvent extraction and liquid
chromatography/ mass spectrometry
The

experimental steps consisted of accelerated solvent

extraction of freeze-dry biosolid sample, and cleanup by
solid-phase extraction followed by simultaneous analysis by
liquid chromatography/tandem mass spectrometry.
The analytes were separated using a Phenomenex Luna
C18column.
This study describes an efficient and reproducible analytical
approach for simultaneous determination of multiple classes
of pharmaceuticals in biosolids.
reference : Journal of chromatography , volume 1218 ,
issue 1 , 7
january 2011 , pages 10-16

2-The Determination of Plasma Ethanol

by Gas Liquid Chromatography using
Trichloracetic acid as the Protein
Precipitant .

Discussion
In

a recent report whole blood was diluted 1: 1

with aqueous isobutanol and injected directly onto
the column.
TCA has previously been used as a precipitating
agent
however it has not been generally
accepted because of the suggested possibility of
alcohol oxidation in the
acid environment .
TCA being volatile gives a peak on Chromosorb
102, but does not affect the separation of
volatile organic compounds. Because of the
possible variable thermal breakdown of TCA to
chloroform during chromatography, TCA itself
cannot be used as an internal standard. Chloroform
and TCA do not separate on this column under the
conditions used for ethanol estimations.