CHROMATOGRAPHIC TECHNIQUES

Aim: Separation and purification of biological molecules from a mixture of

molecules and investigation of their properties.

They are used for the separation of - large amounts (several grams)
- small amounts (micro- or picogram) of materials

Selection of a particular chromatographic method depends on the nature of material

to be isolated. Several chromatographic methods can be used sequentially to
complete purification.

Principle: In all chromatographic systems, there are two phases.

1- Stainary phase
2- Mobile phase
Separation is due to distribution of molecules between two phases.

Partition (distribution coefficient) = is used to describe the distribution of any

compound between two phases.

- between two immiscible liquids (two solvents)

concentration in Solvent A

concentration in Solvent B

- between two phases : solid / liquid

Stationary Mobile

Example: If a substance X has a distribution coefficient of 0.5 in silicic acid and /

benzene; 0.5 = 1/2, This means that [X] in benzene is twice of that
in the silicic acid.
Distribution coefficient is defined for chromatographic purposes as:

concentration of substance in mobile phase

concentration of substance in stationary phase

Stationary phase: may be - solid

- liquid
- solid/liquid mixture (semi-solid)
- gel
and it is immobilized.

Mobile phase: may be - liquid or

- gaseous
and flows through stationary phase.

Example to chromatographic systems with different stationary and mobile phases

Stationary Mobile
Adsorption chromatography solid liquid
Paper chromatography semi-solid liquid
Gas chromatography liquid gaseous
Ion-exchange chromatography solid resin liquid
Gel filtration chromatography solid resin liquid
Affinity chromatography solid resin liquid

The mobile and stationary phases are chosen according to the compound to be
separated.
 General Techniques of Chromatography

There are three forms of chromatographic separations.

1) Column chromatography: Stationary phase is packed into glass or metal
columns.

2) Thin Layer Chromatography: Stationary phase is thinly coated into glass or

plastic surfaces. Stationary phase - thin layer adsorbent

glass

3) Paper Chromatography: Stationary phase is supported by the cellulose fibres

(with H2O) of a paper sheet.

Column Chromatography
All of the major type of chromatographies are carried out using columns:
Adsorption, partition, ion exchange, size exclusion, affinity, gas, high
pressure liquid chromatographies.
Columns: Glass columns are commonly used. Columns should have a support for
the stationary phase.

use of porous glass plate

replaceable nylon net
plug of glass wool
 (Buffer)

A capillary tubing for leading

effluent from the column to the UV-
Visible monitor or collection system

Packing of column: Packing of column is carried out by pouring of slurry of the

stationary phase (adsorbent, resin or gel) into a column. Column outlet must be
closed.
Note: The resin must be equilibrated with the buffer to be used before packing.
Powdered solid particles (resin) were suspended or swelled in buffer.
A layer of solvent should always be maintained above the column surface - do not
dry gel.
Equilibration of column: After packing the column, it is equilibrated with buffer.
Sample application:
Sample is applied to the top of column by -pipette
- capillary tubing (small sample volume)
- peristaltic pump (large sample volume)
Before application of sample - remove solvent above the resin by suction.
Column development (Elution):
The components of the applied sample are separated by continuous passage of
suitable mobile phase through the column.

Rate of flow: is adjusted or maintained constant by - peristaltic pump or

- Mariotte flask

keep the operating pressure constant

Peristaltic
pump

Types of elution:
- use single solvent as the eluant
- Gradient elutions, stepwise or continuous, done by changing the
Δ pH
Δ µ (ionic concentration)
Δ polarity
Ex: two solvents having different µ are mixed in the correct proportions.
Buffer+ 0.8 M KCl Buffer (noKCl)

0.8 M--
S2 S1
KCl conc.

(µ) mix

to column 0

If two chambers have the same dimensions (same volume) called linear gradient.
Fraction collection and analysis:
The eluted fractions are collected in separate tubes using an apparatus called
fraction collector.
Works in time
drop or
volume mode

For continuous monitoring of eluted fractions, UV-Visible monitors are used.

Simple photomers

Ex: Proteins absorb UV light at 280 nm.

B
A

C
Absorbance
at λmax

number of tubes (ml of eluant)

Recorder

Eluants collected in separate tubes using fraction collector. Then each tube is
analyzed for the presence of a desired protein. Ex: total protein
determination and determination of activity of protein due to specific
biological function.

So find that in which tubes (peak or fraction) the protein under interest is eluted.

TLC
It is used for small amount of materials.

Preparation: thin layer adsorbent (0.2-0.5 mm or 5 mm)

glass or plastic

- A slurry of the stationary phase in H2O is applied and coated to a glass, plastic or
foil plate as a uniform thin layer.
- a uniform layer obtained by using a plate spreader starting at one end of
the plate and moving to the other end.
-thickness of the slurry (stationary phase) - 0.25 mm for analytical separations
- 5 mm for preparative separations
- These plates are available commercially.

- Then plates are dried in oven at 100-120°C. This process activates adsorbant
Alumina
Silica adsorbants

Sample Application:
i) Sample is usually low MW molecules
(a.acids, nucleotides, fatty acids, sugars)

solvent front

ii) Sample is applied to the plate by using

a micropipette or syringe.
pipette
iii) Sample is generally applied 2.0-2.5 cm
o 2-2.5 cm from one edge of the plate.
1 2 3 (for analytical separations spot application
for preparative separations band application)
dots streak
(spot) (band)
Development of plate:
 Tank

Slurry (stationary phase)

glass plate

sample
buffer or solvent mixture (mobile phase)

- Separation done in a glass tank which contains the developing solvent (mobile
phase) to a depth of about 1.5 cm.
- Tank is covered by a glass plate for equilibration with solvent vapor.
- After equilibration, thin layer plate is placed vertically in the tank and covered
again.
- Separation of compounds occurs as the solvents travel up the plate. Polar and non-
polar molecules are separated. Q. How? Principle of separation?
- Development time depends on the solvents - 10-60 minutes
- One of the advantage of TLC, it has a short time for separation.

Two dimensional TLC

Aim: to improve resolution
 Direction of second
development
Direction of Solvent
first development system 2 D
90° rotation
B C
A
A
o Origin
o Origin

Solvent system 1

The sample is applied to one corner of a plate as a single spot and plate developed in
one direction in solvent system 1.
Then plate is removed from the tank and dried.
It is developed by another solvent system (system 2) in a direction at right angle to
the first development.
The spot B is further separated into C and D in solvent system 2.

Component Detection:
Qualitative detection methods:
1) Spraying the plate with 50% H2SO4 or 25% H2SO4 in Ethanol
then heating (Δ) burning of compounds and brown spots appear.
2) Examination of the plate under UV light shows the position of UV absorbing
or fluorescent compounds. (use of UV lamp) aromatic compounds
3) Coloured compounds seen with naked eye.
4) Subject the plate to iodine vapor if unsaturated compounds are to be examined.
I
I2|
C=C C-C
|
I
Yellow
5) Spraying the plate with specific colour reagents to develop color. Ex; ninhydrin
for amino acids purple color
6) Autoradiography: used for detection of radioactive compounds. Technique
detects the spots as dark areas on X-ray films.

Quantitative Determination:
The amount of compound may be determined by:
i) On-plate quantification: Quantification of spots still on the plate by
- densitometers: measure the UV or visible absorption of the compound.
- radiochromatogram: used for radio-labelled compounds.

ii) Off-plate quantification:

- It is carried out by cutting off the spot from the plate (plastic).
- then eluting the compound with a suitable solvent.

Samp
cut off le
solvent to release the
compound from adsorbent

O Elution

Detection or quantitation by
----------------------------------------------------- colorimetry

Paper Chromatography
Cellulose fibres of paper act as stationary phase (supporting matrix)
 may be - polar water
- nonpolar material (liquid paraffin)

mobile phase: buffer or organic solvents

Paper Development:
There are two techniques: - ascending
- descending

trough with solvent

Paper o

Glass tank
Sample
application o

Solvent ascending
descending

in both cases, the solvent is placed in the base of glass tank

- Equilibration with solvent vapor
- In ascending technique: - one end of the paper is in contact with the solvent in the
base of tank.
- The sample spots should be just above the surface of the solvent
- Movement of solvent is only due to the capillary action and as the solvent
moves vertically up the paper, separation of sample is
achieved.
- In descending technique: - The paper is allowed to hang vertically in tank. Sample
is on top of the paper and top of the paper is in contact with the solvent in
trough and wetted. The bottom of paper is not in contact with the solvent in
the base of the tank.
 - Movement of solvent downwards is due to gravity and capillary action, so
as the solvent moves down the paper, separation of sample is
achieved.
- Flow of solvent is faster than the ascending technique.
- Resolution is low.

Ascending paper chromatography

Two dimensional chromatography was also used for paper systems similar to TLC.

Q. What is the basis of separation by paper chromatography?

Detection and Identification:

Similar to TLC but H2SO4 is not used. It disintegrates the paper.

Further identification of a compound made on the basis of the Rf value.

Solvent front

the distance moved by

standard
solute Rf =
B the distance moved
by solvent front
A
Rf = A / B
A 1 2 3 S

1 2 3 S is constant for a particular compound

under standard conditions
Solvent: mixture of various liquids
Ex: n-butanol / acetic acid / H2O with a ratio of 50 / 15 / 35
Rf ≤ 1.0