Dr .

Mehdi Hasan Kazmi

SEPERATION
The validity of results from chemical analysis depends initially upon the quality and integrity of the sample
that has been obtained. In the majority of cases, the sample to be analyzed will contain more than one
component and may require some separation and/or concentration to be carried out prior to analysis.
Even pure single substances used as standards rarely exceed 99.99% purity, which still leaves the
possibility of 10 / of another material being present.

SEPERATION TECHNIQUES
For this purpose, separation techniques can be considered to fall into two main groupings.

Bulk Separation
(Physical Method of Separation)
Large scale separations of one component form another, typically they include,

Filtration
Temperature dependant effects ( distillation, evaporation & drying )
Solubility effects ( solvent extraction, crystallization, and precipitation)
Ion exchange dialysis and lyophilisation.

Instrumental Separation
(Chemical Method of Separation)
The most common instrumental separation includes,
Chromatography (GC, HPLC, TLC, Supercritical Fluid chromatography SFC)
Electrophoretic group of separation techniques (Capillary Electrophoresis).

Chromatography
Chromatography is a Greek word,
Chroma means color & Graphy means to write or to analyze.
Chromatography is a way to separate single chemical compounds from mixed substances that depends on
the speed at which they move through special media, or chemical substances.
It is a powerful separation technique which was discovered by a botanist. In order to get familiar with
chromatography, some chromatography terms must be understand which are defined below.

Sample
It is the matter analyzed in chromatography. It may consist of a single component or may be a
mixture of components. When the sample is analyzed, the phase or phases contains analyte of
interests is/are referred to as sample, whereas everything out of interests separated from the
sample before or during analysis is referred to as waste.

O. A. Ansari

CHROMATOGRAPHY TERMS

Dr . Mehdi Hasan Kazmi

Analyte
The analyte is the substance that has to be removed during Chromatography.
Solute
The solute refers to the sample components in partition chromatography.
Solvent
The solvent refers to any substance capable of solubilizing other substance, and especially the
liquid mobile phase in LC.
Mobile Phase
The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC),
a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A better definition:
The mobile phase consists of the sample being separated/analyzed and the solvent that moves the
sample through the column.
The effluent is the mobile phase leaving the column.
Stationary Phase
The stationary phase is the substance which is fixed in place for the chromatography procedure.
A bonded phase is a stationary phase that is covalently bonded to the support
particles or to the inside wall of the column tubing.
An immobilized phase is a stationary phase which is immobilized on the support
particles, or on the inner wall of the column tubing.
Chromatograph
A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic
or liquid chromatographic separation.

EXPLAINATION
Suppose we have a mixture of two solutes, A and B, dissolved in a suitable buffer solution. A column is
filled with beads of an insoluble substance
that is expected to bind differentially and
reversibly to A and B. The column is initially
equilibrated by running a certain volume of
the buffer through it, and the mixture
applied to the top of the column as a
narrow layer. If the stopcock of the column
is carefully opened, the layer of solutes will

O. A. Ansari

Chromatogram
A chromatogram is the visual output of the chromatograph. In the case of an optimal separation,
different peaks or patterns on the chromatogram correspond to different components of the
separated mixture.

Dr . Mehdi Hasan Kazmi

pass into the column, and the solutes can be washed out slowly by pouring more buffer into the column.
This process is called elution, and the solution emerging at the bottom of the column is called the eluate
Let us assume, for the purpose of this illustration, that solute B binds more strongly to the matrix than
solute A. Then, as the mixture travels down the column, the molecules of B will be retarded with respect
to the molecules of A. In time, they will separate into bands, and be eluted at different times.
On the basis of phases, Chromatography can be classified as under

Chromatography

Adsorption

LSC

Partition

GSC

ADSORPTION CHROMATOGRAPHY
Stationary Phase
Mobile Phase
1.
2.
3.
4.
5.
6.

LLC

GLC

Solid

Liquid or Gas

LSC (Liquid-Solid Chromatography) or LCC.

SCC (Simple Column Chromatography).
IEC (Ion Exchange Chromatography).
HPLC (High performance Liquid Chromatography).
GC (Gel Chromatography) or Protein Analysis.
GSC (Gas-Solid Chromatography).

PARTITION CHROMATOGRAPHY
Stationary Phase

Liquid

1.
2.
3.
4.
5.
6.
7.
8.
9.

Liquid or Gas
LLC (Liquid-Liquid Chromatography) or Plane Chromatography.
PC (Paper Chromatography).
TLC (Thin-Layer Chromatography).
Continuous Zinc Electrophoresis.
GLC (Gas-Liquid Chromatography).
HPLC (High pressure/performance Liquid Chromatography).
Reverse Phase Chromatography
BPLC / BPC (Back Pressure Liquid Chromatography).
IPC (Ion-Pair Chromatography).

O. A. Ansari

Mobile Phase

Dr . Mehdi Hasan Kazmi

Stationary Phase
The stationary phase is the substance which is fixed in place for the chromatography procedure. It can
either be solid or liquid.
SOLID STATIONARY PHASE
Solid stationary phase is usually an adsorbent. The adsorbent may be classified with respect to two
different aspects.

ADSORBENTS

1
Inorganic

2
Organic

Column

Thin
Layer

Simple Column
HPLC
GPLC

Gas Plate Coated

with chemical

GSC

Difference between adsorbents used for Column and Thin Layer Chromatography
Simple Column
Adsorbent for column does not require a
binder

Thin Layer
Adsorbent for thin layer must have a
binder

It must be of large mesh size

It must be of small mesh size

Inorganic Adsorbent

Silica Gel (consists of fluorescent indicator)

Alumina
Silicates (Ca, Mg, Phosphates)

Class

Mesh Size (mm)

G40

0.04-0.

G60

0.025-0.04

G100

0.04-0.063
0.063-0.2
0.02-0.5

G60F254

0.63-0.2

O. A. Ansari

Silica Gel

Dr . Mehdi Hasan Kazmi

Alimina (Aluminium Oxide)

Activity according to Brokman Scale
Class
Moisture (%age)
I
0
II
3
III
6
IV
10
V
15
Note: Brokmnan Scale is only for Alumina not for Silica.
Medium

pH

Basic
Neutral
Acidic

Class (mesh mm)

9.5 0.2

60 (0.083-0.2)
90 (0.063-0.2)

7 0.2
4.5 0.2

90 (0.063-0.2)
90 (0.063-0.2)

Powder
Ferric & Chromic Oxides
Zinc Carbonate & Ferro Cyanide
Active Carbon
Zirconium [ phosphate, Hydrous]
Lenthanum Oxides
Combination
LIQUID STATIONARY PHASE
Acetonyl Acetone
Apiezon L, M
Bentone 34
Benzyl Cyanide + AgNO3
Polyethylene Glycol
DOP
Silicone OV-17
Silicone OV-1
Silicone OV-101

Normal Cellulose Powder.

Acetylated Cellulose Powder.
Cellulose Ion-Exchange Powder
Starch
Sucrose
Mannitol
Dextron Gells

O. A. Ansari

Organic Adsorbent

Dr . Mehdi Hasan Kazmi

Mobile Phase
The mobile phase is the phase which moves in a definite direction. On the basis of polarity and di-electric
constant we can grade mobile phase as under,
2 > > > > > > >
> > > > >
> > > > 4 > >

Simple Column Chromatography

INTRODUCTION & WORKING MECHANISM
Column chromatography separates compounds using many chemical actions between the chemical being
tested and the chromatography column (a rod with a blending of special chemicals). The column is run
using either gravity or a pump.
Column chromatography is advantageous over most other
chromatographic techniques because it can be used in both
analytical and preparative applications. Not only can column
chromatography be used to determine the number of
components of a mixture, but it can also be used to separate
and purify substantial quantities of those components for
subsequent analysis.
With all its advantages and preparative power, column chromatography does have its complications.
Properly setting up the column (something that will be done for you prior to experiment) requires some
technical skill, and takes some time. Column chromatography is less foolproof than paper chromatography
and requires constant attention while the experiment is being performed: collection vessels must be
frequently switched and solvent levels need to be topped up.
In short, running a column is time-consuming and tedious, especially for large samples. If it is unnecessary
to preparatively separate large quantities of sample, analytical methods such as paper chromatography
may be more suitable and easier to perform.

First of all select the material of column. Usually Glass or Stainless Steel is used as column
material.
Que: Why we use glass or stainless steel as the material of column?
Ans: We use glass or stainless steel because both of these materials
do not react with the solid or mobile phase.
Column is packed.
Washing is carried out.
Selection of adsorbent is done. It depends upon the sample.
For e.g. Silica with fluorescent or without fluorescent.
Before using the adsorbent, it should be first activated and different adsorbent require different
temperatures for activation.
For e.g. Silica 100oC / hr

O. A. Ansari

EXPERIMENTAL PROCEDURE

Dr . Mehdi Hasan Kazmi

Alumina 320oC / hr
The adsorbent is activated in the oven.
Adsorbent is then cooled in the desiccators.
Close the inner side of column by
Select a non-polar solvent system. For e.g. Hexane
Que: Why we use non-polar solvent i.e. hexane?
Ans: We use hexane because the particles soluble in hexane will
come out.
Fill the column with solvent to the required length, then add adsorbent.
When the column is filled half with the adsorbent, release the solvent drop wise.
The column should be packed carefully there should be no air bubbles. To remove air bubbles
completely, we pass the solvent two times.
Now introduce the sample.
Before adding the sample the surface is covered with cotton, asbestos or glass wool.
Prepare the sample in an organic or inorganic solvent system.
If the sample is organic in nature, organic solvent system can be used. For e.g. Chloroform.
The sample is prepared in a petridish & chloroform is added to the sample.
We form layers
Que: Why we form layers?
Ans: Because particles of sample must not be disturbed.
We pass non-polar solvent i.e. hexane because the particles soluble in hexane will come out.
Then we take 98% Hexane and 2% Chloroform and the particles soluble in 2% Chloroform will
come out.
and so on..
For organic sample, we use non-polar solvent.
For inorganic sample, we use polar solvent.

TERMINOLOGIES
RETENTION TIME (tR)

O. A. Ansari

The maximum time required to separate a particular constituent completely from the column is called
retention time.

Dr . Mehdi Hasan Kazmi

RETENTION VOLUME (VR)

The total volume of a particular constituent (solute) obtained from the column is called retention volume.

O. A. Ansari

Additional Information
The retention time of a solute is taken as the elapsed time between the time of
injection of a solute and the time of elution of the peak maximum of that solute. It is a
unique characteristic of the solute and can be used for identification purposes.
The corrected retention time of a solute is the retention time minus the retention time
of a completely unretained solute. By multiplying the corrected retention time of a
solute by the exit flow rate then the corrected retention volume can be obtained. If the
mobile phase is compressible (i.e. the mobile phase is a gas) a pressure correction must
be applied which is a function of the column inlet-outlet pressure ratio. Values of the
corrected retention volume per ml of stationary phase for a solute measured over a
range of temperatures can provide the standard energy of distribution, the standard
enthalpy of distribution and the standard entropy of distribution for the solute
concerned.