Introduction to Chromatography

Contributed by:
Jonathan James
The highlights are:
1. Definition
2. Terms in chromatography
3. Classification
4. Retardation factor
1. Chromatography
 Introduction

Chromatography means color writing it is
used in many scientific fields, used in
many subjects and applied in all
analytical fields.
 Definition
Chromatography is a method of analysis
and separation technique of organic and
inorganic compound. it is used for large
and small quantities so it is used
quantitatively and qualitatively and proved
to be more effective from the other means
of separation and identification.
2. The separation of mixture of compound in
chromatography to it is components depend on
:the action of two forces
 1) Mobile force (driving force) that will try to move
the components of mixture.
 2) stationary or retardation force that will try to
keep components in it is place depending on many
 a) Solubility in mobile phase.
 b) Adsorption ability of component to be separated.
 c) Ionic forces.
3. There are several important terms in
 1) Stationary phase: also called column, adsorbent,
bed, sorbent, opposing force, and retardation force.
 2) Mobile phase: also called solvent, effluent, and
 3) Chromatographic system: means the whole
conditions of chromatography e.g. stationary phase,
mobile phase, temperature, dimension, method of
 4) Developing: means separating mechanism (addition
of mobile phase that cause separation).
 5) Chromatogram: the result of the separation
4.  There are different types of chromatography:
 A) According to mobile phase (modern type of
 1) Liquid chromatography.
 2) Gas chromatography.
 B) According to the mechanism of separation
(classical type)
 1) Adsorption chromatography.
 2) Partition chromatography.
 3) ion-exchange chromatography.
 4) Gel filtration
 5) Affinity chromatography.
 6) Electrophoresis
 In this case the stationary phase is solid stationary phase (cellulose paper)
composed from cellulose which is a carbohydrate consists of about 1000
molecules of anhydrous glucose units connected by oxygen groups forming
several chains, these chains links with each other forming cellulose net work.
 The mobile phase either single liquid or mixture of liquids and the
mechanism of separation depending mainly on partition in which the
stationary phase is the hydration shell of cellulose fibers.
6. The separation of component of a mixture in
partition chromatography dependent on
differences in partition coefficient of the
component between an aqueous and an
immiscible organic liquid, the aqueous phase
is usually the stationary phase. Strips of
filter paper is used and as the solvent moves
the component also move along the paper at
varying rates, depending mainly on the
differences in their partition coefficient
.between the aqueous and organic phase
7. There are several developing
technique in the paper
 1) Ascending development
 In this technique the paper is
suspended vertically with about 1-2
cm above the surface of the solvent to
prevent diffusion of the sample
downward into the solvent and the
developing by capillary action
8. Descending development)2
 In this technique the solvent
adding in the top of the tank
(jar) and the filter paper is
folded into U- shape and the
developing into down
9. Horizontal development)3
 In this technique the sample spotted around the
center of horizontally filter paper with insert the
wick in the center
 The solvent move and separated the sample
along radial path
10. Two dimensional)4
 1) Chemical detection by using chemical
 2) Physical detection by using UV light.
 3) Radioactive method: specific
detection procedures when use to detect
separated compound having some
radioactivity or labeled compounds.
 4) Biological methods by using certain
microorganisms and are especially used
for the detection of antibiotics.
11. Retardation factor (RF)
 Retardation factor can be defined as the
distance moved or traveled by the compound
to the distance moved by the solvent and it is
constant for each compound when
chromatography is carried out using the same
technique, mobile phase, and the same
conditions. Usually the Rf value is used for the
identification of the separated compound by
comparison with the Rf value of a standard.
 distance moved by sample
 Rf value =
 distance moved by solvent
Circular filter paper chromatography
(Horizontal paper chromatography)
 1) Prepare a circular filter paper and insert a
wick in the center of the paper.
 Mark four pencil dots (starting points)
approximately 1cm from the wick.
 2) Apply the sample on pencil dot (3different
magic colors and ink).
 3) Place a chromatographic paper over the dish
that contains the mobile phase in such a way
that develops to about 4-5 cm
 4) Remove the chromatogram, make the
solvent front and dry at room temperature.
 5) Examine the chromatogram by the daylight
and calculate the Rf value for each separated