Dr.S. H. Burungale

1. Extraction Chromatography
Dr. S. H. Burungale

2. CONTENTS
Introduction to chromatography
Definition of Chromatography
Types of column chromatography
Theory of chromatography
Practical considerations in column
chromatography
Factors affecting efficiency of a column
Applications

3. INTRODUCTION
Chromatography designates the generic name collectively
assigned to host divergent separation techniques that have been
duly recognized right from the early 1900s till date.
Mikhail Tswett , a Russian Botanist first and foremost coined
the terminology “Chromatography”
In 1906 he performed investigations of plant pigments, by using
adsorption chromatography and has successful separated by using
leaf pigments
The term “Chromatography" emerged from Greek words :
‘Chroma’ means Colour and ‘Graphein’ means ‘to write’.

4. DEFINITION
Chromatography is a technique employed for
separation of the components of mixture by
continuous distribution of the components between
two phases.
Ettre(1993) vehemently recommended the
IUPAC definition of chromatography which
defines it as ‘A physical technique of separation
where in the components required to be separated
between the two phases , one of which being
‘stationary’ (stationary phase), while the other
(mobile phase) that moves in a definite direction’

9. COLUMN ADSORPTION CHROMATOGRAPHY
The principle involved in this technique is Adsorption.
When a mixture of compounds (adsorbate) dissolved in the
mobile phase (eluent) moves through a column of stationary
phase (adsorbent) they travel according to the relative
affinities towards stationary phase.
The compound which has more affinity towards stationary
phase travels slower and the compound which has lesser
affinity towards stationary phase travels faster.
In this way, the compounds are separated.

11. COLUMN PARTITION CHROMATOGRAPHY
The principle involved in this technique is partition. When two
immiscible liquids are present, a mixture of solutes will be distributed
according to their partition co-efficients.
When the mixture of compounds dissolved in the mobile phase and
passed through a column of liquid stationary phase, the component
which is more soluble in stationary phase travels slower and the
component that is more soluble in mobile phase travels faster.
The stationary phase used cannot be a liquid. So that a solid support is
used over which a thin film or coating of a liquid is made which acts as a
stationary phase.

16. STATIONARY PHASE
(ADSORBENT)
A good number of solid compounds belonging to
either ‘organic’ or ‘inorganic’ domain are being
extensively employed as adsorbents in column
chromatography.
Examples: 1. Organic substances: Carbon, Starch ,
Cellulose 2. Inorganic substances: Alumina, Silica gel,
Fuller’s earth, Kiesulgur

17. 17
Silica gel
Alumina
Cellulose
Kieselguhr
Silica Gel: It is slightly acidic in nature. The silanol gps (Si-OH) of silica
gel represent adsorption active surface centers which are capable to
interact with solute molecules. The ability of the silanol gps. to react
chemically with appropriate reagents is used for controlled surface
modification
Structure of Silica Gel
Si-OH Silanol Group; Si-O-Si Siloxane Group
Silanol Groups represent adsorption- active surface centre

18. Theoretical Plate
LC - liquid chromatography
packed columns
Silica
beads
Stationary
phase
liquid (mobile phase)

19. Ionic Recognition Extractant based resin
– Analyte retention related to charge and ionic radii Neutral and
Anionic Extractant based resin
– Analytes are directly hydrogen ion dependant, although
anionic extractant based resins tend to show reduced
analyte uptake as the acid competes
Acidic Extractant based resin
– Analyte retention is inverse hydrogen ion dependant
Anionic Extractant

20. General requirements:
Particle size and geometry: uniform size& spherical
shape. (60-200μ)
High mechanical stability
Inert and should not react with the solute or other
compounds
Insoluble in the solvents or mobile phases used
It should allow free flow of mobile phase
Useful for separating a wide variety of compounds
Freely available and inexpensive.

21. Synthesis of Stationary phase
The chromatographic Column was made of a borosilicate glass
tube with 8mm bore , 30cm length and filled glass wool at the
bottom.
0.1M TOPO solution was prepared by dissolving 1.94 g of
reagent in 100 mL of toluene . Silica gel (60 to 120 Mesh ) was
dried at 120 0c for 2 to 3 hours. And stored in a desiccators.
Some of the Silica gel was packed in a U tube through which
was passed a stream of dry nitrogen gas bubbled through
containing a 20 ml Dimethyl dichloro silane DMCS. The passaes
of DMCS vapour was contuned for 3 to 4 H. The silica gel was
then washed with anhydrous methanol and dried.

22. A 5g portion of the silaned silica gel was soaked in a 5 ml
of 0.1 M TOPO in toluene
For 24 h. The solvent was then evaporated till the gel was
nearly dry A slurry of coated silica gel in distilled water
was prepared by centrifugation at 2000 rpm and l lastly
coated silica gel was packed into chromatographic column
to give a height of 6 cm. the bed was covered with glass
wool.

53. 1. Chromatography is a physical method that is used to separate and
analyse __________
a) Simple mixtures
b) Complex mixtures
c) Viscous mixtures
d) Metals
Answer: b
Explanation: Chromatography is a physical method that is used to
separate complex mixtures. The mixture of different components is
flushed through the system at different rates.
2. In which type of chromatography, the stationary phase held in a
narrow tube and the mobile phase is forced through it under
pressure?
a) Column chromatography
b) Planar chromatography
c) Liquid chromatography
d) Gas chromatography

54. Answer: a
Explanation: In Column chromatography, the stationary phase held in a
narrow tube and the mobile phase is forced through it under pressure. It
is carried out in a long glass column having a stop-cock near the bottom.
3. In chromatography, the stationary phase can be ___________
supported on a solid.
a) Solid or liquid
b) Liquid or gas
c) Solid only
d) Liquid only
Answer: a
Explanation: In chromatography, there are two phases namely,
stationary phase and mobile phase. The stationary phase can be solid or
liquid supported on a solid.

55. 4. In chromatography, which of the following can the mobile phase be made of?
a) Solid or liquid
b) Liquid or gas
c) Gas only
d) Liquid only
Answer: b
Explanation: In chromatography, the mobile phase can be composed of liquid or gas. It
cannot be a solid material.
5. Which of the following cannot be used as adsorbent in Column adsorption
chromatography?
a) Magnesium oxide
b) Silica gel
c) Activated alumina
d) Potassium permanganate
Answer: d
Explanation: The given options are all examples of adsorbents in Column adsorption
except potassium permanganate. Some other adsorbents are starch and chromatographic
purified siliceous earth

56. Thank
You