chromatography techniques

1. CHROMATOGRAPHY
Presented by –
Simi Baruah
Roll No-18
Narayan Sarkar
Roll No-6
M.Sc. 3rd semester
Guided by-
Dr. Nabanita Bhattacharyya
Assistant Professor
Dept. of Botany

2. CONTENTS
INTRODUCTION
HISTORY
PRINCIPLE
TYPES OF CHROMATOGRAPHY
COLUMN CHROMATOGRAPHY
PAPER CHROMATOGRAPHY
THIN LAYER CHROMATOGRAPHY
CONCLUSION

3. INTRODUCTION
Chromatography is a Greek word (colour
writing), chroma =“colour” & Graphein=
“to write”
It is the collective term for a set
laboratory techniques for the separation
of mixtures.
Definition:
Chromatography is a technique for the
separation of a mixture by passing it in
solution or suspension through a
medium ,in which the components move
at different rates.

4. HISTORY OF
CHROMATOGRAPHY
To write with colors -- literally translated
from its Greek
roots chroma and graphein
Chromatography: Russian botanist
Mikhail Tswett in 1903 , first developed
this technique.
It has since developed into an invaluable
laboratory tool for the separation and
identification of compounds.

5. PRINCIPLE OF
CHROMATOGRAPHY

6. Chromatography is based on the principle
where molecules in mixture applied onto
the surface or into the solid, and fluid
stationary phase (stable phase) is
separating from each other while moving
with the aid of a mobile phase.
The factors:
Molecular characteristics related to
Adsorption (liquid-solid),
Partition (liquid-solid), and
Affinity or
differences among their molecular
weights.

7.  Because of these differences, some
components of the mixture stay
longer in the stationary phase, and
they move slowly in the
chromatography system, while
others pass rapidly into the mobile
phase, and leave the system faster.

8.  Basis of the chromatography technique:
Stationary phase: This phase is always
composed of a “solid” phase or “a layer of a
liquid adsorbed on the surface solid
support”.
Mobile phase: This phase is always
composed of “liquid” or a “gaseous
component.”
Separated molecules:
The type of interaction between the
stationary phase, mobile phase, and
substances contained in the mixture is the
basic component effective on the
separation of molecules from each other.

11. Eluent: An eluent is a solvent used to carry
the components of a mixture through a
stationary phase. It is alternative term used
for the mobile phase.
Eluate: The mobile phase that exits the
column is termed as eluate.
Elution: The process in which solutes are
washed through a stationary phase by the
movement of a mobile phase.

12. TYPES OF
CHROMATOGRAPHY

13. 1)Adsorption chromatography
Separation is based on differences between the
adsorption affinities of the sample components for
the surface of an active solid stationary phase.
2)Partition chromatography
It is a type of chromatography in which the
components of the mixture get distributed into the
two phases due to differences in partition
coefficients(Kd),which is the ratio of the
concentration of solutes in two phases.
Kd= concentration of solute in phaseA
concentration of solute in phaseB
The distribution of solutes between two phases is based
on solubility differences.

14. 3)Ion exchange chromatography:
 It is applicable for the separation of charged
molecules.
 Stationary phase is an ion exchanger(either cation
exchanger or anion exchanger)
 Solute ions of the opposite charge in the mobile liquid
phase bind reversibly to the ion exchanger.
 The graeter the charge ,the stronger the interaction.
 Neutral solutes show no affinity for the stationary
phase and move with the eluting buffer.
 The bound solutes can be released by eluting the
column with a buffer of increased ionic strength or pH

15. Separation of cation by ion exchange:

16. 4) Size exclusion chromatography:
 It separates molecules on the basis of size and
shape.
 A column matrix filled with porous gel beads made
of insoluble and hydrated polymer such as
polyacrylamide or agarose acts as stationary phase.
 Solution containing molecules of various sizes is
passed through the column
 Molecules smaller than the pores can enter the
pores in the beads whereas larger molecules can
not.
 So larger molecules move faster and elute first.
 Smaller molecules have longer retention time than
the larger molecules.

18. 5)Affinity Chromatography:
It involves the following steps:
Choice of an appropriate ligand
Immobilization of ligand onto a
support matrix
Binding of the molecules of interest
with the ligand
Removal of non specifically bound
molecules
Elution of the molecules of interest in
a purified form.

19. Typical biological interactions used in
affinity chromatography:
Types of ligand Molecules of interest
Enzyme Substrate analogue
Antibody Antigen
Nucleic acid Complementary base
sequence
Avidin Biotin
Calmodulin Calmodulin –binding
protein
Poly(A) RNA containing poly(U)
sequence
Proteins A and G Immunoglobulins

21. PAPER CHROMATOGRAPHY
 What Is Paper Chromatography?
 It was discovered by Synge and Martin in the
year 1943.
 It is the technique that uses paper sheets or
strips as the adsorbent being the stationary
phase through which a solution is made to pass.
 Inexpensive method of separating dissolved
chemical substances by their different migration
rates.
 Powerful analytical tool that uses very small
quantities of material.

22. PAPER CHROMATOGRAPHY
PRINCIPLE
 It is a partition chromatography technique
 Cellulose paper is a supporting medium over
which the solvents flow.
 Water bound to the polar cellulose is the
stationary phase and organic solvent which flows
over it is a mobile phase. Organic solvent moves
over the hydrated cellulose fibres
 As the solvent passes through an area of paper
containing a solute (mixture of components), the
solute begins to partition itself between the
aqueous and organic phases in proportion to its
relative solubility in the two phases

23.  The components of the solute more
soluble in organic phase will be carried
faster along the organic phase.
Conversely, greater the affinity for
water, slower the solute will move
with respect to the solvent front.
 Thus if several compounds possess
different solubility rates, each will
move across the paper at specific rate
which is generally different from that
of any other compound

24.  The distance the solute moves, in
relation to the distance the solvent
moves, serves as a means of identifying
the solute and is called Rf.

26. PROCEDURE OF PAPER
CHROMATOGRAPHY
 Selecting a suitable type of development: It is
decided based on the complexity of the solvent, paper,
mixture, etc. Usually ascending type or radial paper
chromatography is used as they are easy to perform
 Selecting a suitable filter paper: Selection of filter
paper is done based on the size of the pores and the
sample quality.
 Prepare the sample: Sample preparation includes
the dissolution of the sample in a suitable solvent
(inert with the sample under analysis) used in
making the mobile phase

28.  Spot the sample on the paper: Samples should be
spotted at a proper position on the paper by using a
capillary tube.
 Chromatogram development: Chromatogram
development is spotted by immersing the paper in the
mobile phase. Due to the capillary action of paper, the
mobile phase moves over the sample on the paper.
 Paper drying and compound detection: Once the
chromatogram is developed, the paper is dried using
an air drier. Also, detecting solution can be sprayed
on the chromatogram developed paper and dried to
identify the sample chromatogram spots.

29.  Paper Chromatography
Applications
Some of the uses of Paper Chromatography
in different fields are discussed below:
1)To study the process of fermentation and
ripening.
2 )To check the purity of pharmaceuticals.
3)To inspect cosmetics.
4)To detect the adulterants.
5)To detect the contaminants in drinks and
foods.
6)To examine the reaction mixtures in
biochemical laboratories.
7)To determine dopes and drugs in humans
and animals.

30. COLUMN CHROMATOGRAPHY
 What Is Column Chromatography?
 This method is a type of adsorption chromatography technique.
 It is a technique which is used to separate a single chemical
compound from a mixture dissolved in a fluid.
 It separates substances based on differential adsorption of
compounds to the adsorbent as the compounds move
through the column at different rates which allow them to get
separated in fractions.
 This technique can be used on a small scale as well as large scale
to purify materials that can be used in future experiments.

31. Principle of column
chromatography
 When the mobile phase along with the mixture
that needs to be separated is introduced from the
top of the column, the movement of the individual
components of the mixture is at different rates
 The components with lower adsorption and
affinity to stationary phase travel faster when
compared to the greater adsorption and
affinity with the stationary phase.
 The components that move fast are removed
first whereas the components that move
slowly are eluted out last.

32. The adsorption of solute molecules to
the column occurs in a reversible
manner. The rate of the movement of
the components is expressed as:
 Rf = the distance travelled by solute
the distance travelled by the solvent
Rf is the retardation factor.

34. Column Chromatography
Procedure
 Mobile phase – This phase is made up of solvents
and it performs the following functions:
 It acts as a solvent – sample mixture can be
introduced in the column.
 It acts as a developing agent – helps in the separation
of components in the sample to form bands
Some examples of solvents used as mobile phase
based on their polarity are – ethanol, acetone,
water, acetic acid, pyridine, etc.
 Stationary phase – It is a solid material which
should have good adsorption property.

35.  The stationary phase is made wet with the
help of solvent as the upper level of the
mobile phase and the stationary phase
should match.
 In the first step the compound mixture that
needs to be separated, is added from the
top of the column without disturbing the top
level.
 Without disturbing the stationary phase
solvent mixture is added slowly by touching
the sides of the glass column.

36.  The tap is turned on to initiate the
movement of compounds in the
mixture.
 The movement is based on the polarity
of molecules in the sample.
 The non-polar components move at a
greater speed when compared to the
polar components.

37.  For example, a compound mixture consists of
three different compounds viz red, blue, green
then their order based on polarity will be as
follows blue>red>green
 As the polarity of the green compound is less,
it will move first.
 When it arrives at the end of the column it is
collected in a clean test tube.
 After this, the red compound is collected and at
last blue compound is collected.
 All these are collected in separate test tubes.

38. Column Chromatography Applications
 Column Chromatography is used to isolate
active ingredients.
 It is very helpful in Separating compound
mixtures.
 It is used to determine drug estimation from
drug formulations
 It is used to remove impurities.
 Used to isolation metabolites from biological
fluids.

39. THIN LAYER
CHROMATOGRAPHY
What Is Thin Layer Chromatography?
 It is a technique used to isolate non-volatile mixtures.
 The experiment is conducted on a sheet of
aluminium foil, plastic, or glass which is coated
with a thin layer of adsorbent material.
 The material usually used is aluminium oxide,
cellulose, or silica gel.
 On completion of the separation, each component
appears as spots separated vertically.

40.  Each spot has a retention factor (Rf)
expressed as:
Rf = dist. travelled by sample
dist. travelled by solvent
The factors affecting retardation factor
are the- solvent system,
amount of material spotted,
absorbent and temperature.

41.  Thin Layer Chromatography
Principle
 Like other chromatographic techniques, thin-
layer chromatography (TLC) depends on the
separation principle.
 The separation relies on the relative affinity of
compounds towards both the phases.
 The compounds in the mobile phase move
over the surface of the stationary phase.

42. The movement occurs in such a way
that the compounds which have a
higher affinity to the stationary phase
move slowly while the other
compounds travel fast.
 On completion of the separation
process, the individual components from
the mixture appear as spots at respective
levels on the plates.
Their character and nature are identified
by suitable detection techniques.

44. Thin Layer
ChromatographyExperiment
The stationary phase that is applied to the plate
is made to dry and stabilize.
 To apply sample spots, thin marks are made at
the bottom of the plate with the help of a pencil.
 Apply sample solutions to the marked spots.
 Pour the mobile phase into the TLC chamber
and to maintain equal humidity, place a
moistened filter paper in the mobile phase.
 Place the plate in the TLC chamber and close it
with a lid.
 It is kept in such a way that the sample faces the
mobile phase.

45. Immerse the plate for development.
Remember to keep the sample spots
well above the level of the mobile
phase.
Do not immerse it in the solvent.
Wait till the development of spots.
 Once the spots are developed, take out
the plates and dry them.
The sample spots can be observed
under a UV light chamber.

46. Thin Layer Chromatography
Applications
1)The qualitative testing of Various medicines
such as sedatives, local anaesthetics,
anticonvulsant tranquilisers, analgesics,
antihistamines, steroids, hypnotics is done by
TLC.
2)TLC is extremely useful in Biochemical
analysis such as separation or isolation of
biochemical metabolites from its blood plasma,
urine, body fluids, serum, etc.
3)Thin layer chromatography can be used to
identify natural products like essential oils or
volatile oil, fixed oil, glycosides, waxes,
alkaloids, etc.

47. 4)It is widely used in separating
multicomponent pharmaceutical formulations.
5)It is used to purify of any sample and direct
comparison is done between the sample and
the authentic sample.
6)It is used in the food industry, to separate and
identify colours, sweetening agent, and
preservatives
7)It is used in the cosmetic industry.
8)It is used to study if a reaction is complete.

48. Thin–layer chromatography offers several
benefits over the paper chromatographic
separations. Some of the benefits are:
 Time Saving
The biggest advantage offered to the
chromatographer is time- saving. Paper
chromatography can take several hours to develop
the plate whereas development in thin layer
chromatography can be completed in much shorter
time (about half an hour or so)
 Automation
Paper chromatography has not seen much
automation over the years but thin layer
chromatography instruments available have
automation capabilities which include
autosampler, constant volume sample dispenser,
documentation and camera for retaining pictorial
record of separations

49.  Rigid Support
The cellulose paper support in paper chromatography
is flexible whereas the adsorbent in TLC is coated
onto a rigid metal, glass or plastic plate. This
contributes to reproducibility of spots and faster
development. Due to support rigidity there is less
diffusion and as a result well-defined spots are formed.
 Choice of Support
TLC presents a vast choice of support adsorbent
phases including liquid coated adsorbents which can
include fluorescence inducers as well. On the other
hand choice of papers in paper chromatography is
very much limited.
 Development Chamber Design
It is not necessary to suspend the plate as required in
paper chromatography from a rod on top of the
development chamber. It can be simply placed in a slanting
position with its bottom edge resting on the chamber base.

50.  Sample Volume
The quantity of sample applied is small( in
microliters) and can be reproducibly applied with
the help of automated sample dispensers
 Choice of Spray Reagents
Corrosive spray reagents can char the filter
paper and can even deteriorate the sample
spots. Coated plates used in thin layer
chromatography can withstand corrosive spray
reagents to a greater extent.
 Heating
Thin-layer chromatography plates can be heated
if required for spot development. Paper
chromatography sheets cannot withstand heating
beyond a point.


51. CONCLUSION
 Now a days, chromatography is a very
popular biophysical technique for the
separation of bimolecules from both
plants and animals.
 It is used for the separation of plant
pigments such as chlorophyll ,
xanthophyll etc.
 Separation of amino acids is done by
this method.

52. REFERENCES
 A text book of plant physiology ,
biochemistry and biotechnology by
Verma and Verma
 Biophysics and molecular biology by
Pranav Kumar
 https://Byjus.com
 www.biochemden.com

53. THANK YOU