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.

1. COLUMN CHROMATOGRAPHY
Under the guidance of
Ms. K. Veditha
Assistant professor
Dept. of PA&QA
Submitted by
M. Durga Prasad
Regd No:11AB1R0034
VIGNAN PHARMACY COLLEGE
(Approved by AICTE, PCI & affiliated to JNTU-K)
VADLAMUDI, GUNTUR DISTRICT – ANDHRA PRADESH, INDIA
PIN NO: 522213

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’.

5. TYPES OF COLUMN CHROMATOGRAPHY
S.N
o
Types of column
chromatography
Mobile
phase
Stationary phase Sample phase
1 Adsorption
chromatography
Liquid Solid adsorbent Solution
2 Partition
chromatography
Liquid Immiscible solvent on
solid matrix
Solution
3 Ion exchange
chromatography
Liquid Ion exchange resin Solution
4 Gel
chromatography
Liquid Solvent held in the
interstices of a
polymetric solvent
Solution

6. 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.

7. Separation of compounds based upon Affinities

8. 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.

9. • Substances with large differences in their partition coefficients may be
completely separated by simple solvent extraction techniques involving
few (one to three) extractions.
• As differences in partition coefficients of a mixture of substances
decreases, the number of solvent extractions to complete separation
increases.

10. THEORY OF CHROMATOGRAPHY
 Martin and synge in 1941 developed the concept of the ‘theoretical plate’ in
order to establish a satisfactory theory for partition chromatography.
 The column is considered as being made up of large number of parallel
layers of ‘ theoretical plates’.
 When the mobile phase passes down the column distribute themselves
between the stationary and mobile phases in accordance with their partition
coefficients.

11. The rate of movement of the mobile phase is assumed to be such that the
equilibrium is established within each plate.
The equilibrium is dynamic and the components move down the column
at definite rate depending on the rate of movement of the mobile phase.
N= L/H
Where N= Number of theoretical plates,
L = Length of the column
H = Height equivalent of theoretical plate

12. PRACTICAL REQUIREMENTS
 Stationary phase
 Mobile phase
 Column characteristics
 Preparation of the column
 Introduction of sample
 Development techniques
 Detection of components
 Recovery of components

13. 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

14. 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.

15. ADSORPTION PROCESS
 Adsorption is defined as the phenomenon of concentration of
molecules of a gas or liquid at a solid surface.
 When a solid surface is exposed to gas or a liquid, molecules
from the gas or the solution phase accumulate or concentrate at
the surface.
 The substance that concentrates at the surface is called
Adsorbate and the solid on whose surface the concentration
occurs is called the Adsorbent.

16. Adsorbate
Adsorbent
Mechanism of Adsorption
Adsorbate
(Methylene Blue)
Adsorbent
(Charcoal)
Adsorbent atoms or molecules are not surrounded by atoms or
molecules of their kind and they have unbalanced attractive
forces on the surface which can hold adsorbate particles.
Example: Silica gel, Alumina

17. The most commonly used adsorbents are Silica and Alumina.
These are activated at 200 C
Structure of Alumina Structure of Silica

18. ADSORPTION ISOTHERMS
The adsorption isotherms are of three types. They are
(a) Linear adsorption isotherms.
(b) Convex adsorption isotherms.
(c) Concave adsorption isotherms.

19. A. Linear adsorption isotherms :
 The linear adsorption isotherms are obtained when the amount
of adsorbent is proportional to the concentration of solution.
 When a substance moves as a band through a column of
adsorbent there is no tendency for any portion of the band to be
adsorbed more strongly than another.
 Therefore, a symmetrical peak is obtained as the eluate from
the column is examined.

20. B. Convex adsorption isotherms :
 The convex adsorption isotherms are obtained when adsorption
from weak solutions is greater than from strong solutions.
 The pattern of the substance is symmetrical initially.
 But the substance in low concentration, the front of the band is
held strongly by adsorption than in the center of the band.
 Therefore, a sharp leading edge to the band is obtained.

21. C. Concave adsorption isotherms :
 The concave adsorption isotherms are obtained when
adsorption from strong solutions is greater than from weak
solutions.
 So, the peak obtained in this isotherm is concave in shape.

22. Adsorption isotherms and related elution patterns of substances from a
column of adsorbent.
m = weight of substance adsorbed per g of adsorbent.
Cs = Concentration of solution.
Cf = Concentration of each fraction.
f = number of each fraction.

23. Types of Adsorbents
S.No Adsorbent Example
1 Strong adsorbent Alumina, Fuller’s Earth,
Activated charcoal
2 Intermediate adsorbent Calcium carbonate, Calcium
phosphate, Magnesia, Slaked
lime, Silica gel
3 Weak adsorbent Cellulose, Starch, Talc, Sucrose
powder

24. Commonly used adsorbents for separation of
chemical constituents in Column chromatography
S.No Adsorbent Separable chemical constituents
1. Alumina, Magnesia Alkaloids, Sterols, Vitamins
2. Aluminium chloride Sterols
3. Calcium carbonate Carotenoids, Xanthophylls
4. Carbon Amino acids, Carbohydrates, Peptides
5. Magnesium carbonate Porphyrins
6. Magnesium silicate Alkaloids, Glycerides, Sterols
7. Silica gel Amino acids, Sterols
8. Starch Enzymes

25. MOBILE PHASE
Mobile phase is very important and they serve several functions.
They act as solvent, developer and as a eluent.
The functions of the mobile phase are:
 As developing agent
 To introduce the mixture into the column – as solvent
 To remove pure components out of the column – as eluent

26. Choice of the solvent:
•Depend on the solubility characteristics of the mixture.
•Should also have sufficiently low boiling points which permit ready
recovery of eluted material.
• Polarity

27. SOLVENTS POLARITY
Propanol 3.9
Tetrahydro
4.0
furan
Ethanol 4.3
Acetone 5.1
Acetonitrile 5.8
Ethylene glycol 6.9
Dimethyl
7.2
sulfoxide
Water 10.2
Increasing polarity

28. COLUMN CHARACTERISTICS & SELECTION
 Chromatographic columns were made up of good quality of glass
that should be neutral because to avoid the affects of solvents, acids
or alkalies.
Column selection
Multi-component system Long column
Components with similar
affinities
Long column
Components with different
affinities
Short column
More no. of compounds Long column
Weak adsorbent few compounds Short column

29. • The column dimensions are very important for effective separation.
• The length : diameter ratio from 10:1 to 30:1.
• For more efficiency 100:1 can be used.
The length of the column depends upon :
 Number of compounds to be separated.
 Type of adsorbent used.
 Quantity of sample
 Affinity of the compounds towards adsorbent used.
Better separation will be obtained with a long narrow
column than short thick column because number of plates will be more.

31. PREPARATION OF THE COLUMN
 It consists of a glass tube with the bottom portion of the column
packed with glass wool / cotton wool or may contain asbestos pad.
 Above this the adsorbent is packed.
 After packing a paper disc is kept on the top, so that the adsorbent
layer is not disturbed during the introduction of sample.
 Slurry is introduced into the column using funnels.
 The level of solvent must never be allowed to fall below the level of
adsorbent to prevent cracks.

32. Apparatus of column chromatography

33. PACKING OF COLUMNS
 The packing of column is an exceptional art that essentially needs a
lot of skill, wisdom and talent.
 A careful attention should always be given to the perfect uniform
packing of the selected adsorbent into the chromatographic column
so as to achieve the maximum efficiency.
 The packing of column is carried out in two different manners.
Wet packing and
Dry packing.

34. WET PACKING
A thin slurry of the adsorbent with the appropriate
solvent (mobile phase) is prepared in a glass
beaker and is poured slowly into the column
Any air bubbles trapped in the slurry should be
removed by the help of a long glass rod by
agitation.
Adsorbent once gets settled in the column, place
a disc of whatman filter paper on its top layer and
washed sand is added to top of disc.
Solvent is continued to run down unless the
level of liquid attains a height of nearly 1cm
above the top level of the packed column.

35. Wet packing process

36. DRY PACKING
Dry packing involves the pouring of fine powdered
form of the adsorbent into the column.
The column must be tapped while the filling
process is going on so as to maintain the soft
compactness of the adsorbent in the body of the
column.
The column is filled upto 3/4th of the actual height
of the column. The empty head above the surface of
the packed column is filled with the mobile phase.

37. PROCESS OF DRY PACKING

38. INTRODUCTION OF SAMPLE
A graduated pipette is filled up with
the sample mixture and introduced by
touching the top of the adsorbent
layer having a filter paper with a
layer of sand.
The tip of the pipette is placed against
the inside wall of the column just
above surface of the adsorbent.

39. DEVELOPMENT TECHNIQUES
 The development techniques are categorized into three types.
(a) Elution analysis
Isocratic elution technique
Gradient elution technique
(b) Frontal analysis
(c) Displacement analysis

40. ELUTION ANALYSIS
 Elution analysis refers to the specific removal of chemical entities from a
chromatographic support by the aid of solvent.
 This method makes use of a small volume of mixture that need to be
separated and the respective ‘mobile phase’ is permitted to flow through the
column downward due to gravity.
 With the passage of time the ‘mobile phase’ moves down the column and
the mixture of ‘analytes’ undergo resolution into various ‘distinct zones’ by
the fact that the analytes in the mixture get adsorbed to various degree.

41. Isocratic elution technique :
 In this technique, the same solvent composition or solvent of sample
polarity is used throughout the process of separation.
Gradient elution technique :
 In this elution technique, solvents of increasing polarity or increasing
elution strength are used during the process of separation.

42. 6FRONTAL ANALYSIS
 Tiselius (1940) first and foremost developed this method.
 The ‘Frontal analysis’ employs the solution of the ‘respective sample
mixture’ which is incorporated continuously onto the column.
 In this particular instance there is no mobile phase (i.e., eluting
solvent) is used at all for the development of the ‘analytes’ on the
column.
 At first the least adsorbed component passes out of the column and

43. the intermediate component is adsorbed later and next the most adsorbed
component is passed out.
 The graphical representation provides separation profile of the
components. The extrapolation of the various points clearly shows
the presence of other components along with the first one and needs
further separation.

44. DISPLACEMENT ANALYSIS
 The principle involved in this method is that ‘small volume of
mixture of components’ is introduced into the column and the usual
‘elution’ is performed by means of a solvent consisting of a solute
that possesses high degree of adsorptivity for the adsorbent packed
in the column.
 Then the adsorbed components present in the ‘sample mixture’ are
displaced by the ‘added solute’ from the eluting mobile phase.
 Each component present in the sample mixture helps to displace
another solute that is less adsorbed.

45. In this way, the least adsorbed component is flushed out of the column.
 The graphic representation of the plot is obtained by the critical
separation of a sample mixture comprising of three components
(assuming adsorption of X<Y<Z).
 In the event, when D is designated as displacer, the graph is

46. DETECTION AND RECOVERY OF COMPONENTS
 The coloured components are detected by visual examination.
 The colourless components may also be detected visually if they
fluorescence.
Ex : Quinine & Ergotamine.
 Recovery of the components after detection on the column requires
‘extrusion’ of the column of adsorbent and isolation of each zone for
extraction with solvents.
 In case of plastic tubing the zones are isolated by cutting tubing into
sections.

47.  For colourless compounds the eluate is collected as a large number
of fractions, each of small volume.
 Each fraction is examined appropriately for the presence of a
compound.
 The examination may by
 Evaporation of the solvent from each fraction and weighing the residue
 By simple spot tests
 By examination of the fraction by paper or thin layer chromatography
 By spectrophotometry

48. FACTORS AFFECTING EFFICIENCY OF A COLUMN
Factor Effect
Particle size of solid
stationary phase
Decrease in size improves separation
Column dimensions Efficiency increases as ratio length
Column temperature Increase in column temperature results in speed
of elution but does not improve separation
Solvent It should be of low viscosity & high volatility
Solvent flow rate Uniform and low flow rate gives better resolution
Conduction of adsorbent Deactivation of adsorbent decreases separation
Concentration of solutes Substances of high concentration moves slowly

49. APPLICATIONS OF COLUMN CHROMATOGRAPHY
 Column chromatography is best suited to separate active
principle from plant materials.
 To separate impurities along with the important constituents
 Isolation of metabolites from important components
 Used for determination of phytomenadione in tablets and
injections
 Determination of flucinolone, acetonide, betamethasone in
formulations.

50.  Used for separation of inorganic ions like copper ion, cobalt ion,
nickel ion.
 Determination of the percentage w/w of strychnine in syrup of iron
phosphate with quinine and stychnine.
 Determination of quinine in ethanolic solution.
 Useful in the separation of carbohydrates and their derivatives.
 To separate natural compound mixtures like alkaloids, glycosides.

51.  Determination of phenothiazine in the presence of diphenylamine
and carbazole.
The chromatogram for a mixture of diphenylamine and
phenothiazine.

52. ADVANTAGES OF COLUMN CHROMATOGRAPHY
 Any type of mixture can be separated.
 Wider choice of mobile phase.
 Automation is possible
 Any quantity of mixture can be separated.

53. DISADVANTAGES OF COLUMN
CHROMATOGRAPHY
 Time consuming.
 More amount of mobile phase are required.
 Automation makes the technique more complicated and expensive.

54. REFERENCES :
 A.H. BECKETT & J.B. STENLAKE, Practical pharmaceutical chemistry,
4th edition, part two, page no: 86-105.
 ASHUTOSH KAR, Pharmaceutical analysis – II, page no: 161-181.
 Dr . S. RAVI SANKAR, Pharmaceutical analysis, 3rd edition, page no: 13-4
to 13-13.
 B.K. SHARMA, Instrumental methods of chemical analysis, page no : C-8
to C-15.
 Dr. A.V. KASTURE, Dr. K.R. MAHADIK, Dr. S.G. WADODKAR, Dr.
H.N. MORE, Pharmaceutical analysis volume – II, page no: 10-17

55. ACKNOWLEDGEMENT
 Thank you for paying attention.
 I sincerely thank my principal Dr. P. Srinivasa babu sir
for the honoured encouragement.
 I also sincerely thank my guide K. Veditha madam for
the valuable guidance.
 A special thanks for Ch. Devadas sir and Seminar
committee.