1. Types of Chromatography

2. Classification of chromatography
• According to separation mode:
a) Adsorption chromatography
b) Partition chromatography
c) Ion-exchange chromatography
d) Size exclusion chromatography
e) Affinity chromatography

3. Classification of chrom. (cont.)
• According to mobile phase:
a) Gas Chromatography
i- Gas solid chromatography
ii- Gas liquid chromatography
b) Liquid chromatography
i- Paper chromatography
ii- Thin-layer chromatography
iii- Column chromatography

4. Classification of chrom. (cont.)
• According to form of stationary phase
a) Planar chromatography
i- Paper chromatography
ii- Thin-layer chromatography
b) Column chromatography
i- Gas chromatography
ii- Liquid chromatography (LC/HPLC)

5. LIQUID-COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column
packed with solid particles which may or may not be
coated with another liquid.
With the proper solvents, packing conditions, some
components in the sample travel through the column
more slowly than others resulting in the desired
separation.

7. FOUR BASIC LIQUID CHROMATOGRAPHY
The basic liquid chromatography modes are
named according to the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC

8. L C used for samples:
 containing large molecules/ionic
 containing substances with low vapor
pressure (non-volatile substances)
 Substances thermally unstable
 Substances can’t be vaporized without
decomposing

9. LIQUID SOLID CHROMATOGRAPHY
Normal phase LS
Reverse phase LS
δ− δ+
Si - O - H
30 µ Silica Gel
The separation mechanism in LSC is based on the
competition of the components of the mixture sample
for the active sites on an absorbent such as Silica Gel.

10. LIQUID SOLID CHROMATOGRAPHY
OH HEXANE
Si - OH
OH CH
CH3 3
CH3- C C-CH3
CH3 CH3
CH3

11. Adsorption chromatography
 Stationery phase is solid and mobile phase is
liquid
 Distribution between two phases (adsorption and
desorption)
 Attractive forces (ionic, dipole-dipole, dipole
induced dipole)
 Good adsorbent has large surface area and more
active sites
 Equilibration occurs at: surface-solute, surface-
solvent, solvent-solute

12. Adsorption isotherms
 An adsorption isotherm is a plot of the
concentration or amount of analyte on a
surface as a function of its concentration in
the bulk phase.
 In liquid chromatography, the bulk phase is,
of course, the mobile phase.

13. Adsorption isotherms
 Linear (k = Cs/Cm) = 1
 Convex (k = Cs/Cm1/n) where n >1
 Concave

14. Adsorbents
 Adsorbing power depends on
1- chemical nature of the surface
2- area available
3- pretreatment

15. Commonly used adsorbents
 Alumina, Al2O3 ,(Aluminum oxide). It may
be acidic, basic and neutral in nature.
Available in various grades
 Silica gel (silicon dioxide). It is acidic in
nature. Available in various grades.
 CaCO3
 Sucrose
 Starch
 cellulose

16.  Adsorbent should have uniform size and
large surface area
 Weight of the adsorbent should be 20-50
times more than the sample

17. Solvents & adsorbents
 Since adsorbents are polar, non-polar elute first.
Usually, the elusion order is as: alkyl halids <
saturated hydrocarbons< unsaturated
hydrocarbons <ethers < esters < ketones < amines
< alcohols < phenols < acids and bases. Polymeric
compounds and salts often don’t elute
 The solvents in the order of polarity Hexane/Pet
ether < CCl4 < toluene < dichloromethane <
chloroform < diethyl ether < acetone < ethyl
acetate < propanol < ethanol < methanol < acetic
acid < water

18. Columns and packing
 Various sizes are available
 Wet method
 Dry method
 Sample loading and running the column

19. WATER-SOLUBLE VITAMINS
1. Niacinamide 2. Pyridoxine
H 3C N
N
HO CH 2OH
CONH 2 CH 2OH
3. Riboflavin
CH 2OH
HOCH
HOCH 4. Thiamin
HOCH
CH 2
H 3C N N O H 3C N NH 2 S CH 2CH 2OH
NH Cl
H 3C N N N
CH 2 CH 3
O

20. WATER-SOLUBLE VITAMINS
2
3
Inject 4
1
Column: u Bondapak C18
Solvent: MeOH
Sample: Water-Soluble Vitamins
0 5 10 15 20

21. LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH3 CH2 OCH2 CH2 CN(Normal)
CH3 (CH2 ) 16 CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the
Stationary Phase) which is immiscible in the solvent (Mobile) phase.
Partitioning of the sample between 2 phases delays or retains some
components more than others to effect separation.

22. Advantages of Partition
chromatography
– Advantage over adsorption chromatography
 More reproducible and predictable
 Distribution coefficient is constant over a much
greater range of concentration yielding sharper and
symmetrical peaks
 It is also of two types
1-Normal phase
2-Reversed phase

23. Supports for stationary phase
 Silica gel
 Kieselguhr/Celite
 Cellulose

24. steps
 Sample preparation
 Sample loading
 Elution
 Detection: chemical methods; diverse types
of detectors can be used

25. ION-EXCHANGE CHROMATOGRAPHY
- +
SO3 Na
Separation is based on the competition of different ionic
compounds of the sample for the active sites on the ion-
exchange resin (column-packing).

26. Ion exchange chromatography
 A process where ions held by solid matrix
are exchanged for counter ions in the
solution
 Synthetic ion exchange resins are used for
water purification and separation of ions

27. MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
pH2
- + +
SO3 Na H3N
COOH
Ion-exchange Resin
- +
SO3 H3N
-
COO pH4.5
+
Na

28. Chromatography of Amino Acids
Stationary Phase Mobile Phase
+
H3N
-
SO3 Na+
COOH
+
Na
OH
- +
SO3 H3 N
COOH
Exchange Resin
-
SO3 H3N+
COOH
pH3.5
OH
-
SO3 +
H3 N
+ - + -
Na COO H OH = H 2 O
+
Na
-
SO3 H3 N
+
- + -
COO H OH = H 2 O
-
SO3Na+
pH4.5

29. Ions exchange resins
 Consist of three dimensional polymeric
chains, cross linked by short chains, which
carry ionisable functional groups.
 Based on ions these are of two types
– Cation exchangers (weak or strong)
– Anion exchangers (weak or strong)

30. Formation of resin
 Styrene and divinylbenzene
 The number of cross linkers
determine by the ratio of Styrene :
divinylbenzene
 Increasing cross linkers increases
the rigidity and reduces swelling

31. Ion-exchange chromatography can be used to
perform preparative separation of amino acids
Negatively charged resin binds selectively to
positively charged amino acids

32. Behavior of resin
 Important properties which determine
behavior of resin are:
1- size of particles
2- degree of cross linking
3- nature of functional groups
4- number of functional groups

33. Theoretical principles
 Ion exchange equilibrium
 distribution coefficient (KD) indicates affinity of
the resin for ions relative to hydrogen
 Generally, if KD is large, resin will incline to
attract the ion
 Polyvalent ions are more attracted to the resin
compared to mono-valent

34.  In groups where charges are same, the
difference between KD is related to the size
of the ion

35. Uses of ion exchange
chromatography
 Ion separation
 Concentration of trace matters
 Separation of alkali and alkali earth metals
 Separation of amino acids, proteins,
peptides, nucleic acid and nucleotides
 immunoglobulin

36. Steps
 Same to that of the others
 Detection: conduction measuring

38. Size exclusion chromatography
 Molecular gel chromatography, gel
permeation, molecular sieving or molecular
exclusion
 Stationary phase serves as molecular sieve
 Separate molecules based on size via
sieving or filtration
 Adsorption and electrical charge play role
in separation

39. Gels
 Open three dimensional network formed by
cross linking large ploymeric chains
 Polar groups absorb water and swell
 Have an exclusion limit i.e critical size of a
molecule that can just penetrate the interior

40. Theoretical principles
 There are 2 kinds of solvent in the gel
Vi = Volume within gel
Vo = volume outside the beads of the gel
Large molecules will not be able to enter or penetrate the
pores of the gel, hence their elution volume (Ve ) will be
Ve = Vo
Whereas, smaller molecules must be swept through Vo plus
some additional volume which is a fraction of Vi

41. Ve = Vo + KDVi, where KD = distribution
coefficient
KD = Average concentration of solute in gel/
Average concentration of solute outside gel
KD value should be between 0 and 1
If sieving action is the only mechanism of
separation (KD=1) then
Ve = Vo + Vi
If K<1, It indicates solvent interacts with the gel
(adsorption, hydrogen bonding)

42. Types of the gels
 Sephadex, dextran gel (classified by the
amount of water regain)
 Biogel, polyacrylamide gel, inert series of
gels, insoluble in water and common
organic solvents
 Styragel rigid cross linked polystyrene gel
useful at temperature > 150oC with organic
solvents, it can be used under high pressure

43. Gel Filtration

44. Applications
 Desalting (removal of salts and small molecules
from macromolecules)
 Concentrating (concentration of dilute solutions of
macromolecules with MW> exclusion limit
 Fractionation (separation of mixture of closely
related molecules having small difference in KD
values namely proteins, peptides, nucleic acids,
polysaccharides, enzymes and hormones)

45. Types of Chromatography
MOBILE PHASE LIQUID
Liquid-Liquid Liquid-Solid
FORMAT Chromatography Chromatography
(Partition) (Adsorption)
STATIONARY Solid
Liquid
PHASE
Normal Phase Normal Phase Reverse Phase
Reverse Phase
Mobile Phase - Mobile Phase -
Nonpolar Polar
Stationary phase - Stationary phase -
Polar Nonpolar

46. Detectors
1. Ultraviolet Detector
200-400nm
254 nm
2. Reflective Index Detector
Universal Detector

48. Affinity chromatography
 Separation where surface of inert phase has
been modified to selectively bind
compounds having specific functional
group
 Binding force should be strong enough to
effect separation but weak enough to get the
compound when desired

49. Properties of Inert matrix
 Mechanically and chemically stable
 Large surface area
 Easily derivatized
 Good flow characteristics
 Examples (agarose, controlled pore glass,
cellulose)

50. Spacer
 An arm to move active group away from the bead
so that steric hindrances are at minimum
 Effectiveness depend on their
– length
– stability of the attachment to the bead
– hydrophobic nature
– presence of fixed charges and their concentration
 Affi-gel has spacer arm –O- (CH2)3 NH2
[Oxypropylamine]

51. Affinity Chromatography

52. Affinity Chromatography

53. Selected ligands and their affinity
compounds
Ligands Affinity compounds
Diazo-NAD- dehydrogenases
AMP analogues NADP- binding proteins
Blue dextran Yeast phosphofructokinase
2000
Methotrexate Dihydrofolate reductase
B12 Transcobalamin I and II

54. Chromatographic techniques
 Classical LC
 TLC/ paper chromatography
 Modern LC

55. Classical chromatography
technoques
 Glass or plastic columns
 Need skill
 Solvent flow (gravity, suction) and individual
samples collected manually
 Detected using different detectors
 Detection and quantification achieved by manual
analysis of fractions
 Results are recorded in the form of chromatogram
(sample concentration vs fraction number)

56. Disadvantages
 Column packing procedure tedious
 Low column efficiency, long analysis time
 Technique depends on user
 Detection of solutes is labor intensive and
takes a lot of time

57. Plane chromatography
 Plane surface rather than column
 2 dimensional
 Selective properties (use of two solvents)
 Include
– Paper chromatography and
– thin layer chromatography

58. Principles
 Principles are similar to column
 Successive equilibrations of the analyte
between two phases
 Non ideal processes may cause zone
spreading
 Degree of retention is Rf
– Ratio between distance traveled by
solute/distance traveled by solvent

59. Relation between Rf and K
 Rf = number of moles of solute in mobile
phase/total moles in both phases
= Cm Am/CmAm+ CsAs
Am and As are the cross sectional areas of two
phases. By dividing Cm
Rf = Am/Am+AsCs/Cm = Am/Am+ KAs

60. Paper chromatography
 Mainly qualitative and semi quantiative
 Easy to perform
 Mechanisms
1- liquid liquid
2- adsorption
3- hydrogen bonding
4- ion exchange

61. Nature of the paper
 Highly purified cellulose
 Great affinity for water and polar solvents
 Paper may be impregnated with alumina,
silica or ion exchange resin

62. Procedure
 Sample application
 Development
1- ascending
 Simple and popular
 Solvent flow through capillary action
 Slow development
 Slow rate enhances partition, separation

63. 2- descending
 Flow is downward
 Paper folded U shape
 Solvent flow capillary and gravity
 Much faster

64. detection
 Visible
 Application of Reagents
 UV absorbance
 Florescence
 IR
 Radioactivity
 Chemical tests
 Bioautography

65. qualitative
 Based on Rf values
Semi-quantitative
 Extraction and spectroscopy
 densitometry