Chromatography and its types

1. Paper Chromatography

2. Introduction
• First introduced by the German scientist
Christian Friedrich Schonbien in 1865.
• It is a type of simplest Chromatography. It is
the most simplest and widely used type of
chromatography procedures which runs on
specialized paper.

3. Definition
• Analytical method used to separate colored
chemicals and substances.
• Applicability to isolation, identification, and
Quantitative determination of organic and
inorganic compounds.

4. Two Principles of Paper
Chromatography
1). Paper Partition Chromatography.
Paper impregnated with alumina and silica act
as absorbent ( stationary phase ) and solvent
as mobile phase.
2).Paper Adsorption Chromatography.
The moisture or water present in pores of
cellulose fibers present in filter paper act as
stationary phase and another solvent as
mobile phase.

5. Principle of separation
• Principle of separation is mainly partition rather
than adsorption.
Types of paper Chromatography
1). Ascending
2).Descending
3). Ascending-Descending mode.
4).Radial mode
5). Two-Dimensional chromatography

6. Ascending Chromatography
• As the name indicates ,the chromatogram
ascends .Here the development of paper
occurs due to the solvent movement or travel
in upward direction on the paper.
• Descending Chromatography
Development of papers occurs due to solvent
travels downward on the papers.

9. Ascending Descending mode
• In it the solvent first travels upward and then
downward on the paper.
• Radial Mode
Here the solvent travels from center
(midpoint) towards the periphery of Circular
chromatographic paper.

11. Two Dimensional Chromatography
• Here the chromatogram development occurs
in two directions at right angles.

12. Applications
• Used in forensic studies.
• In analytical chemistry for identifying and
separating colored mixtures like pigments.
• Sugars, amino acids, lipids and nucleic acids and
other biomolecules can be easily identified by
spraying with appropriate reagents to detect
these specific compounds.
• Paper chromatography can be reproduced easily
as long as the conditions are controlled and
maintained.

13. Retention Factor
• Indicated by
Rf= Distance travelled by applicant from application point
Distance travelled by solvent from application point

14. APPLICATioNS:
• Among all the chromatography methods paper
chromatography is an inexpensive and rapid method
that provides graphic and clear results.
• Used as a qualitative method for identifying the
components in a mixture.
• Used in several scientific studies in identification of
unknown organic and inorganic compounds from a
mixture.

15. Column Chromatography
• Is a method used to purify chemical compounds
from mixture of compounds on the basis of their
polarity.
• Large scale application.
• This is a solid liquid technique in which
Stationary phase is a solid
Mobile phase is liquid
• Vertical glass columns is used support the
stationary phase

16. Principle
• The principle of column chromatography is adsoption.
• Mixture of components dissolved in M.P is introduced in
column.
• Components move according to their relative affinity.
• Components adsorbed with the statonary phase
Weakly adsorbed components move faster separated
first

17. Apparatus
1. Column
2. Stopcock
• Cotton prevent solid materials to pass
• Sand provide level surface.
• Stationary phase ( silica , alumina )
• Mobile phase ( solvent )

18. Gels
• Silica and alumina are both polar adsorbents.
• Silica gel is less polar than alumina.
• Silicais preferentially retains basic compounds.
• Alumina preferentially retains acidic
compounds.
Non Polar compounds elute first

19. Steps
1. Packing column
 Bottom portion of the column is packed with cotton wool.
 Then sand is added
 Packing can be either
1. Dry
Add dry solid phase to the column and pass buffer to
saturate the solid
2. Wet
Mix both liquid and solid outside of the column and pour it
into the column.

21. 2. Loading column
• is loaded using pipette to make sure even layer on top of silica gel.
• Then mobile phase is poured.
• Stop cock is opened and solvent start moving along sample which will
separat different components according to their relative affinity with
stationary phase.
• components that strongly interacts with the stationary phase retained
in the column thus moves slowly
• But when the interactions are weak the components elutes easily so,
exits first

23. 3. Analyzing
• Depend on the type of molecules separated
• protein or DNA gel
• Plant extracts run a TLC plate

24. Factors Affecting Column Efficiency
1. Dimension of the column (length/width
ratio )
2. Particle size of column packing.
3. Activity of the adsorbent.
4. Temperature of the column.
5. Packing of the column.
6. Quality of solvents (low viscosity )

25. Advantages
Any type of mixture can be separated
 Any quantity of mixture can be separated
Wider choice of Mobile Phase
 Automation is possible

26. Disadvantages
Time consuming
 more amount of Mobile Phase are required
 Automation makes the techniques more
complicated
 Expensive

27. High-Performance Liquid
Chromatography
(HPLC)

28. HPLC
• HPLC stands for High performance liquid
chromatography sometimes referred as High
Pressure Liquid Chromatography.
• HPLC is a powerful tool in analysis because it
yields high performance as compared to
traditional column chromatography.
• It can separate mixture of compounds.

29. Conti...
• High performance liquid chromatography is
improved form of column chromatography.
• It pumps a sample mixture in a solvent (known
as the mobile phase) at high pressure through a
column with chromatographic packing material
(stationary phase).
• HPLC has the ability to separate, and identify
compounds that are present in any sample that
can be dissolved in a liquid in trace
concentrations.

30. Instrumentation
The components use in HPLC system include:
 the solvent reservoir or multiple reservoirs
 a high-pressure pump
 a column
 injector system
 the detector.

32. HPLC work
• A reservoir holds the solvent. A pump is used to
generate a specified flow of the mobile phase.
• An injector is able to inject the sample into the
continuously flowing mobile phase stream that carries
the sample into the HPLC column.
• The column contains the chromatographic packing
material needed to effect the separation.
• This packing material is known as stationary phase.
• Then a detector is needed to see the separated
compound bands as they elute from the high pressure
column.

33. Conti...
• The information is sent to the computer by
the detector, which generates the
chromatogram.

34. • Mobile phase enters the column from the left and exits at the
right.
• when the sample enters the column and begins to form a
band.
• The sample shown here, a mixture of yellow, red, and blue
dyes, appears at the inlet of the column as a single black
band.
• individual dyes have moved in separate bands at different
speeds.

35. • As the separated dye bands leave the column, they pass
immediately into the detector.
• The detector contains a flow cell that detects each separated
compound band.

36. AFFINITY CHROMATOGRAPHY

37. INTRO ...
• Affinity chromatography
separates proteins on
the basis of a reversible
interaction between a
protein and a specific
ligand coupled to a
chromatography matrix.
• AC is designed to purify
a particular molecule
from a mixed sample.

38. • The technique can be
used to separate active
biomolecules from
denatured or functionally
different forms, to isolate
pure substances present
at low concentration in
large volumes of crude
sample and also to
remove specific
contaminants.

39. Principle ...
• Affinity chromatography is principally based
on the molecular recognition of a target
molecule by a molecule bound to a column.
• It relies on the reversible interactions between
the protein to be purified and the affinity
ligand coupled to chromatographic matrix.
• Binding is;
** Reversible, &
** Specific.

40. Components ...
While affinity chromatography is used for the
purification and separation of large
biomolecules from complex mixtures, these
components is to be considered;
• The support (matrix),
• Spacer arms, and
• Ligand.

41. Matrix ...
• chemical inertness; does not react (or bind) with
sample.
• chemical stability; resistance toward microbial and
enzymatic attack.
• Chemical reactivity allowing ligands and spacers to be
introduced .
• Good flow properties for rapid separation
Such as;
 Agarose,
 Polyacrylamide,
 Cellulose, and silica.

42. Ligands ...
• Ligands are ions or neutral molecules attach to
the matrix that bond to a biomolecule
(protein).
The selection of the ligand for affinity chromatography is influenced by
two factors:
o the ligand must exhibit specific and reversible binding affinity for
the target substance
o and it must have chemically modifiable groups that allow it to be
attached to the matrix without destroying binding activity.

43. Spacer Arms ...
• The binding sites of the
target molecule are
sometimes deeply located
and difficult to access due
to steric hindrance, a
spacer arm is often
incorporated between the
matrix and ligand to
facilitate efficient binding
and create a more
effective and better
binding environment.

44. Steps:
Affinity purification involves 3 main steps:
• Incubation of a crude sample with the affinity
support to allow the target molecule in the
sample to bind to the immobilized ligand.
• Washing away non-bound sample components
from the support.
• Elution (dissociation and recovery) of the target
molecule from the immobilized ligand by altering
the buffer conditions so that the binding
interaction no longer occurs.

45. • Step 1. Loading affinity
column

46. • Step 2. Proteins sieve
through matrix of
affinity beads.

47. • Step 3. Proteins interact
with affinity ligand with
some binding loosely
and others tightly.

48. • Step 4. Wash off
proteins that do not
bind and that are
loosely bond.

49. • Step 5. Elute proteins
that bind tightly to
ligand and collect
purified protein of
interest.

50. Kinds of ELUTION ...
• Elution by change in buffer composition that
elutes the bound substance without harming
it .
• pH Elution; high pH applied that elute the
substance but damage it either temporary or
permentaly.
• Competitive elution; by addition of substance
that competes for binding to protein with
ligand.

51. Ion Exchange Chromatography
• The separation of molecules based on their
total charge.
• This technique enables the separation of
similar types of molecules that would be
difficult to separate by other techniques
because the charge carried by the molecule of
interest can be readily manipulated by
changing buffer pH.
• Used in separation of proteins.

52. Protein
• Made of 20 type of Amino Acids
• Amino acid carry different charges based on
their side chains
• Protein carry net –ive or +ive charge
depending on types of AA present
• Protein differ by charge from one another
• separated by ion exchange

53. Stationary phase
• Solid phase
• Gel beads – 0.30 to 0.85nm in diameter
• Carbohydrate polymers
• Cellulose
• Agarose
• Fixed and inert
• Side chains added to make them +ive or –ive
charge

54. Stationary phase

55. • Anion exchange
Matrix
X - A+ ………… B-
Mixture:
D- E+
Ion Exchange:
X - A+ ……. D-
B- E+
• Cation exchange
Matrix
X – A- ………… C+
Mixture:
D- E+
Ion Exchange:
X – A- ……. E+
C+ D-
Elute Elute

56. • Assembly
• Funnel
• Colum filled with gel beads
• Eluent collected at bottom

57. Process(eg cationic exchange)
• Mixture of 3 types of protein
• Coloumn filled with –ively charged resins
• Pour mixture in funnel
• Initially they will collect at top
• After sometime move downward due to gravity

58. • The negative protein repels the resin and
move fastest, collected at bottom
• Slightly positive will bind to resin at middle
and move downward after sometime
• Strongly positive will bind at top
• To seprate bound proteins elution is carried
out

60. Elusion
• Two types
– Salt gradient
– PH gradient

61. Salt gradient
• Principle: increase net +ive ions
• Salt solution poured in column e.g. NaCl
• Na+ Ions produced, compete with protein ions
to bind with resin
• Less strongly bound protein elute first
• Concentration of salt increased and more
strongly bound proteins separate
• Proteins collected at bottom

63. PH Gradient
• Principle:

64. • Mixture of buffer added
– Binding Buffer, low pH
– Elusion Buffer, high pH
• pH gradient produced
– cationic exchange: low pH to High pH buffer
– Anionic exchange: high pH to low pH buffer

65. Example of cationic exchange
• Low pH buffer added first, causes formation of
more +ive charge on protein
• Then high pH buffer added, it generates pH
gradient
• protein moves to region that is equal to their
isoelectric point
• More elusion buffer added that cause increase in
pH and protein now carry negative charge
• Proteins desorb and elute from column