chromatography

1. Chromatography
• Topic: Chromatography
• Source: Wilson and Walker’s Principles and
Techniques of Biochemistry and Molecular
Biology (8th Edition)
• Prepared strictly from textbook content for
classroom teaching

2. Introduction to Chromatography
• Chromatography is a separation technique
used to separate components of a mixture.
• The separation is based on differential
distribution of components between two
phases.
• Chromatography is widely used in
biochemistry and molecular biology.

3. Principle of Chromatography
• Chromatography involves a stationary phase
and a mobile phase.
• Components distribute themselves between
these phases.
• Differences in distribution result in separation.

4. Stationary Phase
• The stationary phase is the immobile phase in
chromatography.
• It may be a solid or a liquid supported on a
solid matrix.
• The nature of the stationary phase determines
separation behaviour.

5. Mobile Phase
• The mobile phase moves through the
stationary phase.
• It may be a liquid in liquid chromatography.
• Solutes are carried at different rates by the
mobile phase.

6. Distribution Coefficient
• The distribution coefficient describes solute
partitioning.
• It governs the retention of molecules.
• Different solutes have different distribution
coefficients.

7. Resolution
• Resolution describes how well two
components are separated.
• High resolution indicates good separation.
• It depends on efficiency and selectivity.

8. Factors Affecting Chromatography
• Flow rate, temperature and solvent
composition affect separation.
• Stationary phase properties influence
retention.
• Sample load also affects performance.

9. Classification of Chromatography
• Chromatography is classified based on
separation mechanism.
• Major types include adsorption, partition, ion-
exchange, size-exclusion and affinity
chromatography.
• Each type has distinct applications.

10. Adsorption Chromatography
• Separation is based on adsorption to a solid
surface.
• Different solutes adsorb with different
strengths.
• Stronger adsorption leads to slower migration.

11. Partition Chromatography
• Separation is based on partitioning between
two liquid phases.
• Stationary phase is a liquid coated on a solid
support.
• Solubility differences drive separation.

12. Ion-Exchange Chromatography
• Separation is based on charge interactions.
• Stationary phase contains charged groups.
• Oppositely charged molecules bind to the
matrix.

13. Cation-Exchange Chromatography
• Negatively charged matrices bind cations.
• Positively charged molecules interact with the
matrix.
• Elution is achieved by salt or pH changes.

14. Anion-Exchange Chromatography
• Positively charged matrices bind anions.
• Negatively charged molecules are retained.
• Elution conditions depend on ionic strength.

15. Size-Exclusion Chromatography
• Separation is based on molecular size.
• Large molecules elute first.
• Small molecules enter pores and elute later.

16. Gel Filtration
• Gel filtration is a type of size-exclusion
chromatography.
• It is commonly used for protein purification.
• It is a gentle and non-denaturing technique.

17. Affinity Chromatography
• Affinity chromatography uses specific
biological interactions.
• Ligands are immobilised on the stationary
phase.
• Target molecules bind selectively.

18. Elution
• Elution refers to removal of solutes from the
stationary phase.
• Changes in solvent composition are commonly
used.
• Elution may be isocratic or gradient-based.

19. Isocratic Elution
• Mobile phase composition remains constant.
• Simple to perform.
• Suitable for simple separations.

20. Gradient Elution
• Mobile phase composition changes gradually.
• Improves separation of complex mixtures.
• Widely used in protein chromatography.

21. Column Chromatography
• Column chromatography is widely used for
preparative separations.
• Sample is applied to the top of the column.
• Fractions are collected at the outlet.

22. Chromatographic Media
• Media consist of solid matrices.
• They provide support and interaction sites.
• Common materials include agarose and silica.

23. Flow Rate
• Flow rate influences resolution and time.
• High flow rate reduces resolution.
• Optimisation is essential.

24. Detection Methods
• Eluted components are detected using
suitable detectors.
• UV absorbance is commonly used.
• Detector response is recorded as a
chromatogram.

25. Chromatogram
• A chromatogram is a graphical output of
separation.
• Peaks represent individual components.
• Peak area correlates with concentration.

26. Applications
• Chromatography is used for purification of
biomolecules.
• It is essential in research and biotechnology.
• It underpins many laboratory techniques.

27. Advantages
• High resolution and versatility.
• Applicable to a wide range of samples.
• Many techniques are non-destructive.

28. Limitations
• Can be time-consuming.
• Requires specialised equipment.
• Method optimisation is necessary.

29. Summary
• Chromatography is a fundamental separation
technique.
• Different methods exploit different molecular
properties.
• Proper selection ensures effective separation.

30. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

31. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

32. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

33. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

34. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

35. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

36. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

37. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

38. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

39. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

40. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

41. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

42. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

43. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

44. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

45. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

46. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

47. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

48. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

49. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

50. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

51. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

52. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

53. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

54. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

55. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

56. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

57. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

58. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

59. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter

60. Reference
• Wilson and Walker’s Principles and Techniques
of Biochemistry and Molecular Biology
• Chromatographic methods described in
Preparative Protein Biochemistry chapter