1. Seperation Techniques for Biomolecules
Humaira Rafique
2011-ag-1158

2. Contents:
Chromatography
Electrophoresis
Microfilteration

3. Chromatography

4. Chromatography
Technique to separate components of a mixture by passing them through a matrix.
Separation occurs because each compound in a mixture interacts differently with the matrix.

5. Types of Chromatography:
Column Chromatography
Size Exclusion Chromatography
Affinity chromatography
Ion Exchange chromatography
High Performance Liquid Chromatography
Fast Performance Liquid Chromatography
Gas Chromatography

6. Column chromatography
Compounds are separated through
differential intermolecular forces between the components of the mixture with the mobile phase and between the components with the stationary phase.

7. COLUMN CHROMATOGRAPHY
Polar components (b) adsorb more strongly to the polar silica and elute after the less polar components (a), which move more quickly with the non-polar (relative to silica) solvent.

8. Column chromatography
Applications:
Column chromatography (CC) is an extremely valuable technique for purification of
Synthetic or
Natural products.

9. Size Exclusion Chromatography
Size Exclusion chromatography is a separation based on size. It is also called molecular exclusion, Gel Filteration or gel permeation chromatography.

10. Size Exclusion Chromatography
Separation mechanism is not based on adsorption.
Resolution depends on particle size, pore size, flow rate, column length and diameter, and sample volume.
Three steps
Equilibration
Sample loading
Elution

12. Size Exclusion Chromatography
Applications:
Fractionation and molecular weight determination of proteins,
nucleic acid separation,
plasmid purification,
polysaccharide fractionation

13. Affintiy Chromatography
Affinity chromatography (AC) is a technique enabling purification of a biomolecules with respect to biological function or individual chemical structure.
The substance to be purified is specifically and reversibly adsorbed to a ligand (binding substance), immobilized by a covalent bond to a chromatographic bed material (matrix).

14. Affinity Chromatography
Two important things to consider;
Finding a ligand specific enough to allow step elution.
Finding conditions for safe binding and release within the stability window of the target molecule and the ligand.

16. Affinity Chromatography
Applications:
It is used to separate and purify the
Proteins
Peptides
DNA fragments, etc

17. Ion Exchange chromatography
Separates compounds based on their net charges. Negatively or positively charged functional groups are covalently bound to a solid support, yielding either a cation or anion exchanger, respectively.

18. Ion Exchange chromatography
Applications:
It is used to separate the
4 Heamoglobin variants
Proteins, etc

19. High Performance Liquid Chromatography (HPLC)
HPLC is also called high-pressure liquid chromatography. 
It is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture.

20. High Performance Liquid Chromatography

21. High Performance Liquid Chromatography
Applications:
HPLC has many uses including
Medical
.Detecting vitamin D
Concentrations in blood serum
Legal
Detecting performance enhancement drugs in urine

22. High Performance Liquid Chromatography
Applications:
Research
Purifying substances from a complex biological sample Separating similar synthetic chemicals from each other Manufacturing (e.g. during the production process of pharmaceutical and biologic products

23. Fast Performance Liquid Chromatography (FPLC)
Liquid chromatography is a term which refers to all chromatographic methods in which the mobile phase is liquid.
The stationary phase may be a liquid or a solid, in the form of a matrix
A type of liquid chromatography where the solvent velocity is controlled by pumps. The pumps control the constant flow rate of the solvents

24. Fast Performance Liquid Chromatography
Applications:
It is used for
Purification of proteins
Rapid purification of RNA
Analyzing lipoprotein profiles
In pharmaceutical (eg, Drugs)
The analysis of chemical components of Chinese herbal medicines

25. Gas Chromatography
The mobile phase of a chromatographic system is gaseous and stationary phase is a liquid coated on inert solid particles, the technique is called Gas Liquid Chromatography or simple Gas chromatography.

26. Gas Chromatography Applications:
Analyze the contents of chemical products
Volatile mixtures are separated
Quality assessment of alcoholic beverages
Analysis of food
Analysis of biomolecules(eg, melanins)

27. Other Types of Chromatography
Hydroxyapatite chromatography
Reverse-Flow Chromatography
Hydrophobic interaction chromatography
Immobilized metal ion affinity chromatography

28. Hydroxyapatite chromatography
Ca5(PO4)3(OH)2 is a form of calcium phosphate that can be used for the separation and purification of
proteins
enzymes
nucleic acids
viruses, and other macromolecules.

29. Reverse-Flow Chromatography
Flow to the column is then reversed and the bound proteins elute from the top of the column in very concentrated form, which helps prevent denaturation.
Especially useful when purifying antibodies using Protein A or other affinity columns.

30. Hydrophobic interaction chromatography
Separates molecules based on their hydrophobicity. Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer.
Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity.

31. Immobilized metal ion affinity chromatography
Most widely used – nickel: also zinc and cobalt
Nickel binds molecules rich in electrons – such as histidine (from the His-tag fame!)
IMAC uses the affinity of histidine’s imidazole side chains for metal ions.

32. Electrophoresis

33. Electrophoresis
s Electrophoresis is a separation method based on the differential rate of migration of charged species in a buffer solution across which has been applied a dc electric field. The rate of migration of a given species depends upon charge and size of ions. dc electric is a separation method based on differential rate of migration of charged species in a buffer solution across which has been applied a dc electric field.

34. Types of Electrophoresis
Gel Electrophoresis
SDS-PAGE Electrophoresis
Immuno Electrophoresis
Capillary Electrophoresis

35. Gel Electrophoresis
Gel electrophoresis separates molecules on the basis of their charge and size.The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to retard the passage of molecules according to their size and shape.

36. How does gel electrophoresis work?
The gel is made from agar
DNA is a negative molecules
Molecules sort based on
Charge
Size
shape

37. The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration.
% agarose: 2% % %
500 bp
200 bp
50 bp
500 bp
200 bp
50 bp
500 bp
200 bp
50 bp
The concentration of gel/buffer will affect the resolution of fragments of different size ranges.

38. Gel Electrophoresis Types: Slab Gel Electrophoresis (SGE)
Polyacrylamide Gel Electrophoresis (PAGE)
Agarose Gel Electrophoresis (AGE)
Pulsed Field Gel Electrophoresis (PFGE)

39. Slab Gel Electrophoresis
Slab separation are carried out on a thin flat layer or slab of a porous semisolid gel containing an aqueous buffer solution within its pores

40. Slab Gel Electrophoresis
Slab gel electrophoresis can have either a horizontal or vertical format.
Sample is introduced into wells at the top of the gel.

41. Slab Electrophoresis Limitations: Slow Labor intensive
Difficult to automate
Does not yield very precise
Quantitative information
Joule heating

42. Polyacrylamide Gel Electrophoresis
Acrylamide, in combination with a cross linker, methylene bis-acrylamide
Synthetic, consistent polymer
Polymerization catalysts: ammonium persulfate (APS) plus N,N,N',N'-tetramethylethylenediamine (TEMED), or light activation

43. PAGE Resolves 1 bp difference in a 1 kb molecule (0.1% difference),
Tracking and loading dyes are used.
Different buffers are used for protein separation.
Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), Tris Phosphate EDTA (TPE) used most often for DNA.

44. Native PAGE (non-denaturing PAGE)
Types of PAGE
Native PAGE (non-denaturing PAGE)
The proteins are resolved in the absence of SDS
SDS-PAGE (Denaturing PAGE)
The proteins are resolved in the presence of SDS

45. Feature of PAGE High resolving power.
Acceptance relatively large sample size.
Minimal interaction b/w migrating molecules & matrix.
Physical stability of matrix.
Separation basis
Molecular sieving
Electrophoresis mobility

46. Agarose Gel Electrophoresis
Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA.
It is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.
We will be using agarose gel electrophoresis to determine the presence and size of PCR products.

47. Agarose Gel Electrophoresis

48. SDS-PAGE What is SDS-PAGE?
Based on the migration of charged molecules in an electric field
Separation technique
Uses the Polyacrylamide gel as a “support matrix”. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run.

49. Role of SDS
Denatures proteins by wrapping around the polypeptide backbone.
SDS binds to most proteins in amount roughly proportional to molecular weight of the protein-about one molecule of SDS for every two amino acids
In doing so, SDS creates a large negative charge to the polypeptide in proportion to its length

50. Conclusion
SDS PAGE is a useful method for separating and characterising proteins, where a researcher can quickly check the purity of a particular protein or work out the different number of proteins in a mixture.
Since we did not obtain results for the experiment,
we have to rely on sample results
Cannot validate the experimental technique

51. Pulsed Field Gel Electrophoresis
Very Large DNA Molecules are Separated by Pulsed Field Gel Electrophoresis (PFGE).

52. Immuno Electrophoresis/ID
Ag-Ab reaction
Affinity based
Agarose 1 %

53. Capillary Electrophoresis
Capillary electrophoresis yields high-speed, high-resolution separation on exceptionally small sample volumes

54. Modes of Capillary Electrophoresis
Capillary Zone Electrophoresis
Capillary Gel Electrophoresis
Capillary Isotachophoresis
Capillary Isoelectric Focusing

55. Capillary Electrophoresis

56. Electrophoresis Applications:
It has been applied to a variety of difficult analytical separation problems
Inorganic anions and cations
Amino acids
Vitamins
Drugs

57. Electrophoresis Applications: Carbohydrates Peptides Nucleotides
Proteins
Polynucleotides

58. Summary
Electrophoresis is used to separate molecules by size and/or charge.
Nucleic acid fragments can be resolved on agarose of polyacrylamide gels.
PFGE is used to resolve very large DNA fragments.
CGE is more rapid and automated than slab gel electrophoresis.
The choice of electrophoresis method depends on the type and size of sample.

59. Microfilteration

60. Microfiltration
Separates soluble contaminants remaining within the supernatant
Supernatant may include:
Other proteins
Bio-molecules
Un-used growth media
Microfiltration image from:

61. Microfiltration
MF membranes are easily tested by direct examination, as their pores can be observed by electron microscopy.
Large areas of microfiltration membrane can be tested and verified by a bubble test: Pores of the membrane are filled with liquid, then a gas is forced against the face of the membrane.

62. How does Microfiltration work?
Pressure driven process
Separates:
Components in a solution or suspension based on molecular size
Particles size range:
10mm (starches) to aprx mm (DNA, Viruses, and globular proteins)
Microfiltration image from:

63. Microfiltration

64. Microfiltration Proteins act as the permeate
Applications:
Proteins act as the permeate
Separates larger particles
For example-
Colloids
Fat globules
Cells

65. Microfiltration Applications: Pharmaceutical, Biotechnology,
Medical app’s (used on the vents of sterile water tanks to prevent microbial contamination; protect autoclaves and freeze-dryers during the admission of gas after the duty cycle; etc.)

66. THANKS