Centrifugation

1. Satish Pradhan Dnyanasadhana College, Thane.Department of ChemistryS.Y.B.Sc. Analytical ChemistryPaper-III Sem-IVSeparation Techniques in Analytical Chemistry By Dr.g.r.bhagure2017-2018

4. Centrifugation

5. Decantation

7. Distillation

9. Electrophoresis B - +

13. ION EXCHANGE CHROMATOGRAPHYH+ Na+ • H+ R

14. ION EXCHANGE CHROMATOGRAPHYH+ Cl- • OH- R

16. Father of electrophoresis: Arne Tiselius (Nobel Prize in 1948) • When we place any charged molecules in an electric field, they move toward the positive or negative pole according to the charge they are having. • Proteins do not have any net charge • whereas nucleic acids have a negative charge so they move towards the anode when electric field is applied .

17. Introduction • Electrophoresis is a method whereby charged molecules in solution, chiefly proteins and nucleic acids, migrate in response to an electrical field. • Their rate of migration through the electrical field, depends on the strength of the field, on the net charge, size, and shape of the molecules, and also on the ionic strength, viscosity, and temperature of the medium in which the molecules are moving. • As an analytical tool, electrophoresis is simple, rapid and highly sensitive. • It can be used analytically to study the properties of a single charged species or mixtures of molecules. It can also be used preoperatively as a separating technique

18. 3.4 Factors Affecting gel Electrohoresis • Electrophoretic velocity depends on: • Inherent Factors •  How much charge the particles have •  What is the molecular weight •  Secondary structures (i.e., its shape). • External Environment •  pH of solution •  Electric field •  Solution viscosity •  Temperature

19. Electrophoresis is usually done with gels formed in tubes, slabs, or on a flat bed. • In many electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes, so that the only electrical connection between the two chambers is through the gel.

20. In most electrophoresis units, the gel is mounted between two buffer chambers containing separate electrodes so that the only electrical connection between the two chambers is through the gel.

21. The Technique

22. The Technique

23. The Technique

25. Separation of ProteinsThe principle behind the separation of proteins is similar to that of nucleic acids. Proteins can be separated by paper or cellulose acetate electrophoresis by simply placing a protein sample on a strip of filter paper or cellulose acetate saturated with a buffer, dipping the ends of the strip into chambers of buffer, and subject the strip to an electric field. The separation of most proteins, however, is performed in a polyacrylamide gel. The gel is cast and submerged in a vertical chamber of buffer.Proteins can be separated on the basis of size (molecular weight) alone, net charge alone, or size and charge together. A common technique for separating proteins by size only is sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In this type of separation, a protein mixture is treated with the detergent sodium dodecyl sulfate. The detergent binds and causes the proteins to dissociate into polypeptides and become negatively charged. The proteins thereafter separate into bands according to their sizes alone. Bands are then visualized by staining with silver stain or a protein dye called coomassie blue.Proteins can be separated on the basis of charge alone, using a method called isoelectric focusing. The separation is performed in a glass tube of polyacrylamide gel in which a pH gradient has been established. 

32. Paper ChromatographyL+L • Principle: In paper chromatography stationary phase is liquid as well as mobile phase is also liquid. In paper chromatography solute undergoes partition between the two liquid phases. The rate of transfer of solute and its effective separation on paper will depend on partition coefficient of the solute between the two phases. The solutes from the original mixture will have migrated along paper at different rates, forming a series of separated spots. For identification purposes spots are characterized by Rf values.

33. Distance traveled by solute Rf = --------------------------------------------- Distance traveled by solvent Solvent Front S O L V E N t F L O W Solute front (B) 8 Rf (A) = -------=0.5 16 Solute front (A) 12 Rf(B)= ----= 0.75 16 Original Line

34. Solvent Front S O L V E N t F L O W Solute front Solute front Original Line

36. The paper used for chromatography • Whatman no.1 is strong, medium fast, pure cellulose paper that is widely used. • For the separation of polar substances special ion exchange paper (containing ion exchanging groups) • For the separation of the component which is hydrophilic in nature, esters of cellulose can be used.

37. Preparation of Paper 15-20 Cm Sample application 3-5 Cm

38. Preparation of Paper 15-20 Cm Sample application 3-5 Cm

39. Solvent system:- • In paper chromatography the solvents used as stationary phase and mobile phase should have following characteristics; • The solvents should not react with any component during separation. • The chemical compositions of the solvents should not change with time. • The Rf value for the component should be any where between 0.06 to 0.95. • The distribution ratio of the component should be independent of its concentration. • The solvents used may be miscible or immiscible but one of the solvent should be polar that can work as stationary phase. • The paper shows affinity with polar solvent that can work as stationary phase. • If water is used as stationary phase then no special impregnation is necessary. If polar solvent other than water is used then it is necessary to remove the water from the paper. • Ex. Water and Ethanol

40. Mobile Phase • Solvent system used: one main organic liquid saturated with distilled water. • Polar solvent which is adsorbed on paper is used • The solvent should be cheap, • very pure, • should not volatile by temperature • Its rate of flow should not affected by temperature

41. Preparation of the sample. • The solid sample is dissolved in organic solvent having low boiling point. The percentage of the sample in the solution should be 0.1—1%. About 10 micro liter of the sample are transferred to the paper by using capillary or micro syringe. If the sample is of biological origin, then proteins, lipids and inorganic ions present in excess are to removed for better separation.

42. Preparation of Paper 15-20 Cm Sample application 3-5 Cm

43. Solvent system:- • In paper chromatography the solvents used as stationary phase and mobile phase should have following characteristics; • The solvents should not react with any component during separation. • The chemical compositions of the solvents should not change with time. • The Rf value for the component should be any where between 0.06 to 0.95. • The distribution ratio of the component should be independent of its concentration. • The solvents used may be miscible or immiscible but one of the solvent should be polar that can work as stationary phase. • The paper shows affinity with polar solvent that can work as stationary phase. • If water is used as stationary phase then no special impregnation is necessary. If polar solvent other than water is used then it is necessary to remove the water from the paper. • Ex. Water and Ethanol

44. Mobile Phase • Solvent system used: one main organic liquid saturated with distilled water. • Polar solvent which is adsorbed on paper is used • The solvent should be cheap, • very pure, • should not volatile by temperature • Its rate of flow should not affected by temperature

45. Preparation of the sample. • The solid sample is dissolved in organic solvent having low boiling point. The percentage of the sample in the solution should be 0.1—1%. About 10 micro liter of the sample are transferred to the paper by using capillary or micro syringe. If the sample is of biological origin, then proteins, lipids and inorganic ions present in excess are to removed for better separation.

46. Application of the sample. • The point of the application of the sample or the origin is marked with pencil on the paper. The sample should be applied by micro pipette or capillary. 10-20 micro liter sample is to be applied. After application of sample on the marked spot solvents associated with sample solution is evaporated by using hair drier or current of hot air.

48. Ascending Paper Chromatography Mobile Phase

49. Identifying the Spots • Location of the substances: • The substances separated by paper chromatography are colorless. • These separated substances are detected by using visualizing agent. • The paper is dried at temperature 100Oc. • The spots are detected by means of physical or chemical method.