1. Satish Pradhan Dnyanasadhana College, Thane. Department of Chemistry S
Satish Pradhan Dnyanasadhana College, Thane. Department of Chemistry S.Y.B.Sc. Analytical Chemistry Paper-III Sem-IV Separation Techniques in Analytical Chemistry By Dr.g.r.bhagure
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2. Based on Solubilities
Precipitation,
Filtration
Crystallization

3. Based on Gravity
Centrifugation
Sedimentation
Decantation

4. Centrifugation

5. Decantation

6. Fractional Distillation
Based on volatility
Distillation
Fractional Distillation

7. Distillation

8. 1.3.4. Based on Electrical effects
Electrophoresis

9. Electrophoresis B - +
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10. 1.3.5. Based on retention capacity of a Stationary Phase
Chromatography

11. 1.3.6. Based on distribution in two immiscible phases
Solvent Extraction

12. Based on capacity to exchange with a resin
1.3.7.
Based on capacity to exchange with a resin
Ion Exchange

13. ION EXCHANGE CHROMATOGRAPHY H+
Na+ H+ R
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14. ION EXCHANGE CHROMATOGRAPHY H+
Cl-
OH- R
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15. 1.4. Electrophoresis: Principles, Basic Instrumentation, Working and Application in separation of biomolecules like enzymes and DNA. (02L)

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 Proteins The 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. 

27. 1.6. Chromatography : (05L)
Introduction to Chromatography
Classification of chromatographic methods based on stationary and mobile phase

28. Adsorption Chromatography S+L OR S/G Partition Chromatography
Classification
of Chromatography
Adsorption Chromatography
S+L OR S/G
Partition Chromatography
L+L OR L+G
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29. Adsorption Chromatography Column Chromatography
S+L OR S/G
Thin Layer
S+L
Column Chromatography
Ion Exchange
Gas Solid Chromatography
S+G
Gel Chromatography
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30. Partition Chromatography
L+L OR L+G
Paper Chromatography
L+L
HPLC
Gas Liquid
L+G
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31. Applications of paper chromatography
Principle
Techniques
Applications of paper chromatography

32. Paper Chromatography L+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.
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33. Solvent Front S O Solute front (B) L V E N t F Solute front (A) W
Distance traveled by solute
Rf =
Distance traveled by solvent
Solvent Front S O L V E N t F W
Solute front (B) 8
Rf (A) = =0.5 16
Solute front (A) 12
Rf(B)= = 0.75 16
Original Line
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34. Solvent Front S O L V Solute front E N t F W Solute front
Original Line
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35. EXPERIMENTAL PROCEDURE
Preparation of the Paper.
Solvent system used
Preparation of the sample.
Application of the sample.
Development of the Chromatograms.
Identifying the Spots
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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.
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37. Preparation of Paper
15-20 Cm
Sample application
3-5 Cm
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38. Preparation of Paper
15-20 Cm
Sample application
3-5 Cm
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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
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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
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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.
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42. Preparation of Paper
15-20 Cm
Sample application
3-5 Cm
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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
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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
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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.
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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 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.
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47. Development of the Chromatograms
Ascending
Descending
Horizontal
Radial
Multiple
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48. Ascending Paper Chromatography
Mobile Phase
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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.
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50. Physical Method
In this method paper is exposed to ultraviolet light in the wave length range nm.
The compound produces florescent spots. Compounds which do not produce florescent spots are exposed to high pressure mercury vapour lamp.
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51. Physical Method
Sun rays
U.V.Light
Paper
Chromatogram
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52. Unsaturated Compounds
Chemical Method
H2S gas
Iodine vapors
Acid –base indicators
Ninhydrin
Carboxylic acids
Unsaturated Compounds
Metal ions
Cu+2
Amino acids
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53. Chemical Method H2S Gas Separated metal ions Paper Chromatogram
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54. Detection of Carboxylic acids
Chemical Method
Acid-Base Indicators
Detection of Carboxylic acids
Paper
Chromatogram
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55. Qualitative Analysis Or Interpreting the Data
The Rf value for each spot should be calculated. Rf stands for "ratio of fronts" and is characteristic for any given compound. Hence, known Rf values can be compared to those of unknown substances for the identifications.
Distance traveled by solute
Rf =
Distance traveled by solvent
Note: Rf values often depend on the temperature, solvent, and type of paper used in the experiment; the most effective way to identify a compound is to spot known substances next to unknown substances on the same chromatogram.)
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56. Application of Paper Chromatography1
For the separation of organic, inorganic, biochemical and natural products 2
Metal ions having same chemical properties or belonging to the same group can be detected and separated 3
The technique is useful for the separation of metal ligand chelate or complexes, it’s also useful for the separation of isomer. 4.
The purity of a sample can be tested .In this case pure sample can produce only one spot where as impure sample can produce two or more. 5
It is useful in the field of biochemistry. 6
It is very useful technique in the field of food, drugs and cosmetics.
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57. 1.6.4. Thin layer Chromatography Principle
technique
Application: Determination of Purity of substance
Application in determining progress of a given reaction

58. Principle: The sample is dissolved in in a volatile solvent
Sample is applied with the help of capillary on to the base line drawn on the solid adsorbent.
The plate is dipped in to the solvent working as mobile phase.
As the mobile phase rises up the TLC plate by capillary action, the components dissolve in the solvent and move up the TLC plate.
In adsorption separation will occur on the basis of differences in adsorption (preferential adsorption). Weekly adsorbed component will separate first and strongly later on.
In partition , if the component is soluble in the mobile phase it will move with the solvent and vise versa .
The movement of the solute is measured relative to that of solvent is expressed as the retardation factor.

59. This technique manipulates POLARITY
More polar substances bind strongly to the adsorbent and elute SLOWER
Less polar substances bind weakly to the adsorbent and elute FASTER
The strength of interactions between the adsorbent and eluting components vary approximately in this order:
Salt formation > coordination > H-bonding >
dipole-dipole > van der Waals
Less Polar
More Polar
Polarity decreases

60. OH OH
Silica Gel OH
Silica Gel OH OH OH
Mobile
phase OH

61. Sample to be applied on this area
Less Polar
More Polar
Adsorbs weakly and separate very fast
Adsorbs stronger and separate very slowly
Sample to be applied on this area

62. . distance traveled by substance . distance traveled by solvent front
The Rf Value
A given compound will always travel a fixed distance relative to the distance the solvent travels
This ratio is called the Rf value and is calculated in the following manner:
. distance traveled by substance . distance traveled by solvent front

63. Materials used in TLC Glass Plate Adsorbents
Oven for activation of plate
Developing chamber
Mobile Phase
A device for applying the adsorbent layer
Storage facility for the prepared plate

64. A device for applying the adsorbent layer
Materials used in TLC
Glass Plate
Hooper
A device for applying the adsorbent layer
Mobile phase
Developing chamber

65. Stationery phase Alumina (Al2O3) Stationery phase Description
Application
Silica gel G
Silica gel with average particle size 15µm containing ca 13% calcium sulfate binding agent
Used in wide range pharmacopoeial test
Silica gel G254
Silica gel G with fluorescence added
Same application with Silica gel G where visualization is to be carried out under UV light.
Alumina (Al2O3)
Cellulose
Cellulose powder of less than 30µm particle size.
Identification of tetracycline's

66. MOBILE PHASE TLC Solvents or Solvent Systems.
A single solvent or mixture of two solvents can work as mobile phase in TLC .Ex. petroleum ether, carbon tetrachloride, chloroform, ethyl acetate, hexane can used as mobile phase.
The ability of mobile phase to move up is depend on the polarity itself
Volatile organic solvents is preferably used as mobile phase.

67. Experimental Procedure
Thin layer Chromatography

68. Methods used to apply adsorbent Dipping plate in slurry
TLC Plate Preparation
Methods used to apply adsorbent
Spreading
Spraying
Dipping plate in slurry

69. TLC Plate Preparation
TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility.
They are prepared by mixing the adsorbent, such as silica gel , with a small amount of inert binder like calcium sulphate (gypsum) and water. This mixture is spread as thick slurry on an uncreative carrier sheet, usually glass, thick aluminum foil, or plastic.
The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes
Around 0.5 – 2.0 mm for preparative TLC.

70. Spreading the slurry by Hooper
Materials used in TLC
Glass Plate
Spreading the slurry by Hooper
Mobile phase
Developing chamber

71. Activation of plate Glass Plate
Plate is kept for drying in oven at 100OC.
This step is called as activation of plate.
By doing this surface area of the adsorbent increases.

72. Drawing a Line and circle to apply the sample
Glass Plate
Circle to apply sample
Mobile phase
Developing chamber

73. Experimental Procedure
TLC Chamber Preparation
Cut the filter paper so that it fits in the jar, touching the bottom and reaching a height of about 1cm from the top of the jar
To ensure that the filter paper will work, put it in the jar, and then place an unused TLC plate in the jar. If the above criteria are met and the plate doesn’t make any contact with the filter paper, the setup should work
Remove the TLC plate, and then completely saturate the filter paper with the development solvent using a pasteur pipet.
Fill the jar with development solvent to a depth no greater than 0.5cm
Put the lid on the jar to preserve the saturated conditions

74. Application of sample Spotting the TLC Plate
Dip the open end of a capillary tube into the solvent containing the compound to be eluted
Touch the end of the capillary tube lightly and very briefly to the coated surface of the TLC plate
Your spots should be made on the line drawn across the plate in the correct lanes and shouldn’t have a diameter much larger than the capillary tube
After spotting the plate, place it in the saturated chamber and close the lid
Substances should be eluted until the solvent front reaches a height of about 0.5cm from the top of the TLC plate

75. Materials used in TLC Glass Plate Circle to apply sample Mobile phase
Developing chamber

76. Methods of Plate Development
Ascending development
Descending Development
Two dimensional Development

77. TLC Visualization Methods
Physical Methods:
Ultraviolet Light—some organic compounds illuminate or fluoresce under short-wave UV light:

78. Chemical methods
Iodine Vapor—forms brown/ yellow complexes with organic compounds
Fluorescent Indicators—compounds fluoresce when placed under UV light
Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon exposure to light
Sulfuric Acid Spray + Heat—permanent charred spots are produced

79. Applications of TLC Qualitative analysis: -
If the separated components are colored then identification is very easy. All the visualizing agents used in paper chromatography (Detecting agents or indicators) can be used in TLC.
From Rf value qualitative analysis can be performed.

80. Thank you