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 2017-2018 11/16/2018

1.3.1. Based on Solubilities Precipitation, Filtration Crystallization

1.3.2. Based on Gravity Centrifugation Sedimentation Decantation

Centrifugation

Decantation

Fractional Distillation 1.3.3. Based on volatility Distillation Fractional Distillation

Distillation

1.3.4. Based on Electrical effects Electrophoresis

Electrophoresis B - + 11/16/2018

1.3.5. Based on retention capacity of a Stationary Phase Chromatography

1.3.6. Based on distribution in two immiscible phases Solvent Extraction

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

ION EXCHANGE CHROMATOGRAPHY H+ Na+ H+ R 11/16/2018

ION EXCHANGE CHROMATOGRAPHY H+ Cl- OH- R 11/16/2018

1.4. Electrophoresis: Principles, Basic Instrumentation, Working and Application in separation of biomolecules like enzymes and DNA. (02L)

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 .

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

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

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.

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.

The Technique

The Technique

The Technique

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. 

1.6. Chromatography : (05L) 1.6.1. Introduction to Chromatography 1.6.2. Classification of chromatographic methods based on stationary and mobile phase

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 11/16/2018

Adsorption Chromatography Column Chromatography S+L OR S/G Thin Layer S+L Column Chromatography Ion Exchange Gas Solid Chromatography S+G Gel Chromatography 11/16/2018

Partition Chromatography L+L OR L+G Paper Chromatography L+L HPLC Gas Liquid L+G 11/16/2018

Applications of paper chromatography Principle Techniques Applications of paper chromatography

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. 11/16/2018

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 11/16/2018

Solvent Front S O L V Solute front E N t F W Solute front Original Line 11/16/2018

EXPERIMENTAL PROCEDURE Preparation of the Paper. Solvent system used Preparation of the sample. Application of the sample. Development of the Chromatograms. Identifying the Spots 11/16/2018

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. 11/16/2018

Preparation of Paper 15-20 Cm Sample application 3-5 Cm 11/16/2018

Preparation of Paper 15-20 Cm Sample application 3-5 Cm 11/16/2018

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 11/16/2018

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 11/16/2018

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. 11/16/2018

Preparation of Paper 15-20 Cm Sample application 3-5 Cm 11/16/2018

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 11/16/2018

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 11/16/2018

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. 11/16/2018

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. 11/16/2018

Development of the Chromatograms Ascending Descending Horizontal Radial Multiple 11/16/2018

Ascending Paper Chromatography Mobile Phase 11/16/2018

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. 11/16/2018

Physical Method In this method paper is exposed to ultraviolet light in the wave length range 240-260 nm. The compound produces florescent spots. Compounds which do not produce florescent spots are exposed to high pressure mercury vapour lamp. 11/16/2018

Physical Method Sun rays U.V.Light Paper Chromatogram 11/16/2018

Unsaturated Compounds Chemical Method H2S gas Iodine vapors Acid –base indicators Ninhydrin Carboxylic acids Unsaturated Compounds Metal ions Cu+2 Amino acids 11/16/2018

Chemical Method H2S Gas Separated metal ions Paper Chromatogram 11/16/2018

Detection of Carboxylic acids Chemical Method Acid-Base Indicators Detection of Carboxylic acids Paper Chromatogram 11/16/2018

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.) 11/16/2018

Application of Paper Chromatography 1 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. 11/16/2018

1.6.4. Thin layer Chromatography Principle technique Application: Determination of Purity of substance Application in determining progress of a given reaction

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.

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

OH OH Silica Gel OH Silica Gel OH OH OH Mobile phase OH

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

. 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

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

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

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

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.

Experimental Procedure Thin layer Chromatography

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

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.

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

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.

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

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

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

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

Methods of Plate Development Ascending development Descending Development Two dimensional Development

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

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

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.

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