Introduction to separation methods In analytical chemistry:

1. Dnyanasadhana College, Thane.Department of ChemistryM.Sc. Analytical Chemistry Sem-I Introduction to Separation Methods in Analytical ChemistryDr.G.R.Bhgaure

2. Introduction to separation methods In analytical chemistry: Precipitation, filtration, distillation, extraction and chromatography, modes Of separation in chromatography: adsorption, partition, ion exchange, size exclusion And electro chromatography.

3. PRECIPITATION, • Precipitation is an ionic reaction carried out • Solution in which positive ion from one substance combines with negative ion of another substance to form sparingly soluble salt is called as precipitation

4. Other types of reactions can be used to chemically separate an analyte and interferent, including precipitation, • Two important examples of the application of precipitation are the pH-dependent solubility of metal oxides and hydroxides, and the solubility of metal sulfides. • Separation of cations in to groups (Semi-Micro qualitative Inorganic analysis )

5. Filtration • Physical method of separation technique

6. Distillation • SEPARATIONS BASED ON A CHANGE OF STATE • Since an analyte and interferent are usually in the same phase, a separation often can be effected by inducing a change in one of their physical or chemical states. • Changes in physical state that have been exploited for the purpose of a separation include liquid-to-gas and solid-to-gas phase transitions. Changes in chemical state involve one or more chemical reactions.

7. Changes in Physical State When the analyte and interferent (matrix) are miscible liquids, a separation based on distillation may be possible if their boiling points are significantly different. • Changes in Chemical State Distillation, sublimation, and recrystallization use a change in physical state as a means of separation.

9. Solvent Extraction • Separations Based on a Partitioning Between Phases • The most important class of separation techniques is based on the selective partitioning • of the analyte or interferent between two immiscible phases. • When a phase containing a solute, S, is brought into contact with a second phase, the solute partitions itself between the two phases.

10. Solute (phase -2) Solute (phase-1)

11. Partition coefficient: • An equilibrium constant describing the distribution of a solute between two phases; only one form of the solute is used in defining the partition coefficient (KD). [solute]phase-2 • KD= -------------------- [solute]phase-1 • If KD is sufficiently large, then the solute will move from phase 1 to phase 2. The solute will remain in phase 1, however, if the partition coefficient is sufficiently small.

12. Liquid -Liquid Extractions • A liquid–liquid extraction is one of the most important separation techniques used in environmental, clinical, and industrial laboratories. • Two examples from environmental analysis serve to illustrate its importance. • Public drinking water supplies are routinely monitored for trihalomethanes (CHCl3, CHBrCl2, CHBr2Cl, and CHBr3) because of their known or suspected carcinogeneity. Before their analysis by gas chromatography, trihalomethanes are separated from their aqueous matrix by a liquid–liquid extraction using pentane. • A liquid–liquid extraction is also used in screening orange juice for the presence of organophosphorous pesticides. A sample of orange juice is mixed with acetonitrite and filtered. Any organophosphorous pesticides that might be present in the filtrate are extracted with petroleum ether before a gas chromatographic analysis.

13. Chromatography • Chromatography, • Modes Of Separation in chromatography: adsorption, partition, ion exchange, size exclusion And electro chromatography.

14. Chromatography • Defination • No.of Phases involved Involved Chromatography • Classification of Chromatography • Adsorption Chromatography • Partition Chromatography

18. Size exclusion Chromatography • Separation of molecule takes place according to molecular size of an analyte. • Stationary phase : Small, approximately 10-mm, porous particles of cross-linked dextrin or polyacrylamide. • Mobile Phase: Liquid

19. Size-exclusion chromatography, which also is called gel permeation or molecular exclusion chromatography, in which separation takes place according to molecular size based on size. • In this technique a column is packed with small, approximately 10-mm, porous particles of cross-linked dextrin or polyacrylamide. The pore size of the particles is controlled by the degree of cross-linking, with greater cross-linking resulting in smaller pore sizes. The sample to be separated is placed into a stream of solvent that is pumped through the column at a fixed flow rate. Particles too large to enter the pores are not retained and pass through the column at the same rate as the solvent. • Those particles capable of entering into the pore structure take longer to pass through the column. Smaller particles, which penetrate more deeply into the pore structure, take the longest time to pass through the column. • Size-exclusion chromatography is widely used in the analysis of polymers and in biochemistry, where it is used for the separation of proteins.

20. Electro chromatography. • Electrophoresis is another class of separation techniques in which analyte's are separated based on their ability to move through a conductive medium, usually an aqueous buffer, in response to an applied electric field. • In the absence of other effects, cations migrate toward the electric field’s negatively charged cathode, and anions migrate toward the positively charged anode. • More highly charged ions and ions of smaller size, which means they have a higher charge-to-size ratio, migrate at a faster rate than larger ions, or ions of lower charge. Neutral species do not experience the electric field and remain stationary. As we will see shortly, under normal conditions even neutral species and anions migrate toward the cathode. • In either case, differences in their rate of migration allow for the separation of complex mixtures of analyte's.

21. Gel Electrophoresis • Electrophoresis is the movement of molecules by an electric current. • Nucleic acid moves from a negative to a positive pole.

22. Capillary electro chromatography :A form of capillary electrophoresis in which a stationary phase is includedwithin the capillary column. • Capillary Electro-chromatography Another approach to separating neutral species is capillary electrochromatography (CEC). In this technique the capillary tubing is packed with 1.5–3-mm silica particles coated with a bonded, nonpolar stationary phase. • Neutral species separate based on their ability to partition between the stationary phase and the buffer solution (which, due to electroosmotic flow, is the mobile phase). Separations are similar to the analogous HPLC separation, but without the need for high-pressure pumps. Furthermore, efficiency in CEC is better than in HPLC, with shorter analysis times.

23. Electrophoresis Buffers • Carry current and protect samples during electrophoresis. • Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), Tris Phosphate EDTA (TPE) used most often for DNA. • 10 mM sodium phosphate or MOPS buffer used for RNA. • Buffer additives modify sample molecules. • Formamide, urea (denaturing agents)

24. ++ + + - - + = = + Capillary Electrophoresis (CE) • Separates solutes by charge/mass ratio. • Capillary gel electrophoresis is used to separate nucleic acids.

25. Capillary Electrophoresis + - + - - + - Battery

26. Electrophoresis Equipment Horizontal or submarine gel

27. Applications • 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.