Separation Techniques

• Chromatography

   

  •It involves the separation of a mixture on the basis of specific differences of the physical-chemical characteristic of the different components on a supporting medium called adsorbent or sorbent.
• The constituents of the mixture are separated by a continuous redistribution between two (2) phases:

   

  1.Stationary phase

  2.Mobile phase (also called eluent or carrier fluid)
• Paper Chromatography

  • Principle

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  A spot of the substance to be fractionated is placed on the paper just above the solvent level.

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  The organic solvent (mobile phase) moves up through the paper by capillary action and various fractions in the sample move at different rates.
• Paper Chromatography

  • Sorbent
  • Special grade of filter paperExample: Whatman Phase Separating Paper
• Paper Chromatography

  • Basis of Separation-Rate of diffusion-Solubility of the solute-Nature of the solvent
• Paper Chromatography

  • Clinical Use

  Fractionation of sugars, amino acid and barbiturates
• Gel Chromatography

  • Other names

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  Gel Permeation

  Size Exclusion

  Molecular Sieve

  Gel Filtration

  Molecular Exclusion
• Gel Chromatography

  • Principle
  • Where a mixture of small and large molecules is allowed to pass over small particles in a column, the smaller molecules diffuse into the gel, whereas larger molecules tend to pairs rapidly in the column and appear in the eluate first.
• Gel Chromatography

  • Sorbent
  • Gels with poares of accurately controlled size
  • Types of Gel Chromatography

  1.Hydrophilic gels (water-loving)

  2.Hydrophobic gels (water-fearing)
• Gel Chromatography

  • Hydrophilic Gels (water-loving)
  • Used in the separation of solute in aqueous medium such as enzymes, antibodies and other proteins.

  Examples: dextran; agarose; polyacrylamide
• Gel Chromatography

  • Hydrophobic Gels (water-fearing)

  Used in the separation of solute soluble only in organic solvents such as

  triglycerides and fatty acids.

  Examples: methylated Sephadex; polystyrene beads
• Gel Chromatography

  • Hydrophobic Gels (water-fearing)1.Basis of Separation2.Molecular weight and size3.Charge of the ionsHydrophobicity of the molecules
• Gel Chromatography

  • Clinical Use

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  Fractionation of polysaccharides, nucleic acids, proteins including enzymes and isoenzymes
• Ion-Exchange Chromatography

  • Principle
  • Substances to be separated are passed on the ion exchange column and depending on the net charge and pH of the solution, the substance is absorbed from solution in the ion-charge resin.
  • Ions with greatest charge densities will be held most strongly on an ion-exchange material.
• Ion-Exchange Chromatography

  • Sorbent
  • Anion or cation resin with functional group
  • Basis of Separation

  Differences in sign and ionic charge densities.
• Ion-Exchange Chromatography

  • Clinical Use
  • For the separation of unwanted substances present in a solution mixture.
  • Concentration of solute of interest suspended in highly diluted samples can be determined.
• Ion-Exchange Chromatography

  • Example
  • Natural purification of water as it percolates through soil
• Thin Layer Chromatography

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  • It is used for drug screening (semiquantitative screening test)
  • Sample components are identified by comparison with standards on the same plate
  • Each drug has a retention factor (Rf) value and it must match the Rf of the drug standard
• Thin Layer Chromatography

  • When all drug spots including the standards have migrated with the solvent front, it is cause by incorrect aqueous to nonaqueous solvent mixture.
  • Extraction of the drug is pH dependent - the pH must be adjusted to reduced the solubility of the drug in the aqueous phase.
  • Biological samples such as blood, urine and gastric fluid can be used for the test.
• Thin Layer Chromatography

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  • Sorbent
  • Thin plastic plates impregnated to a layer of silica gel, alumina, polyacrylamide gel or starch gel
• Thin Layer Chromatography

  • Basis of Separation

  • Rate of diffusion

  Solubility of the solute

  • Nature of the solvent
• Liquid-Liquid Chromatography

  • Principle
  • A highly polar substance tends to be more soluble in a highly polar solvent (water), while the less polar substance tends to be more soluble in a less polar solvent (organic solvent). Thus following the "like dissolves like" principle.
• Liquid-Liquid Chromatography

  • Basis of Separation
  • Differences in solubility between two liquid phases1.Aqueous phase2.Organic solvent phase
• Liquid-Liquid Chromatography

  • Clinical Use
  • Fractionation of barbiturates

  Lipid studies
• Column Chromatography

  • Basis of Separation
  • Difference in pH

  Polarity of solvent

  • Clinical Use
  • Fractionation of sugars
• Gas Chromatography

  • Principle
  • It is capable of separating and measuring nanogram and picogram amounts of volatile substances.
• Gas Chromatography

  • Kinds of Gas ChromatographyGas SolidChromatography

  Sorbent is a solid of large surface

  Gas Liquid

  Chromatography

  • Sorbent is nonvolatile liquid
• Gas Chromatography

  • Basis of Separation

  Sample volatility

  • Rate of diffusion into liquid layer of the column packing
  • Solubility of sample gas in the liquid layer
• Gas Chromatography

  • Clinical Use
  • Drug screening and drug analysisFractionation of steroids, lipids, barbiturates, blood, alcohol and other toxicologic substances
• High Performance Liquid Chromatography

  • PrincipleIt follows the concept of selective adsorption. It applies 4,000 - 10,000Ibs/square inch pressure for the rapid identification and separation of high molecular weight components and many labile biologic compounds such as peptides, drugs, hormones, barbiturates, lipids, steroids and antibiotics.
• Electrophoresis

  • It refers to the migration or movement of charged particles in an electric field.
  • A charged particle or ion will migrate towards the anode or cathode depending on the isoelectric pH (pl) of the solution under the influence of an externally applied electric field.
• Electrophoresis

  • Definition of Terms

  lontophoresis - migration of small charged ions

  • Zone electrophoresis - migration of charged macromolecules
• Electrophoresis

  • Definition of Terms

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  Amphoteric - is a molecule whose net charge can be either positive or negative depending on pH conditions

  Electroendosmosis/Endosmosis - movement of buffer ions and solvent relative to the fixed support
• Net Electric Charge

  • The higher the electric charge, the faster the electrophoretic separation will be; the lower the electric charge, the slower is the electrophoretic separation
• Size and Shape of the Molecule

  • The bigger molecules, the slower the separation.
  • Molecular shape also plays a role in determining how far a given proteins moves, however, general statements cannot made about molecular shape since various techniques are affected in different ways by the protein shape (whether it is globular, fibrous, or something in between.
• Electric Fluid Strength

  The actual rare omigrapan is determiners directly hy the radius.

  The higher the ionic strength, the slower the movement.

  • Mobility also depends greatly on buffer type and buffer concentration
• Electric Fluid

  Strength

  • Buffers

  Barbital (Veronal) buffer with pH 8.6

  • Tris-boric EDTA buffer with pH 8.7
• Nature of Supporting

  Media

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  • Zone electrophoresis - electrophoresis on a supporting media

  Paper Electrophoresis

  Earliest supporting media

  Disadvantages: paper is fragile and easily damaged staining of protein in variable
• Nature of Supporting

  Media

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  Starch Gel Electrophoresis

  Using starch base, larger samples could be employed

  Disadvantages: fragile and unable to store results permanently
• Nature of Supporting Media

  Cellulose Acetate Electrophoresis

  A strip consists of a clear plastic backing with a coating of cellulose acetate particles attached to it