chromatography, HPLC

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<ol><li> 1. Chromatography, HPLC Dr.ANSHUL </li><li> 2. History Mikhail Tswett, Russian, Botanist In 1906 used chromatography to separate plant pigments. He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column. Chroma means color and graphein means to write </li><li> 3. Importance Chromatography has application in every branch of the physical and biological sciences 12 Nobel prizes were awarded between 1937 and 1972 alone for work in which chromatography played a vital role </li><li> 4. Chromatography is a physical method of separation in which the components to be separated are distributed between two phases one of which is stationary (stationary phase) while the other (mobile phase) moves in a definite direction. The chromatographic process occurs due to differences in the distribution constant of the individual sample components. </li><li> 5. Chromatography Technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried faster. </li><li> 6. Classification of chromatography according to mobile phase: 1- Liquid chromatography: mobile phase is a liquid. (LLC, LSC). 2- Gas chromatography : mobile phase is a gas. (GSC, GLC). </li><li> 7. Classification according to the packing of the stationary phase: 1- Thin layer chromatography (TLC): stationary phase is a thin layer supported on glass, plastic or aluminium plates. 2- Paper chromatography (PC): stationary phase is a thin film of liquid supported on an inert support. 3- Column chromatography (CC): stationary phase is packed in a glass column. </li><li> 8. Classification according to the force of separation: 1- Adsorption chromatography. 2- Partition chromatography. 3- Ion exchange chromatography. 4- Gel filtration chromatography. 5- Affinity chromatography. </li><li> 9. Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. The stationary phase: a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase: a developing liquid which travels up the stationary phase, carrying the samples with it. Components of the samples will separate on the stationary phase according to- how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase. Thin Layer Chromatography (TLC) </li><li> 10. Thin Layer Chromatography (TLC) </li><li> 11. Procedure A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, mobile phase, slowly rises up the TLC plate by capillary action. </li><li> 12. As the solvent moves, an equilibrium is established for each component of the mixture between the molecules of that component which are adsorbed on the solid and the molecules which are in solution. components differ in solubility and in the strength of their adsorption to the adsorbent so components will be carried farther up on the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. </li><li> 13. Identification of the Spots If the spots can be seen, outline them with a pencil. If no spots are obvious after staining- hold the plate under a UV lamp Many organic compounds can be seen using this technique. many commercially made plates also contain a substance which aids in the visualization of compounds. </li><li> 14. Visualizing Agents Ninhydrin- Amino acids: Rhodamine B- Lipids. Antimony trichloride- Steroids Aniline phthalate- Sugar. </li><li> 15. Interpreting the Data The Rf (retention factor) value for each spot should be calculated. It is characteristic for any given compound on the same stationary phase using the same mobile phase. Hence, known Rf values can be compared to those of unknown substances to aid in their identifications. </li><li> 16. Application Identification of drugs in urine. Analysis of amino acids &amp; carbohydrates in urine. Analysis of lipids in amniotic fluid.(L/S ratio in RDS.) Limitation- Two different substance may exhibit same Rf value. </li><li> 17. Paper Chromatography A method of partition chromatography using filter paper strips as carrier or inert support. The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases. One is usually water adsorbed on cellulose fibres in the paper (stationary phase). The second is the organic solvent flows and migrate the sample on the paper (mobile phase). </li><li> 18. Paper Chromatography </li><li> 19. Paper Chromatography Application- Detects amino acid, sugar and pigments. Identification of type of aminoaciduria- Cystinuria, PKU. </li><li> 20. Gas Liquid Chromatography Type of partition chromatography used for qualitative and quantitative analysis of large no. of compounds. It provides high speed of resolution very good reproducibility and high sensitivity. It is based on partition of a compound between liquid and gas phase. </li><li> 21. Gas Liquid Chromatography Principle : Volatile molecule (lowest boiling point) swept rapidly along with carrier gas. Molecules soluble in liquid phase retained in liquid . Volatility and solubility affect rate of flow of sample molecule in gas column. </li><li> 22. Gas - Liquid Chromatography Sample introduced by syringe. Column separates components. (Heated in oven) Detector monitors compounds emerging from outlet. Recorder plots signals as a chromatogram. </li><li> 23. Uses Of GLC Volatile organic compound separation. Non volatile substance separation (converting into volatile by oxidation, acylation, alkylation) Alcohol, ester, fatty acid and amines present in biological sample can be separated. Concentration of individual element such as carbon and hydrogen - determined accurately. Functional group can be identified. Position of double bond can be determined by cleaving the bond by oxidation. </li><li> 24. Adsorption Chromatography Principle : It is differential adsorption of solute on the surface of stationary phase. Certain solid material have ability to hold molecule at their surface. This adsorption forces involves weak &amp; non ionic forces such as hydrogen, Vanderwals forces. This is used for separation of organic compound. </li><li> 25. Adsorption Chromatography Adsorbent : Should be inert (should not react with eluting solvent). Should be stable for long peroid. Should be cheap. Most commonly used adsorbent are : Silica gel- AA and steroids. Alumina -small organic molecule . Activated carbon carbohydrate and protein Most strongly adsorb component forms top most band while least adsorb component forms lower most band on adsorbent media </li><li> 26. Adsorption Chromatography Factors Affecting : Nature of adsorbent. Nature of solvent. Rate of flow of solvent through column. Dimension of column. Temperature of column during separation. </li><li> 27. Adsorption Chromatography Applications : Separation of Aromatic compound phenol, amine etc. Aliphatic hydrocarbon from aromatic hydrocarbon Nucleic acid </li><li> 28. Ion Exchange Chromatography Principle : Based on exchange of ions b/w charges stationary phase and ions of opposite charge in mobile phase. Soluble mixture to be separated, allowed to pass through a column of ion exchange resins which are insoluble polymer containing ionic group. Ion exchange resin made of-cellulose, dextran, acrylamide polymer etc. </li><li> 29. Ion Exchange Chromatography Ion exchange resin : two type 1: Cation exchange resin- contain negatively charge ions (-carboxyl, -phosphate ions and bind covalently to positively charge ion in mobile phase. 2: Anion exchange resin-contain positively charge ion &amp; binds to negatively charged ions in mobile phase </li><li> 30. Ion Exchange Chromatography Application: Separation of Amino acid Glycated hemoglobin Oligonucleotides Removal of inorganic ions from aqueous mixture. Disadvantage : Different substance with same ionic charge can not be separated </li><li> 31. Gel Filtration Chromatography Syn.(size exclusion, gel permeation, molecular exclusion chromatography) Principle : Separation based on molecular size, shape, weight. A mixture of molecules of different size placed on top of gel filtration column large molecule pass through interstitial space b/w beads without any resistance Small molecule enter in pores of beads and effectively removed from stream of eluting solvent. Movement of small molecule is retarted- eluting out slowly </li><li> 32. Gel Filtration Chromatography Advantage Separation of substance - independent of ph, temperature, ionic strength and buffer composition. No adsorption- labile substance (enzyme) not affected Elution volume- directly related to molecular weight. </li><li> 33. Gel Filtration Chromatography Application : Use to purifying enzymes and other proteins and fractionating nucleic acid. To determine molecular weight of protein. Agarose gel various type of RNA and viruses are fractionated and purified. </li><li> 34. Affinity Chromatography Principle : Based on specific and non covalent binding of substance like protein &amp; enzyme to specific ligand which is attached to gel matrix. Ex. Separation of LDH from RBC- using NAD+ cofactor as ligand which is linked to affinity gel. </li><li> 35. Affinity Chromatography Application : Isolation of albumin, DNA polymerase, coagulation factors. Isolation of glycoproteins. Purifying human immunoglobulins. Purification of bone collagen, HBsAg, ribosome. </li><li> 36. High Pressure Liquid Chromatography (HPLC) Method in which the solution to be analyzed is passed, under high pressure, through a long, thin column packed with tiny beads such that analyses are completed in minutes rather than hours and with improved resolution. </li><li> 37. (HPLC) Columns are packed in strong stainless steel cylinder - withstand high pressure. Non compressible resin - used as supporting media. UV absorption, fluorescence, regenerative index detectors are used. All physiochemical mechanism(adsorption, partition, affinity, ion exchange) are possible in HPLC. Entire HPLC process can be completed in a few minutes of effective separation and detection. </li><li> 38. HPLC </li><li> 39. HPLC Uses : Spectrophotometer detector- identification of drugs in urine and serum. Flurometer- amino acids &amp; other primary amine can be detected. Electrochemical detector- urinary catecholamine &amp; compounds(unsaturated FAs &amp; PGs) can analyzed. Urinary VMA(vanillylmandelic acid), metanephrins, homovanillic acid can be analyzed. </li><li> 40. THANK YOU </li></ol>

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