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Chromatography-DR SALMAN KHAN
Chromatography is a technique used to separate mixtures into individual components. It allows scientists to analyze, identify, purify, and quantify mixtures and their components. The main types of chromatography are partition chromatography, adsorption chromatography, ion exchange chromatography, gel filtration chromatography, affinity chromatography, and high pressure liquid chromatography. Each type uses different properties of the components and stationary and mobile phases to separate mixtures.
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Chromatography-DR SALMAN KHAN
- 1. Chromatography chromatography DR SALMAN KHAN SRISAI DENTAL COLLEGE ,VKB 1st Year Student
- 2. What is Chromatography? Chromatographyis a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components. Separate • Analyze • Identify • Purify • QuantifyComponentsMixture
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- 5. Uses for Chromatography Chromatographyis used by scientists to: • Analyze – examine a mixture, its components, and their relations to one another • Identify – determine the identity of a mixture or components based on known components • Purify – separate components in order to isolate one of interest for further study • Quantify – determine the amount of the a mixture and/or the components present in the sample
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- 11. Preparing the ChromatographyStrips ● Take a strips of filter paper (Whatman No. 1 or 3) ● Draw a line 2 cm above the bottom edge of the strip with the pencil ● Label each strip with its corresponding solution ● Place a spot from each pen on your starting line
- 12. Preparing the Solventmixture solvent mixture consisting of •BUTANOL: ACETIC ACID :WATER 4 : 1 : 5
- 13. Developing the Chromatograms ● Placethe strip in the beaker ● Make sure the solution does not come above your start line ● Keep the beakers covered ● Let strips develop until the ascending solution front is about 2 cm from the top of the strip ● Remove the strip and let it dry
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- 17. Rf = distancemoved by substance distance moved by solvent front compounds
- 18. Thin Layer Chromatography ● Thinlayer of silica gel is spread on a glass plate ● TLC takes 2-4hrs in whereas paper chromatography takes 14-16 hrs ● Can be performed as two dimensional
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- 20. Gas Liquid Chromatography Herethe mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube. And the stationary phase is an involatile liquid held on particles of a solid support.
- 22. In the animationbelow the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.
- 24. 2.Adsorption chromatography ● Separation isbased on differences in adsorption at the surface of solid stationary medium ● Alumina,silica gel are used ● These are packed into columns ● Components get adsorbed at different affinity
- 25. 3.Ion Exchange chromatography ● Separationof molecules on the basis of their electrical charges ● Ion exchange resins – cation exchangers and anion exchangers are used for this purpose
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- 28. 4.Gel filtration chromatography Alsocalled molecular sieving Hydrophilic cross linked gels –agarose ,dextran ,acrylamide are used Technique is used for (1)seperation ,purification of proteins (2)mol.wt determination Eg:insulin,immunoglobulins
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- 31. 5.Affinity chromatography ● Based onhigh affinity of specific protein for specific chemical group ● Eg:co-enzymes can be used to purify enzymes
- 32. 6.High pressure liuidchromatography (HPLC) ● Require use of non compressible resin material and strong metal column ● Widely used for separation of all types of compounds ● This allows high flow rates ● Seperation is achieved with better resolution and high speed