Chromatography Lecture PDF

CHROMATOGRAPHY Dr. Rajagopalan Prasanna  CHROMATOGRAPHY It is an analytical technique that deals with the separation of closely related compounds from the mixture. This technique is used to separate the mixture of proteins, peptides, amino acids, lipids, carbohydrates, vitamins a...

CHROMATOGRAPHY Dr. Rajagopalan Prasanna  CHROMATOGRAPHY It is an analytical technique that deals with the separation of closely related compounds from the mixture. This technique is used to separate the mixture of proteins, peptides, amino acids, lipids, carbohydrates, vitamins and drugs  Physical Basis of Separation The individual compounds (within a sample) are separated on the basis of differences in their physical characteristics, e.g. Molecular size Shape Charge Volatility Solubility and / or Absorptivity  Essential Components of Chromatographic system 1. Stationary phase: – It is a porous solid matrix through which the sample dissolved in the mobile phase, percholates. 2.Mobile phase: – It is the mixture of substances(to be separated) dissolved in a LIQUID or GAS  Physical Basis of Separation All forms of chromatography work on the same principle They all have – A stationary phase – A mobile phase The mobile phase flows through the stationary phase and carries the components of the mixture with it The compounds from a mixture are separated by a continuous redistribution between TWO (mobile and stationary) phases  Definition: Eluent: fluid entering a column Eluate: fluid exiting the column Elution: the process of passing the mobile phase through the column. Gel Exclusion (Gel filtration, Gel permeation) – separates molecules by size; sieving - not real interaction, small molecules travel longer Ion Exchange – Attraction of ions of opposite charges (for ionic compounds -anions or cations)  Thin Layer Chromatography Stationary Phase: uniform layer of silica gel, cellulose or alumina coated onto a piece of glass or metal (It is the type of “Solid-liquid adsorption Thechromatography”) separation relies on the relative affinity of compounds towards both the phases. After sample has been applied on the stationary phase, a solvent ( mobile phase) is drawn up the plate via capillary action Compounds which have a higher affinity to the stationary phase move slowly while the other compounds travel fast. On completion of the separation process, the individual components from the mixture appear as spots at respective levels on the plates. The different spots can be seen under the UV-lamp  Thin Layer Chromatography Measuring Rf values Each spot has a retention factor (Rf) expressed as: Rf = dist. travelled by sample / dist. travelled by solvent For example, if the red component travelled 2.1 cm from the base line while the solvent had travelled 2.8 cm, then the Rf value for the red dye is: Rf = 2.1/2.8 = 0.75 TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical metabolites from blood plasma, urine, body fluids, serum, etc  Paper Chromatography Paper chromatography: It is the type of “Solid-liquid partition chromatography” Stationary phase is the cellulose filter paper and the mobile phase is liquid, where the particles are separated on the basis of their polarity towards both the phases. Paper Chromatography: Process  Paper Chromatography: Process After sufficient migration of solvent front across 80 to 90% of its length (about 1 cm below from the top edge), paper chromatogram is removed, dried and developed for identification of specific spots Example : alfa-AMINO ACIDS – NINHYDRIN, which forms PURPLE complex with alfa- AMINO ACIDS is used as a coloring reagent. – The chemical nature of the individual spots(amino acids) can be identified by running the known standards with the UNKNOWN mixture. TLC is used to measure the amino acid concentration in the following conditions: 1.Phenylketonuria: Genetic disorder that prevents the normal breakdown of a protein found in some foods 2.Alkaptonuria: very rare inherited disorder that prevents the body fully breaking down two protein building blocks (amino acids) called tyrosine and phenylalanine 3.Maple syrup disease: A rare but serious inherited condition. Body cannot process certain amino acids (Leucine, isoleucine, and valine), causing a harmful build-up of substances in the blood and urine Thank You