Download as PDF, PPTX
More Related Content
Similar to CHROMATOGRAPHY 1. vxssdllllllllllllllllllllllllllllllllll
![POSTERIOR PITUTARY HORMONES AND THEIR ACTIONS [Autosaved].pptx](https://cdn.slidesharecdn.com/ss_thumbnails/posteriorpitutaryhormonesandtheiractionsautosaved-250727051631-c85075b8-thumbnail.jpg?width=640&height=640&fit=bounds)
CHROMATOGRAPHY 1. vxssdllllllllllllllllllllllllllllllllll
- 1. CHROMATOGRAPHY Dr. Sobiya Afroz1st year PG Department of Physiology
- 3. Learning Objectives • Definition •History • Introduction • Types • Principles • Development • Procedure • Applications • Advantages • Disadvantages
- 4. • The termis derived from the Greek word“chroma”, meaning color. • The method was first employed by Tswett, a botanist in 1903,for the separation of plant pigments using a column of alumina. • HPLC is used nowadaysto separatealmost all biological substances, including proteins, carbohydrates,lipids and nucleic acids
- 6. CHROMATOGRAPHY • Chromatography meanscolor writing • Chromatography is a method of analysis and separation technique of organic and inorganic compounds • Used for large and small quantities • Quantitative and qualitativeanalysis
- 7. Introduction • Proteins differin their molecular size and charge . • They can be separated, purified, isolated according to the following properties • Molecular weight • Solubility • Electric charge • Adsorption properties • Bioaffinity
- 8. Types of chromatography •Planar chromatography • Eg.thin layer chromatography • Column chromatography / flash chromatography • Gas chromatography • Liquid chromatography • High performance liquid chromatography HPLC
- 10.
- 11. Principle • Chromatography isa technique used to separate a group of similar substances on the basis of certain physical characteristics. • A continuous redistribution between two phases namely stationary and moving (mobile) is done. • The stationary phase selectivity retards the substances relative to mobile phase by virtue of number of attractive forces such as van der waals interactions , hydrogen bonds and dipole moments.
- 12. • ADSORPTION • PARTITION •MOLECULAR SIEVING • ION EXCHANGE • AFFINITY
- 13. ADSORPTION CHROMATOGRAPHY • Inthis technique the separationis based on differencesin adsorption at the surface of a solid stationary medium. • The common adsorbingsubstances used are alumina, silicates or silica gel. • These are packed into columns and the mixture of proteinsto be separated is applied in a solvent on the top of the column. • The components get adsorbed on the column of adsorbent with different affinity. • The fractions slowly move down; the most weakly held fraction moves fastest; followed by others, accordingto the orderof tightness in adsorption. • The eluent fromthe column is collected as small equal fractions and the concentrationof each is measured, in each fraction
- 15. PARTITION CHROMATOGRAPHY • Separationof mixture of amino acids and peptides • Get separated depending on distribution ratios or partition coefficient between two immiscible liquids • Stationary phase : Inert solid support like filter paper or silica gel • Mobile phase : flowing through that support • Used in paper and thin layer chromatography
- 16. PAPER CHROMATOGRAPHY • Sampleis applied as a spot on the filter paper • The edge of this paper is dipped in the solvent with ascending or descending flow • The constituents of the mixture get separated by their different rates of migration on the paper • Different spots are then identified by spraying the paper with a suitable colour reagent
- 17. TYPES OF DEVELOPMENT •ASCENDING CHROMATOGRAPHY • DESCENDING CHROMATOGRAPHY • RADIAL CHROMATOGRAPHY • 2D CHROMATOGRAPHY
- 19. • The fundamentalmeasurement in chromatography is Rf value which is a constant for a given compound in a particular solvent system • Mixture of compounds separated by this method is based on the difference in their Rf value Rf value = distance moved by solute distance moved by solvent Rf is Ratio of front Rf is a constant for a particular solvent system at a given temperature It is always less than 1
- 20. Development • AIM :To separate the given mixture of amino acids and compare the Rf value with the standard by developing a chromatogram • Apparatus: Whatmann filter paper no. 1 chromatography chamber solvent capillary tubes spraying apparatus dryer
- 21. Preparation of sample •1% of solution of amino acids in distilled water • 1gm each of amino acid in 100 ml of distilled water • 1Pinch each of amino acid 3ml of distilled water • REAGENTS: • SOLVENT SYSTEM : • n Butanol: Acetic acid : water = 60:15:25 • SPRAYING REAGENT: • 1 % NINHYDRIN 10 mg in 10 ml acetone • Paper size 12*27 • Standard amino acid solution : 1ml N HCl + pinch of AA powder
- 22. Procedure • Keep aqueoussolvent in the chamber for saturation • Take Whatmann no.1 filter paper and draw horizontal line 2.5 cm above the lower end of paper using a capillary tube • Spot the test sample , standard amino acid solution at a distance of 2.5 cms from each other , 4 in one line • Use capillary tubes to add solution • Immediately dry it with a dryer • Suspend the filter paper with lower end of paper dips into solvent but the horizontal line lies above the surface of solvent
- 23. • After thesolvent front has reached the desired level ( 2-3 hrs ) remove the paper and mark the solvent front with pencil • Dry the paper • Spray the ninhydrin solution uniformly with spraying apparatus on backside of filter paper and dry thoroughly • Observe the purple coloured spots and yellow coloured spots • Mark the centre of the spots with the pencil • Measure the distance of spots moved and the distance of solvent front • Calculate the Rf values • Compare the results with Rf values of the standards
- 24.
- 25. THIN LAYER CHROMATOGRAPHY •Paper is replaced by a thin layer of inert absorbent such as silica gel , alumina or cellulose spread over a glass plate • Better separation can be achieved by two dimensional chromatography • Separation in one direction first and then in a direction perpendicular to it
- 27. Ion Exchange Chromatography •Separation is based on the overall charge on the proteins • Ion exchange resins are cross linked polymers containing ionic groups as part of their structure • Can be cationic or anionic exchangers • The ion exchange resin is packed into a column in a suitable form • Mixture to be separated is allowed to percolate through the column and washed down with elution buffer.
- 30. GEL FILTRATION CHROMATOGRAPHY •Separation of molecule takes place on the basis of molecular size and shape • Stationary phase : column of gel with openings and micro- channels • Smaller molecules retained , move slowly • Larger molecules move out faster • Removal by eluting • Used to determine the molecular size
- 32. AFFINITY CHROMATOGRAPHY • Highaffinity of specific chemical groups called ligands • Mixture of proteins is passed through the column • Unbound proteins are removed by washing with buffer • Protein bound to ligand (specific)retained in column • Elution : concentrated soluble ligand or buffer , competes with immobilised ligand • Used for purification of antigens, antibodies or enzymes
- 34. GAS LIQUID CHROMATOGRAPHY •Stationary phase : liquid • Supported by a column of an inert material as silica • Mobile phase : gas • Mixture separated by volatile at one end of column and vapours are swept over the column by an inert gas such as argon or nitrogen • Particles are detected and quantified • Used for separation of fatty acids
- 36. HPLC • Stationaryphase: usuallyisa bed of fine solid particles with narrow size distribution, densely packed in a metal, glass or plastic tube – a chromatographiccolumn. • The stationaryphase maybe either a bulkcolumn packing or only a part of it deposited on or, more frequently,chemically bonded to a more or less inert supportmaterial. • The mobile phase: (eluent) is a liquid, usuallya mixture of two or more solvents, often containing suitable additives,forced through the column by applying elevated pressure. • These featuresof HPLC are especially usefulin pharmaceutical, biomedical and clinical analysis.
- 37. • HPLC hasbecome one of the most powerful tools in the contemporary organic analysis for the separation and determination of even very complex samples containing nonpolar, moderately or strongly polar and ionic compounds, simple species, high-molecular synthetic polymers or biopolymers
- 40. Medical Applications • InbornErrors Of Metabolism: detection of carbohydrates in urine, serum , faeces • Amino Aciduria: separation of amino acids • Vitamins: A, D , E ,K Separation of vitamins • Checking the purity of samples
- 41. • Applications ofchromatography in the pharmaceutical analysis: • Qualitative and quantitative analysis of molecules. • As chromatographyis easy, precise,rapid, and accurate. • It can be adopted successfullyand efficiently in bulk and pharmaceutical dosage formfor routine quality controlanalysis of drugs. • Chromatographyplays a significantrole in the development of a new molecule, as it determines the structure,and monitorsthe synthesis or reaction process. • It is used to find out the amount of the drug fromthe pharmaceuticaldosage forms. • analyze purified compounds for trace contaminants. • It is used to separate the chiral compounds in pharmaceutical analysis. • Chromatographyis a robustand efficientquantitative tool for the diagnosis and evaluation of diseasesin drug and biomarker research.
- 42. • Applications ofchromatography in the food industry: • Chromatography is used in the processing of the food industry for quality control, by isolating proteins, preservatives, vitamins, and amino acids. • The presence of chemical additives, the nutritional value of processed foods such as junk food, fast food can be determined with the help of a variety of chromatographic techniques. • Many beverage industries use chromatography to confirm that every batch of their product is the same. • The chromatography is broadly used by food manufacturers in determining the contents of processed food and other foodstuffs. • It is used to determine food shelf-life, it is analyzed by which food spoils.
- 43. • Applications ofchromatography in the chemical industry: • The major application of chromatography in the chemical industry is that it is used to monitor the reaction and synthesis of a chemical and its products. • It is used in pesticide and oil industries to detect different contaminants present in the products. • It is used in the chemical industry for determining the pollutants in water, air, and chemicals. • The chemical industries are used chromatography to monitor the quality and purity of the samples.
- 44. • Applications ofchromatography in forensic science: • The chromatographyis used in forensic science to collect the evidence and catch criminals. • Chromatographyis used in blood tests that can determine the amount of alcohol, drugs,or toxic substances in the body after death. • Chromatographyis also to determine if their poison in the body. • Gas chromatographyis used to test samples of blood, urine, and clothes, to recognize criminals and to help them bring justice. • Since chromatographycan reliably determine substancesin the bloodstream,it is used for screening of athletes for doping or performance-enhancing drugsin sports.
- 45. • Application ofchromatography in the environmental analysis: • Chromatography is a widely used separation technique to determine the organic trace components in environmental samples. • Chromatography used for determining and identification environmentally ubiquitous pollutants. • Liquid chromatography with mass spectrometry (LC-MS) can be used to examine many different pesticide residues. • It is also used to determine the volatile and non-polar organic compounds (odors and non-odorous) in the air. • It is used to identify carcinogenic contaminants found in drinking or wastewater. • Some other applications of chromatography includes, applications in the life sciences, molecular biology, nanotechnology, petroleum industries, and diagnosis of disease, etc
- 46. Advantages • The advantagesof chromatography include, • it provides simple and rapid analysis with high resolution, • it requires a very small amount of sample (Gram, PPM, and ng/ml) • it has a broad range of mobile phase and stationary phase to separate the components.
- 47. Disadvantages • Chromatography equipmentcan only be operated by a trained person. • Chromatography instruments are expensive. • An error occurs due to the overloading of the samples. • Chromatography equipment must be handled with care because of these parts are expensive and sensitive. • Some of the chromatography techniques require more solvent to separate the analytes.
- 48. Learning Objectives • Definition •History • Introduction • Types • Principles • Development • Procedure • Applications • Advantages • Disadvantages