Chromatography & Column chromatography
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Chromatography is a laboratory technique used to separate mixtures into individual components. Column chromatography is a type of chromatography that uses a column filled with a solid stationary phase. As a liquid or gas mobile phase passes through the column carrying the sample mixture, the mixture's components separate as they travel through the column at different rates depending on how strongly they interact with the stationary phase. Column chromatography is commonly used to purify compounds by removing impurities based on differences in their adsorption to the stationary phase material in the column.
Chromatography & Column chromatography
- 2. Chromatography, The word Chromatography is derived from two Greek words - 1. chroma which means "color" 1. graphein, which means "to write". “Color writing”
- 3. Chromatography Chromatography is a laboratory separation technique by which a mixture can be separated into its individual components
- 4. TYPES OF CHROMATOGRAPHY Column chromatography Paper chromatography Thin layer chromatography High performance liquid chromatography Gas chromatography Ion exchange chromatography Gel chromatography Affinity chromatography
- 5. Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction.
- 6. REQUIREMENTS OF CHROMATOGRAPHY 1.Mobile phase (Liquid) 2.Stationary phase (Liquid/Solid)
- 7. TECHNIQUES/PRINCIPLES OF CHROMATOGRAPHY 1.Partition chromatography 2.Adsorption chromatography
- 8. 1.Partition chromatography The separation of components between two liquid phases viz original solvent and the film of solvent used in the column. The separation of the components from the sample mixture is carried out by the process of partition of the components between 2 phases. Both phases are in liquid form.
- 9. Partition chromatography Mobile phase Liquid or Gas Stationary phase Liquid
- 10. 2. Adsorption chromatography Mobile phase Liquid Stationary phase Solid
- 12. Column chromatography Column chromatography is a technique which is used to separate a single chemical compound from a mixture dissolved in a fluid. It separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allow them to get separated in fractions. This technique can be used on a small scale as well as large scale to purify materials that can be used in future experiments. This method is a type of adsorption chromatography technique.
- 13. Principle When the mobile phase along with the mixture that needs to be separated is introduced from the top of the column, the movement of the individual components of the mixture is at different rates. The components with lower adsorption and affinity to stationary phase travel faster when compared to the greater adsorption and affinity with the stationary phase. The components that move fast are removed first whereas the components that move slowly are eluted out last.
- 15. Procedure Mobile phase – This phase is made up of solvents and it performs the following functions: 1. It acts as a solvent – sample mixture can be introduced in the column. 2. It acts as a developing agent – helps in the separation of components in the sample to form bands. 3. It acts as an eluting agent – the components that are separated during the experiment are removed from the column 4. Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone, water, acetic acid, pyridine, etc.
- 16. Stationary phase – It is a solid material which should have good adsorption property and meet the conditions given below: 1. Shape and size of particle: Particles should have uniform shape and size in the range of 60 – 200μ in diameter. 2. Stability and inertness of particles: high mechanical stability and chemically inert. Also, no reaction with acids or bases or any other solvents used during the experiment. 3. It should be colourless, inexpensive and readily available. 4. Should allow free flow of mobile phase 5. It should be suitable for the separation of mixtures of various compounds.
- 17. Applications ● Column Chromatography is used to isolate active ingredients. ● It is very helpful in Separating compound mixtures. ● It is used to determine drug estimation from drug formulations ● It is used to remove impurities. ● Used to isolation metabolites from biological fluids.