Chromatography Part-II
- 1. Dr. BASAVARAJAIAH S. M. Assistant Professor and Coordinator P.G. Department of Chemistry Vijaya College Bangalore-560 004 CHROMATOGRAPHY-II (TLC, Paper, and Column)
- 2. Thin LayerChromatography Paper chromatography Column chromatography CONTENTS:
- 3. ThinLayerChromatography Thin-Layer Chromatography (TLC) is a solid-liquid chromatography and is based on the principle of adsorption. In this technique of chromatography, the moving liquid phase is allowed to ascend a thin layer of adsorbent coated onto backing support like a glass or aluminum strip, or plastic strip. Thin Layer Chromatography (TLC) is a quick and easy method for analyzing mixtures of organic compounds. Introduction:
- 4. Thin Layer Chromatography Principle The separation principle of the TLC procedure is based on the given compound’s relative affinity towards the mobile and the stationary phase. The process begins here by moving the mobile phase over the stationary phase’s surface. During this movement, the higher affinity compounds gain less speed as compared to the lower affinity compounds. This results in their separation. The compounds that are more attracted towards the stationary phase secure their position at lower levels while others move towards the higher levels of the surface. So their spots can be seen accordingly.
- 5. ThinLayerChromatographyCOMPONENTS TLC Plates or Stationary Phase: These are used for applying the thin layer of stationary phase. They are inert or stable in nature. The layer of stationary phase is kept even throughout these plates for better analysis. Usually, ready-to-use plates are preferred by the people conducting experiments. Mobile Phase: This comprises a solvent (or solvent mixture). The taken solvent needs to be chemically inert, of the highest possible purity, and particulate-free. Only then can the TLC spots be able to develop.
- 6. TLC Chamber: This is where the thin layer chromatography procedure takes place. It keeps the dust particles away from the process and does not let the solvent evaporate. In order to develop the spots appropriately, a uniform environment is maintained inside this chamber. Filter Paper: This gets placed inside the chamber after being moistened with the mobile phase solution. It ensures that the mobile phase rises uniformly throughout the TLC plate’s length. Detection of the spots or Visualizing agents: If the components separated on the TLC are colored, then these can be visualized easily. But if the components are colorless, then these are made visible by the use of some reagent called visualization reagents.
- 7. ThinLayerChromatographyProcedure Developing the chromatogram: It involves three steps 1. Spotting: First the sample to be analyzed is dissolved in a volatile (easily evaporated) solvent to produce a very dilute (about 1%) solution. Spotting consists of using a micropipette to transfer a small amount of this dilute solution to one end of a TLC plate, in this case, a thin layer of powdered silica gel that has been coated onto a plastic sheet. The spotting solvent quickly evaporates and leaves behind a small spot of the material.
- 8. Development: Development consists of placing the bottom of the TLC plate into a shallow pool of a development solvent, which then travels up the plate by capillary action. As the solvent travels up the plate, it moves over the original spot. Different components in the original spot, having different polarities, will move different distances from the original spot location and show up as separate spots. When the solvent has traveled almost to the top of the plate, the plate is removed, the solvent front marked with a pencil, and the solvent is allowed to evaporate (Drying).
- 9. 3. Visualization: The spots can be directly observed after development. Because most compounds are colorless, however, a visualization method is needed. The silica gel on the TLC plate is impregnated with a fluorescent material that glows under ultraviolet (UV) light. While under the UV light, the spots can be outlined with a pencil to mark their locations. The second method of visualization is accomplished by placing the plate into iodine vapors for a few minutes. A= Distance travelled by solvent front. B= Distance travelled by solute (Analyte). Rf =B / A
- 10. Reagents Substances Iodine vapor General organic and unsaturated compounds Phosphomolybdic acid General organic compounds Fluorescein/Bromine General organic compounds UV light General organic compounds Sulphuric acid General organic compounds 2,4-Dintrophenylhydrazine Ketones and aldehydes Bromocresol green Acids Potassium hydrogen platinate Basic nitrogen or sulfur compounds Antimony pentachloride in CCl4 Terpeniods Ninhydrin Amino acids Dragendroff’s reagent Alkaloids Aniline phthalate Sugars Bromthymol blue Lipids List of some common visualizing agents used in TLC
- 11. The checking of purity of samples. The identification of organic compounds. The separation of inorganic ions. Monitoring the progress of a reaction. Monitoring a column chromatographic separation. Determining the appropriate solvent for a column chromatographic separation. Estimation of biomolecules. Applications of Thin Layer Chromatography:
- 12. PAPER CHROMATOGRAPHY In paper chromatography, the stationary phase is a sheet of paper of suitable texture and thickness, which may sometimes be impregnated with a liquid phase that is immiscible with the mobile phase. Chromatographic separations on paper are usually considerably slower than on thin-layer plates and the method is, in general, not as versatile as thin-layer chromatography since the degree of variation of the stationary phase is much more restricted. The concept of Rf value referred to in the discussion on thin-layer chromatography applies equally well to paper chromatography.
- 13. There are three types of paper chromatography: (i). Ascending Paper Chromatography (ii). Descending Paper Chromatography and (iii). Circular or Horizontal Paper Chromatography. (i). Ascending Paper Chromatography: Here the solvent travels in an upward direction of the chromatographic paper. It is used for the separation of organic and inorganic substances.
- 14. (ii). Descending Paper Chromatography: The development of the chromatogram is downwards by allowing the solvent to travel down the paper. This requires the solvent to be kept at the upper portion in a trough and the end of paper close to the spotting line is dipped into a solvent. The solvent flows downwards.
- 15. (iii). Circular or Horizontal Paper Chromatography: Here a circular filter paper is taken and the sample is given at the center of the paper. After drying the spot, the filter paper is tied horizontally on a Petri dish containing solvent. The solvent rises through the wick and the component gets separated in form of a concentrate circular zone.
- 16. Rf = Distance travelled by solute Distance travelled by solvent front Applications of Paper Chromatography: The separation of amino acids. Structural analysis. Determination of purity of the compounds. Separation of inorganic cations or complexes.
- 17. COLUMN CHROMATOGRAPHY Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids when carrying out small-scale experiments. The separation can be liquid/solid (adsorption) or liquid/liquid (partition) in column chromatography. The stationary phase, a solid adsorbent, is usually placed in a vertical glass column and the mobile phase, is added from the top and let flow down through the column by either gravity or external pressure.
- 18. Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. It can be used to determine the number of components of a mixture and as well as the separation and purification of those components.
- 19. Column Chromatography principle The main principle involved in column chromatography is the adsorption of the solutes of the solution with the help of a stationary phase and afterward separates the mixture into independent components. At the point when the mobile phase together with the mixture that requires to be isolated is brought in from the top of the column, the movement of the individual components of the mixture is at various rates. The components with lower adsorption and affinity to the stationary phase head out quicker when contrasted with the greater adsorption and affinity with the stationary phase. The components that move rapidly are taken out first through the components that move slowly are eluted out last.
- 20. Rf = The distance traveled by solute/ The distance traveled by the solvent
- 21. Steps involved in the column chromatography: The steps included in the column chromatography are: 1. Preparation of the column Mostly the column is comprised of a glass tube with an appropriate stationary phase. The bottom end of the column is packed with a glass wool/cotton wool or an asbestos pad after which the stationary phase is packed. After packing the column, a paper disc is placed on the top to avoid the disturbance of the stationary phase during the introduction of the sample or mobile phase. The disturbance in the stationary phase (adsorbent layer) leads to the irregular bands of separation.
- 22. Two types of preparing the column, known as packing techniques namely: Dry packing technique – The amount of absorbent needed is added as a fine dry powder in the column and the solvent flows freely through the column until equilibrium is achieved. Wet packing technique – The slurry of adsorbent is prepared along with the mobile phase and is poured into the column. It is regarded as the ideal technique for packaging. The column should be properly washed and completely dried before in-use.
- 23. 2. Introduction of the sample The sample (a mixture of components) is dissolved in the minimum amount of the mobile phase. At one instant, the sample is introduced into the column and on the top portion of the column, it is absorbed. Through the elution process, the individual sample can be isolated from this zone. 3. Elution technique Through this technique, the individual components are separated completely from the column. The process of elution can be carried out by employing two techniques: Isocratic elution and Gradient elution .
- 24. Isocratic elution technique – Throughout the procedure, a solvent of the same polarity or same solvent composition is utilized. Example: Use of chloroform alone Gradient elution technique – Throughout the separation procedure, solvents of gradually increased polarity or increased elution strength are utilized. Example: Benzene → Chloroform → Ethyl acetate → Methanol 4. Detection of Components In case the mixture separated in a column chromatography procedure are colored compounds, then monitoring the separation progress is simple.
- 25. In case the compounds undergoing separation are colorless, then small fractions of the eluent are sequentially collected in tubes that are labeled. Thorugh TLC, the composition of each fraction is determined.
- 26. Types of Column chromatography: Adsorption column chromatography Partition column chromatography Gel column chromatography Ion exchange column chromatography Gas Chromatography (GC) High-Performance Liquid Chromatography (HPLC)
- 27. Column chromatography uses: To isolate active constituents. To separate compound mixtures. To remove impurities or carry purification process. To isolate metabolites from biological fluids. To estimate drugs in drug formulations or crude extracts.
- 28. Thank you