THIN LAYER CHROMATOGRAPHY- Power point presentationpptx

Thin layer chromatography is used in various fields as in chemistry, botany and helpful in research of medicinal plants.

1. THIN LAYER CHROMATOGRAPHY - P.TAMIZHARASI, 2 ND PROFESSIONAL BSMS.

2. CONTENTS: INTRODUCTION THIN LAYER CHROMATOGRAPHY PRINCIPLE COMPONENTS OF TLC PROCEDURE APPLICATIONS ADVANTAGES LIMITATIONS

3. WHAT IS CHROMATOGRAPHY ? A technique used to separate molecules from mixture .( chroma – colour , graphien – to write ). Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. .

4. What Is Thin Layer Chromatography? Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent material. The material usually used is  aluminium oxide , cellulose, or silica gel.

5. Who discovered thin layer chromatography? It was developed by Izmailov in the year 1938 considering Mikhail Tswett’s description on column chromatography.

6. PRINCIPLE Like other chromatographic techniques, thin-layer chromatography (TLC) depends on the separation principle. The separation relies on the relative affinity of compounds towards both the phases. The compounds in the mobile phase move over the surface of the stationary phase. The movement occurs in such a way that the compounds which have a higher affinity to the stationary phase move slowly while the other compounds travel fast. Therefore, the separation of the mixture  is attained. On completion of the separation process, the individual components from the mixture appear as spots at respective levels on the plates. Their character and nature are identified by suitable detection techniques.

7. COMPONENTS OF TLC: Before starting with the Thin Layer Chromatography Experiment, let us understand the different components required to conduct the procedure along with the phases involved. Thin Layer Chromatography Plates – ready-made plates are used which are chemically inert and stable. The stationary phase is applied on its surface in the form of a thin layer. The stationary phase on the plate has a fine particle size and also has a uniform thickness. Thin Layer Chromatography Chamber – Chamber is used to develop plates. It is responsible to keep a steady environment inside which will help in developing spots. Also, it prevents the solvent evaporation and keeps the entire process dust-free.

8. Thin Layer Chromatography Mobile phase – Mobile phase is the one that moves and consists of a solvent mixture or a solvent. This phase should be particulate-free. The higher the quality of purity the development of spots is better. Thin Layer Chromatography Filter Paper – It has to be placed inside the chamber. It is moistened in the mobile phase.

9. PROCEDURE: The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. These days, however, ready-made plates are more commonly used.

10. With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots. Then, samples solutions are applied on the spots marked on the line in equal distances. The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom.

11. A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect). Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid.

12. The plate is then immersed, such that the sample spots are well above the level of mobile phase (but not immersed in the solvent) for development. Sufficient time is given for the development of spots. The plates are then removed and allowed to dry. The sample spots are then seen in a suitable UV light chamber, or any other methods as recommended for the given sample.

13. Some common techniques for visualizing the results of a TLC plate include UV light Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with Iodine KMnO4 stain (organic molecules) Ninhydrin Reagent: often used to detect amino acids and proteins

14. APPLICATIONS In monitoring the progress of reactions Identify compounds present in a given mixture Determine the purity of a substance. Analyzing  ceramides  and fatty acids Detection of pesticides or insecticides in food and water Analyzing the dye composition of fibers in forensics Assaying the radiochemical purity of radiopharmaceuticals Identification of medicinal plants and their constituents 

15. ADVANTAGES It is a simple process with a short development time. It helps with the visualization of separated compound spots easily. It helps in isolating of most of the compounds. The separation process is faster and the selectivity for compounds is higher (even small differences in chemistry is enough for clear separation). The purity standards of the given sample can be assessed easily. It is a cheaper chromatographic technique.

16. LIMITATIONS OF TLC : It cannot tell the difference between enantiomers and some isomers. In order to identify specific compounds, the Rf  values for the compounds of interest must be known beforehand. TLC plates do not have long stationary phases. Therefore, the length of separation is limited compared to other chromatographic techniques.