Thin layer chromatography(tlc)

refrence: biophysics & molecular biology by pranav kumar
wilson & walker : instrumentation & biotechniques
biology discussion

1. Presented By:- Shweta Gupta & Neha Gautam THIN LAYER CHROMATOGRAPHY(TLC)

2. History Basic introduction Principle Components Method Advantages Disadvantages Applications What we’ll study?

3. History

4. Chromatography is a technique for separating mixtures into their components in order to analyze, purify, and quantify the component. All the forms of chromatography work on same principle. They all have a stationary phase ( a solid, or a liquid supported on a solid) and a mobile phase ( a liquid or a gas). The mobile phase flows through the stationary phase that carries the component of the mixture with it, different component travel at different rate. What is Chromatography ?

5. THIN LAYER CHROMATOGRAPHY (TLC) PRINCIPLE Thin layer chromatography (TLC) is a technique used to identify the components/compounds present in a mixture by separating them using a thin stationary phase (silica gel) supported on an inert substrate and a mobile phase (solvent). To study the purity of the compound To study the progress of a reaction To identify the various components present in the mixture

6. Stationary phase- silica gel, alumina etc. over glass metal, plate, plastic sheet etc Silica gel- acidic -steroids, amino acids, hydrocarbons etc. Alumina- basic -amines , bile, lipids etc. Cellulose- neutral -carbohydrates , sugars etc Mobile phase - developing solvent/mixtures of solvents(reasonably volatile) Sample- sample containing mixture of compounds and/or individual compounds Components of TLC

7. Why silica gel is used ???

8. Developing - developing solvent should be below the level of spotted samples. Mixtures of strongly polar compounds -ethyl : acetate: butanol : acetic acid: water(80:10:5:5) Polar compounds -10 % methanol in DCM Strongly basic compounds -10%NH4OH in methanol in dichloromethane

9. Visualization : Colored samples- just dry to vaporize the solvent Uncolored samples Iodine chamber- place iodine crystals in the chamber Fluorescent substance added to stationary phase(silica gel), which will fluoresce/glow in UV light(254nm) leaving the dark spots.

10. SYSTEM COMPONENTS OF TLC

11. FIG: TLC SETUP

12. Method

13. FIG: A- Distribution of spots after migration, B- Sample spots seen in UV transilluminator

14. Separation of multiple mixtures

15. The rate of migration of the various substances being separated are governed by their relative solubilities in the polar stationary phase & the non polar mobile phase. During the separation process , a given solute is distributed between the mobile & stationary phases according to their partition coefficient (K d ). Kd= Concentration of solute in phase A Concentration of solute in phase B The migration rate of a substance during TLC is usually expressed as the dimension term R f (relative front), which is the ratio of the distance traveled by substances and solvent front. R f = Distance traveled by substance Distance traveled by solvent front

16. FIG : Example of calculating relative front

17. It is a simple process with short development time . It helps with the visualization of separated compound spots easily. The method helps to identify the individual compounds. 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. ADVANTAGES

18. Stationary phase is short High detection limit Open system – temperature and humidity can affect the results DISADVANTAGES

19. APPLICATIONS OF TLC

20. Pharmaceutical industries utilize TLC technique for qualitative analysis or detect   impurities in various medicines like hypnotics, sedatives, anticonvulsants, tranquillizers, anti-histamines, analgesics, local anesthetics, steroids, etc. Yet another important application of TLC is to separate multi-component  pharmaceutical formulations  into its individual components . Contd… In food and cosmetic industry Any artificial color, preservatives, sweetening agent, and other impurities in food and cosmetic products can be detected and isolated by TLC technique.

21. https://youtu.be/yoYECMP_1iI