Thin layer chromatography.pptx [Autosaved].pptx

thin layer chromatography detailed study.

1. Thin layer chromatography Submitted by Abhijit padhi

2. Chromatography   is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. In this physical method of separation, the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction. Depending upon the stationary phase and mobile phase chosen, they can be of different types.

3. What is Thin Layer Chromatography (TLC)? Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a plate and liquid as a mobile phase.

4. Michael tswett is credited as being the father of liquid chromatography. Tswett developed his ideas in early 1900’s.

5. The two most common classes of TLC are:- -Normal phase -Reversed phase

6. Normal phase Normal phase is terminology used when the stationary phase is polar ; for example silica gel and the mobile phase is an organic solvent or a mixture of organic solvents which is less polar than the stationary phase.

7. Reversed phase Reversed phase is a terminology used when the stationary phase is a silica bounded with an organic substrate such as a long chain aliphatic acid like C-18 and the mobile phase is a mixture of water and organic solvent which is more polar than the stationary.

8. Principle of Thin Layer Chromatography (TLC) Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminum oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character is identified by means of suitable detection techniques.

9. Components of Thin Layer Chromatography (TLC) TLC system components consists of: TLC plates , preferably ready made with a stationary phase: These are stable and chemically inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary phase on the plates is of uniform thickness and is in a fine particle size. TLC chamber-  This is used for the development of TLC plate. The chamber maintains a uniform environment inside for proper development of spots. It also prevents the evaporation of solvents, and keeps the process dust free. Mobile phase-  This comprises of a solvent or solvent mixture The mobile phase used should be particulate-free and of the highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, a stationary phase. A filter paper-  This is moistened in the mobile phase, to be placed inside the chamber. This helps develop a uniform rise in a mobile phase over the length of the stationary phase.

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. 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. 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. Procedure of Thin Layer Chromatography (TLC)

11. PRACTICAL REQUIREMENTS STATIONARY PHASE Adsorbents mixed with water or other solvents→ slurry Silica gel H (Silica gel with out binder) Silica gel G (Silica gel + CaSO4) Silica GF (Silica gel + binder + fluorescent indicator) Alumina, Cellulose powder

12. 2. GLASS PLATE Specific dimensions- 20cm X 20cm, 20cm X 10cm, 20cm X 5cm -Microscopic slides can also be used -Plates should be of good quality & withstand high temperatures. PREPARATION & ACTIVATION OF TLC PLATES 1.Pouring (simplest methods) 2.Dipping (used for small plates) 3.Spraying (difficult to get uniform layers) 4.Spreading (best technique) TLC Spreader

13. 4.APPLICATION OF SAMPLE Using capillary tube or micropipette Spotting area should not be immersed in the mobile phase 5.DEVELOPMENT TANK Better to develop in glass beakers, jars to avoid more wastage of solvents When standard method is used, use twin trough tanks Do chamber saturation to avoid "edge effect"

14.   6. MOBILE PHASE M.P used depends upon various factors Nature of the substance Nature of the S.P Mode of Chromatography Separation to be achieved, Analytical/Preparative e.g.→ pyridine, pet. ether, carbon tetrachloride, acetone, water, glycerol, ethanol, benzene....

15. THIN LAYER CHROMATOGRAPHY DRYING OF CHROMATOGRAM After the solvent has moved a certain distance for certain time the chromatogram is taken out from the tank & position of the solvent front is marked with a pencil. They are dried by cold or hot air depending on volatility of solvents. A simple hair dryer is a convenient device to dry chromatograms.

16. THIN LAYER CHROMATOGRAPHY DETECTING/VISUALISING AGENTS If the substance are colored they are visually detected easily. But for colorless substance, Physical and chemical methods are used to detect the spot. (a) Non specific methods (Physical methods) E.g. iodine chamber method, UV chamber for fluorescent compounds - at 254 or at 365nm.

17.   (b) Specific methods (Chemical methods) or Spraying method EXAMPLES Ferric chloride-Phenolic comp. & tannins Ninhydrin in acetone-Amino acids Dragendroff's reagent-Alkaloids 3,5 dinitro benzoic acid-Cardiac glycosides

18. QUANTITATIVE ESTIMATIONS The method can be divided into two main groups 1 Direct techniques- 2 Indirect techniques-

19. THIN LAYER CHROMATOGRAPHY Direct Measurement Method ( i ) Comparison of visible spots A rough quantitative measurements Component in a mixture can be carried out by comparing the intensity and size of the spot with a standard substance. (ii) Photo densitometry The method is used with the chromatograms of colored compound, instrument which measures quantitatively the density of the spots.

20. Retention Factor (R f  ) Value The behavior of a compound on a TLC is usually described in terms of its relative mobility or Rf value. Rf or Retention factor is a unique value for each compound under the same conditions. The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant: solvent system adsorbent thickness of the adsorbent amount of material spotted temperature Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered. Relative Rf” means that the values are reported relative to a standard.

21. Applications of Thin Layer Chromatography (TLC) 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 .

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