Thin layer chromatography by khaleel S.G

THIN LAYER CHROMATOGRAPHY IN BRIEF

1. THIN LAYER CHROMATOGRAPHY Presented by: Khaleel Basha S.G M.Pharmacy ACHARYA NAGARJUNA UNIVERSITY 1

2. Introduction to Chromatography Thin layer chromatography (TLC) Definition History Principle Involved Experimental Techniques Of TLC Chromatographic Parameters Factors Affecting Resolution Applications Conclusion References 2 CONTENTS

3. Chromatography is a physical method of separation in which 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 . Types of Chromatographic techniques : 3 CHROMATOGRAPHY Technique Stationary Mobile Phase Column/Adsorption Chromatography Solid Liquid Partition Chromatography Liquid Liquid Paper Chromatography Liquid Liquid Thin Layer Chromatography (TLC) Solid Liquid Gas - Liquid Chromatography (GLC) Liquid Gas Gas - Solid Chromatography (GSC) Solid Gas Ion Exchange Chromatography Solid Liquid

4. Thin Layer Chromatography (TLC) can be defined as a method separation or identification of mixture of components into individual components by using finely divided adsorbent solid spread over a glass plate and liquid as a mobile phase. TLC is often named by other names such as drop, strip, spread layer, surface chromatography and open column chromatography. 4 THIN LAYER CHROMATOGRAPHY (TLC) DEFINITION:

5. Simple Equipment Short Development Time Wide Choice Of Stationary Phase Early Recovery of Separated Components Easy Visualization of Separated components Sensitivity 5 Perfection of TLC over other chromatographic techniques:

6. The history of TLC dates back to 1938 when IZMAILOV and SHRAIBER separated plants extracts using 2mm thick and firm layer of alumina set on glass plate. In 1944 CONSDEN , GORDEN & MARTIN used filter papers for separating Amino acids . In 1950, KIRCHNER identified terpenes on filter paper and later glass fiber paper coated with Alumina. In 1958, STAHL developed standard equipment for analysing by TLC . 6 HISTORY

7. Principle of separation is “ADSORPTION” 7 PRINCIPLE

8. Based on principle of separation The separation depends on the relative affinity of compounds towards the stationary phase. The components with more affinity towards stationary phase travels slower. The components with less affinity towards stationary phase travels faster. 8

9. TLC system components : TLC plates : Should be stable and chemically inert Stationary phase on the plate is of uniform thickness, consists of fine particles. TLC chamber : Should maintain uniform Environment inside Prevent evaporation of solvent Development solvent Mobile phase Filter paper 9 Experimental Techniques :

10. 1. Coating material ( Adsorbent ) : Large no. Of coating material are commercially produced as thin layer adsorbent. Popularly Used are : Silica gel Alumina Kieselghur Cellulose powder Calcium phosphate Magnesium trisilicate Ferric oxide hydrate 10 Experimental techniques

11. Generally adsorbent do not adhere satisfactorily to the glass plate to overcome this binders like gypsum, starch, hydrated silicon oxide. Etc.. Are added to the adsorbents. Gypsum is most widely used. In silica gel G And alumina G the G indicates gypsum . Characteristics To be checked while choosing an adsorbent : Solubility To as certain whether it is basic, acidic or amphoteric. Chemical reactions with adsorbent or chemical changes due to the action of adsorbent or binders. 11

12. 2 . Preparation of thin layer In plates : The various methods of preparing layers are as follows : Pouring - measured amount of slurry is put on a given size of plate which is kept on a level surface. This method is simple but not used to a great extent. Dipping - plates are prepared by dipping at a time back to back in chloroform or chloroform-methanol slurries of adsorbent. Not much used. Spraying - small portion of slurry is separated on the glass plate. Difficult to obtain uniform layers on single plate and there may be variation from plate to plate. 12

13. Spreading : - slurry placed in an applicator. This is either moved over the stationary plate or it is held stationary and the plate is pushed or pulled through. It gives layer thickness from 0.2 to 2.0 mm. This method is most important now-a-days to obtain thin and uniform layers. 13

14. Precoated plates :- available on glass or plastic sheets. Quite Expensive Avoid purchase of expensive coating equipment. Thickness of precoated plates usually varies from 0.1 to 2.0 mm. Contain no gypsum. It may be safely activated at 110-120° C Ex: Ready-for-use TLC plates with cellulose-F Ready-for-use TLC plates with Silica Gel-F-254 14

15. 5 . Development tank : Ascending chromatography is most common technique in TLC. Plate placed in the chamber at 45• angle. Bottom of the chamber covered up-to 1mm by solvent. Top of the chamber covered tightly with the lid for saturation With solvent vapors to avoid "edge effect ". Descending TLC is also possible. 15

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17. 6. Solvent systems : Trail and error Method is used to found best eluent if one doesn't know the nature of components to be separated. If chemical nature is known then suitable solvent is selected using STAHL'S triangle. Mixture of two or more solvents of different polarity give better separation than chemically homogeneous solvents. Solvents used must be as pure as possible. 17

18. Elutropic series of selected one or two component solvent systems with increasing polarity : High boiling paraffin's Cyclohexane Benzene Cyclohexane: ethyl acetate( 95:5 ) Chloroform : acetone (9:1) Benzene : methanol ( 95:5 ) Ethyl acetate Ethyl acetate : methanol ( 99:1 ) Acetone Methanol Water. 18

19. 7. Plate development : A drop of sample placed near one edge of the plate ( 20 ×5) or 20×20cm) position marked with pencil. After sample solvent has evaporated, the plate is placed in closed chamber saturated with vapors of development chamber. After the development has transversed one half to two third the length of the plate the latter is removed and dried. 19

20. 8. Detection of Components : Colored substances can be seen visually. Colorless compounds can be detect either under UV or by treating with a visualizing reagent. Specific detecting reagents : Aniline-phthalate reagent for carbohydrates Ninhydrin reagent for Amino acids Dragendroff’s reagent for alkaloids. Ferric chloride for phenols Iodine for unsaturated and aromatic compounds Chromic acid or sulphuric acid . 20

21. 21 TLC Profile Of Extracts Visible light b. short Wavelength (274 nm) c. Long Wavelength (375 nm)

22. 9. Evaluation of chromatogram Evaluation may be either qualitative or quantitative : Qualitative : R f values for TLC show inferior reproducibility. Depends upon factors such as : Quality of the layer material Activation of grade of the layer Quality of solvent Equilibration of chamber Presence of impurities 22

23. Quantitative : Can be carried out in two ways 1. Direct methods 2. Indirect methods 1. Direct methods : Quantitative determination is undertaken directly on the layer. Methods involves : Visual assessment of chromatograms Determination by measurement of spot areas Quantitative TLC incorporating densitometry Direct spectrophotometry on thin layer chromatograms 23

24. 2 . Indirect Methods The substances removed from the adsorbent and then determined after elution. These methods also know as elution techniques. Colorimetry Flourimetry Radiometry Flame photometry Gravimetry UV spectrophotometry 24

25. The position of migrated spots on the chromatogram are indicated by different terms such as Rf, Rx, Rm, evaluated. Rf : The R is related to the migration of the solute front relative to the solvent front as : Rf = Distance travelled by solute from the original line Distance travelled by solvent from the original line Rf defines the movement of the substance relative to the solvent front in a given chromatographic system. Rf values are less than 1. Higher the Rf value the substance is very non-polar, low Rf value refers to a substance that is very polar. 25 Chromatographic parameters :

26. 26 Diagrammatic representation of retention factor Rf : Rx : I n some cases, the solvent front runs off the end of the filter paper, the movement of a substance in such cases is expressed as Rx but not Rf. Distance travelled by the substance from the origin line / Distance travelled by the standard substance x from the original line

27. Retention factor : absolute movement of the chemical depends on how far solvent travels. Thin layer chromatography plate : affect the Rf value for a given chemical Thickness ,change in solvent, uniformity of the layer of adsorbent can greatly change the Rf value. Solvent impact retention factors Thickness of layer Temperature Mobile phase Too much of sample. 27 Factors affecting resolution :

28. As a check on processes :- checking distillation fractions and for checking the progress of purification by molecular distillation. In organic chemistry : For checking the purity of sample As a purification process Examination of reaction For identifying organic compounds like acids, alcohols, alkaloids etc,. For separation of inorganic compounds 28 Applications :

29. Foreign alkaloid substances present in alkaloidal drugs. e.g.: atropine sulphate, codeine Foreign steroids present in steroidal drugs E.g.: betamethasone valerate Separation of amino acids In Quantitative analysis Biochemical analysis: TLC is extremely useful in isolation or separation of biochemical metabolites or constituent from its body fluids, blood plasma, serum, urine etc. One of the most important application of TLC is in separation of multi component pharmaceutical formulations. In food and cosmetic industry, TLC method is used for separation and identification of colours, preservatives , sweetening agent, and various cosmetic products . 29

30. Due to its simplicity, sharpness of separations, high sensitivity, speed of separation and ease of recovery of separated compounds, TLC has found increasing applications in all branches and this technique has been proved especially versatile process for the analytical investigation of substances that available in very small quantities. 30 Conclusion :

31. Gurdeep R. Chatwal, Sham K Anand, ( 2107), Fifth revised edition , Instrumental methods of Chemical Analysis, 2.599-2.615. Asuthoshkar, Third revised edition, Pharmaceutical Drug Analysis, 473-491. Michael judge (June 10 2018) Factors that affect Thin layer chromatography Sciencing.com. 31 REFERENCES:

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