HPTLC method

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY- MODERN TECHNIQUE OF TLC

1. High Performance Thin Layer Chromatography (HPTLC) Archana Chavhan M.Pharm I st year Rajashri Shahu college of pharmacy, buldana 1 Department of QA

2. Introduction Chromatography is a physical process of separation in which the components to be separated are distributed between two immiscible phases i.e. stationary phase which has a large surface area and mobile phase which is in constant motion through the stationary phase. 2 Department of QA

3. HPTLC is a improved method of TLC which utilizes the conventional techniques of TLC in more optimized way. It is also known as planar chromatography. HPTLC is very popular for many reasons such as, Shorter analysis time Greater efficiency Multiple Sample handling Visual chromatogram Detection limit in the range of nanogram with the UV absorption detection & in the picogram range with fluorimetric detection 3 Department of QA

4. Principle Similar to TLC method, HPTLC is also based on the principle of separation. Separation depends on the relative affinity of compounds towards stationary and the mobile phase. Compounds under the influence of the mobile phase (driven by capillary action) travel over the surface of the stationary phase. During this movement, the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus, the separation of components in the mixture is achieved. Once separation occurs, the individual components are visualized as spots at a different level of travel on the plate. Their nature or character are identified using suitable detection techniques.   4 Department of QA

5. Instrumentation 5 Department of QA

6. Difference between HPTLC & TLC HPTLC TLC Chromatographic plates Pre-coated handmade Layer of Sorbent 100μ m 250μ m Efficiency High due to smaller particle size Less Sample spotting Auto sampler Manual spotting Development chamber More amount of solvent Less amount of solvent Scanning Use of UV/ Visible/ Fluorescence scanner Not possible 6 Department of QA

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8. Steps involved are, Selection of chromatographic layer Layer pre-washing Sample & standard preparation Activation of pre-coated plates Application of sample & standard Selection of mobile phase Pre-conditioning (chamber saturation) Chromatographic development & drying Detection & visualization Documentation 8 Department of QA

9. 1. Selection of chromatographic layer Pre-coated plates with different support materials & sorbent layers Support materials : Glass, polyester/polyethylene, aluminum. Sorbents used: silica gel GF, Aluminum oxide, cellulose, sugars and alkaloids, fatty acids. 9 Department of QA

10. 2. Layer Prewashing It is purification step Main purpose is to remove impurities which includes water vapor & other volatile substances Solvents used for pre-washing : Methanol Chloroform: methanol (1:1) Chloroform: methanol: ammonia (90:10:1) 10 Department of QA

11. 3. Sample & standard preparation Sample & reference substances should dissolved in same solvent to ensure comparable distribution at starting zones Dry the plates and store in dust free atmosphere Solvents used are: Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid 11 Department of QA

12. 4. Activation of pre-coated plates Freshly open box of HPTLC plates do not require activation. Plates exposed to high humidity or kept on hand for long time need to be activated by removing moisture. By placing in an oven at 110-120ºc for 30’ prior to spotting Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15 minutes. 12 Department of QA

13. 5. Application of sample and standard Usual concentration range is 0.1-1µg /µl Applicators used, Capillary tubes Micro syringes Micro bulb pipette Automatic applicator- nitrogen gas sprays sample and standard from syringe on TLC plates as bands 13 Department of QA

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15. 6. Selection of mobile phase Chemical properties of analytes & sorbent layer should be considered. Various components of mobile phase should be measured separately & then placed in a mixing vessel. 15 Department of QA

16. 7. Pre- conditioning (Chamber saturation) Un- saturated chamber causes high Rf values Saturated chamber by lining with filter paper for 30 minutes prior to development gives uniform distribution of solvent vapors - less solvent for the sample to travel - lower Rf values . For low polarity mobile phase no need of saturation 16 Department of QA

17. 8. Chromatographic development & drying Plates are spotted with sample & air dried and placed in developing chambers After development, remove the plate and mobile phase is removed from the plate to avoid contamination of lab atmosphere Dry in vacuum desiccators 17 Department of QA

18. 9. Detection and visualization Detection under UV light is first choice non destructive and spots of fluorescent compounds can be seen at 254 nm or at 366 nm Spots of non fluorescent compounds can be seen by using fluorescent stationary phase silica gel GF Non UV absorbing compounds like ethambutol, dicylomine dipping the plates in 0.1% iodine solution When individual component does not respond to UV-derivatisation required for detection. 18 Department of QA

19. Detectors 19 Department of QA

20. Detectors consists of following Lamp Entrance slit Monochromator slit Grating Mirror Beam splitter Reference photomultiplier Measuring photo multiplier Photo diode for transmission measurement 20 Department of QA

21. 10. Scanning & Documentation Scanning Scanner converts band into peak & peak height or area is related to the concentration of substance on spot Peak height & area under spot are measured by instrument & recorded 21 Department of QA

22. Documentation Labeling every single chromatogram Plates with imprinted identification code supplier name, Item number, batch number and individual plate number to avoid manipulation of data at any stage coding automatically gets recorded during photo documentation. 22 Department of QA

23. Thank you 23 Department of QA