HPTLC BY HK@PHARMA 2007

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Presentation Transcript

Slide 1: 

HPTLC- HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY sophisticated and automated form of TLC. HARSHIT SHETH

CONTENTS : 

CONTENTS INTRODUCTION PRINCIPLE OF HPTLC DIFFERENCE OF HPTLC AND TLC INSTRUMENTATION STEPS INVOLVED IN HPTLC APPLICATION REFERENCE

INTRODUCTION : 

INTRODUCTION CHROMATOGRAPHY: Method used for separation of the multi-component mixture.

PRINCIPLE OF HPTLC : 

PRINCIPLE OF HPTLC Same theoritical principle of TLC i.e ADSORPTION CHROMATOGRAPHY. Separation of the components based upon their relative affinity towards the stationary phase.

Difference of HPTLC and TLC : 

Difference of HPTLC and TLC

FEATURES OF HPTLC : 

FEATURES OF HPTLC Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard Several analysts work simultaneously Lower analysis time and less cost per analysis Low maintenance cost Simple sample preparation - handle samples of divergent nature No prior treatment for solvents like filtration and degassing Low mobile phase consumption per sample No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination

Slide 11: 

Visual detection possible - open system Non UV absorbing compounds detected by post-chromatographic derivatization

INSTRUMENTATION : 

INSTRUMENTATION

SELECTION OF CHROMATOGRAPHIC LAYER : 

SELECTION OF CHROMATOGRAPHIC LAYER · Precoated plates - different support materials - different Sorbents available · 80% of analysis - silica gel GF · Basic substances, alkaloids and steroids - Aluminum oxide· Amino acids, dipeptides, sugars and alkaloids - cellulose · Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18· Preservatives, barbiturates, analgesic and phenothiazines- Hybrid plates-RPWF254s

SAMPLE AND STANDARD PREPARATION : 

SAMPLE AND STANDARD PREPARATION SOLVENTS USED ARE: Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Chloroform: Methanol: Ammonia (90:!0:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid

ACTIVATION OF PRE-COATED PLATES : 

ACTIVATION OF PRE-COATED PLATES Plates exposed to high humidity or kept o­n hand for long time to be activated 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.

APPLICATION OF SAMPLE AND STANDARD : 

APPLICATION OF SAMPLE AND STANDARD Usual concentration range is 0.1-1µg / µl · Above this causes poor separation Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe on TLC plates as bands Band wise application - better separation - high response to densitometer

SELECTION OF MOBILE PHASE : 

SELECTION OF MOBILE PHASE Trial and error - one’s own experience and Literature Normal phase - Stationary phase is polar - Mobile phase is non polar Reversed phase - 3 - 4 component mobile phase should be avoided - Multi component mobile phase once used not recommended for further use and solvent composition is expressed by volumes (v/v) and sum of volumes is usually 100 Twin trough chambers are used only 10 -15 ml of mobile phase is required -· Components of mobile phase should be mixed introduced into the twin - trough chamber

PRE- CONDITIONING (CHAMBER SATURATION) : 

PRE- CONDITIONING (CHAMBER SATURATION) Un- saturated chamber causes high Rf values · Saturated chamber by lining with filter paper for 30 minutes prior to development - uniform distribution of solvent vapours - less solvent for the sample to travel - lower Rf values.

CHROMATOGRAPHIC DEVELOPMENT AND DRYING : 

CHROMATOGRAPHIC DEVELOPMENT AND DRYING After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere Dry in vacuum desiccators

DETECTION AND VISUALIZATION : 

DETECTION AND VISUALIZATION Detection under UV light is first choice - non destructive · Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length) Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF-254 Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution When individual component does not respond to UV - derivatisation required for detection

Application of HPTLC in evalution of herbal drug : 

Application of HPTLC in evalution of herbal drug To detect andrographolide in andrographis paniculate among other substance. Sample preparation:5gm powd.+methanol in soxhlet appa.(6.8 h).then evaporate methanol and dissolve the residue in 1 ml methanol. Pre-coated plate:silica gel 60F (0.25mm). Mobile phase:chloroform:methanol(8:1) Detection:uv at 254 nm.

Slide 23: 

Trophane alkaloids: Sample pre: 1% alcoholic solution. Sample application: 10 µl. Detection: uv at 254 nm Rauvolfia alkaloids: Sample pre: 0.5%alcoholic solution. Sample application: 10 µl. Detection: uv at 365 nm Colchicine: Sample pre: 0.5% solution in 70% ethanol Sample application: 10 µl. Detection: uv at 365 nm

Books To Be Referred: : 

Books To Be Referred: PHARMACEUTICAL ANALYSIS BY A.V.KASTURE PLANT DRUG ANALYSIS BY H. WAGNER HERBAL DRUG TECHNOLOGY BY S.S AGRAWAL

UNIVERSITY QUESTIONS : 

UNIVERSITY QUESTIONS Mention the chromatographic methods based on the principales. 1)adsorption 2)absorption Differentiate the TLC/HPTLC.(MMMMIMP) What is chromatography discuss the application of various chromatographic methods in the evalution of herbal drugs. OR In the standardization of herbal products.

Slide 27: 

Good ideas are not adopted automatically. They must be driven into practice with courageous patience. ) 27

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