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HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY(HPTLC)

Presented By : Swati .V. Sahani First Year M.Pharm(sem-1) Orential College Of Pharmacy

Under the guidance of Mr Sayyed Mateen Sayyed Moin

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CONTENTSIntroduction.Principle of HPTLC.Difference between TLC & HPTLC.Steps involved in HPTLC.Materials used for HPTLC plates.Mobile phase.Sample application.HPTLC plate development.Application of HPTLC.2

INTRODUCTIONChromatography is physical method of separation in which the components to be separated are distributed between two phase, one of which is stationary phase while the other mobile phase moves in a definite directionTypes of Chromatographic Techniques

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HPTLC- sophisticated form of thin layer chromatography it involves the same theoretical principle of thin layer chromatography .

Traditional thin layer chromatography & its modern instrumental quantitative analysis version HPTLC are very popular for many reason such as

Visual chromatogram.Simplicity.Multiple sample handling.

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PrincipleSeparation my result due to adsorption or partition or by both phenomenon depending upon the nature of adsorbents used on plates and solvents system used for development. The mobile phase flows through the plate because of capillary action. the components move according to their affinites toward the adsorbent .

The component with more affinity towards stationary phase travels slower. the component lesser affinity towards stationary phase travel faster 5

Difference between TLC & HPTLC6

Features of HPTLC Simultaneous processing of sample and standard better analytical. precision and accuracy, less need of internal standard.Low analysis time and less cost per analysisLow maintenance cost.Simple sample preparation.Low mobile phase consumption per sample .No interference for previous analysis fresh stationary and mobile. phase for each analysis-no contamination.Visual detection possible.

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Steps involved in HPTLC

Selection of chromatographic plates (HPTLC plates)Selection of chromatographic layerPre washing of platesActivation of HPTLC platesLayer pre-conditioning Application of sample and standard Chromatographic developmentDetection of spotsscanning8

Steps Involving in HPTLCSample PreparationApplication of sampleChromatography DevelopmentDetection of SpotsSelection of Chromatography layerPre -washing Pre- Conditioning

Scanning and Documentation

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Selection Of Chromatography PlatesHand Made PlatesPre Coated Plates

Hand Made PlatesCelluloseCellulose with binder starchSilica gel with starchAcetylated cellulose + CaSO4 H2O10

Precoated Layer Of HPTLCDifferent support materials are used GlassPolyester sheetsAluminiumSilica gel 60FAluminium oxide: Basic substances ,alkaloid and steroids.RP,RP8,RP18: Nonpolar substances ,fatty acids,carotenoids,cholesterol.Preservatives,barbiturates,analgesic and phenothiazines-Hybrid plates RP18WF25S

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Sample And Standard PreparationSample and standard should dissolved in the same solvent to ensure comparable distribution at stationary zones.

For normal phase chromatography solvent for dissolving the sample should be non polar.

For reverse phase chromatography polar solvents are used.12

Pre Washing Of Pre Coated PlatesTo avoid any possible interference due to impurities it is recommended to wash the plates is called pre washing.

Methods used for pre washing are : Dipping Ascending Continous Solvents used for washing are:Chloroform in methanol(1:1)Methylene chloride methanol(1:1)1%ammonia or 1% acetic acid 13

Activation Of Pre-coated PlatesFreshly open box of plates do not require activation. Plates exposed to high humidity or kept on hand for long time to be activated By placing in oven at 110-120c for 30 minutes prior to spotting.

Aluminium sheets should be kept in between two glass plates and placing in oven at 110-115c for 15 minutes. 14

Application of standard and sample

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Application Of Standard And SampleSelection of sample application and devices used depends on Sample volume Number of samples to be appliedSamples is applied by use of automatic devices and graduated capillaries.Volume recommended for HPTLC 0.5-5lSample should not excess or not lowOver loading can be over come by applying sample as band.

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NANOMAT AND CAPILLARY DEVICESThe Nanomat serves for easy application of samples in the form of spots on HPTLC plates.

The Nanomat is suitable for HPTLC plates 110 cm and 2010 cm.

capillaries of 0.5, 1.2 and 5l volume available.

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Selection Of Mobile PhaseNormal phase Stationary phase is polarMobile phase is non polar Non polar compounds eluted first because of lower affinity with stationary phase . polar compound retained because of higher affinity with the stationary phaseReversed phase Stationary phase is non polar Mobile phase is polar polar compound eluted first because of lower affinity with stationary phase non polar compounds retained because of higher affinity with the stationary phase.

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Pre Conditioning ( Chamber Saturation)Un-saturated chamber causes high Rf values Saturated chamber by lining with filter paper for 30 minutes prior to development.Lead to uniform distribution of solvent vapours and low Rf value19

Chromatographic Development And Drying

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Chromatographic Development And DryingAfter development remove the plate and mobile phase is removed from the plate to avoid contamination of lab atmosphere.Dry in vacuum desiccator

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Chamber DevelopmentTwin trough and flat bottom chamber

Horizontal development chamber

HPTLC various system

Automatic developing chamber(ADC2)

Automated multiple development (AMD)22

Twin Trough Chambers

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Horizontal Developing Chamber

It is developed from both opposing sides towards the middle.

HPTLC Various SystemDevelopment with six different solvents can be tested side by side.Six different conditions of pre-equilibration , including relative humidity , can be tested simultaneously.

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Automatic Developing Chamber (ADC2)The Automatic Developing Chamber offers convenience , safety and reproducibility for isocratic development of TLC/HPTLC plates.

Automated Multiple Development (AMD)Employed for reproduciblegradient elution.

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DerivatizationFor proper execution of the dipping technique, the chromatogram must be immersed and withdrawn at a controlled uniform speed.

HPTLC SPRAYERThe TLC/HPTLC sprayer consist of a pump unit with two kinds of spray heads.Spray head type A is for spray solutions.

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Detection and visualizationDetection under UV light is first choice non destructive spots of fluorescent compounds can be seen at 254nm(near UV range)

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QuantificationSample and standard should be chromatographed on sample plate after development chromatogram is scannedCamag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode.Scanning speed is selectable up to 100 mm/s spectra recording is fast -36 tracks with up to 100 peak windows can be evaluated.Calibration of single and multiple levels with linear or nonlinear regressions are possible. When target values are to be verified such as stability testing and dissolution profile single level calibration is suitable.

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TLC Scanner 3It can also be used for densitometer measurements of other planar objects such as electrophoresis gels.Key featuresScanning speed 1-100mm/sSpectrum recording up to 100 mm/s.

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Application of HPTLCPharmaceutical Industry : quality control, content uniformity, identity/purity check.Food Analysis: quality control, additives, pesticides, stability testing.Clinical Application :metabolism studies, drug screening, stability testing etc.Industrial Application: process development and optimization, In-process check, validation etc.Forensic : poisoning investigation.Finger print analysis.30

Reference:-

Sethi PD HPTLC High performance thin layer chromatography, First edition, CBS Publisher and Distrributers.Reich, E.and Schhibli A.(2007)High performance liquid chromatography for analysis of medicinal plant, Thieme.Sherma J.Review of HPTLC in Drug Analysis:1996-2009. J AOAC Int.2010;93:754-64.Arup U, Ekman S , Lindblom L, Mattsson JE.High performance Thin Layer Chromatography(HPTLC),1993;25:61-71. 31

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