ADVANCED SPECTRAL ANALYSIS (MPC201T)
TOPIC OF PRESENTATION: HPTLC
PRESENTED BY
SAHID AHMED
M. PHARM II SEM
2312023007
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CONTENTS
High Performance Thin Layer Chromatography (HPTLC)
PRINCIPLE
INSTRUMENTATION
APPLICATIONS
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PRINCIPLE
Principle of HPTLC have similar approach and employ the same physical principles of
TLC (adsorption chromatography) i.e. the principle of separation is adsorption.
The mobile phase solvent flows through because of capillary action.
The components move according to their affinities towards the adsorbent.
The component with more affinity towards the stationary phase travels slower.
The component with lesser affinity towards the stationary phase travels faster. Thus,
the components are separated on a chromatographic plate.
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Normal phase
Stationary phase is polar (eg Silica)
Mobile phase is non polar (eg Hexane)
Non-polar compounds eluted first because of lower affinity with stationary phase
Polar compounds retained because of higher affinity with the stationary phase
Reversed phase
Stationary phase is non polar (eg C18)
Mobile phase is polar (eg methanol)
Polar compounds 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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INSTRUMENTATION
Selection of Chromatography plates
Sample and Standard Preparation
Pre-washing of Pre coated plates
Activation of pre coated plates
Application of Standard and Sample
Selection of Mobile phase
Chromatographic development and
drying
Detection and visualization
Quantification – TLC Scanner 3
Selection of Chromatography plates
HPTLC plates have a uniform, high-quality sorbent layer coated on a glass or 6
aluminum backing. The thickness of the sorbent layer is typically around 100-200 µm.
Sample and Standard Preparation
Sample and reference substances should be dissolved in the same solvent to ensure
comparable distribution at starting zones.
Pre-washing of Pre coated plates
The main purpose of the pre-washing is to remove impurities which include water
vapours and other volatile substances from the atmosphere when they get exposed in
the lab environment.
Activation of pre coated plates
Plates are placed in oven at 110o -120oc for 30 min prior to the sample application.
Application of Standard and Sample
Automated sample application device. Sample is loaded in micro syringe (Hamilton
Syringe) 1μl capacity. Sample can apply either as spot or band by programming the
instrument with parameters like spotting volume ,band length etc. (LINOMAT
APPLICATOR)
Selection of Mobile phase
Chemical properties, analytes and sorbent layer factors should be considered while
selection of mobile phase. Twin trough chambers are used in which smaller volumes of
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Mobile phase usually 10-15 ml is required.
Chromatographic development and drying
The different methods used for development of chambers are likeAscending,
descending, 2-dimentional, horizontal, multiple overrun, gradient & forced flow planar
chromatography. Drying of chromatogram should be done in vacuum desiccators with
protection from heat and light.
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.
Quantification – TLC Scanner 3
Camag 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.
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COMPARISON
TLC VS HPTLC
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APPLICATIONS
Pharmaceutical industry: Quality control, content uniformity, uniformity test,
identity/purity check.
Food Analysis: Quality control, additives, pesticides ,stability testing ,analysis of
sub-micron levels of aflotoxins etc
Clinical Applications: Metabolism studies, drug screening ,stability testing etc
Industrial Applications: Process development and optimization, In-process
check, validation etc.
Forensic : Poisoning investigations
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REFERENCES
Sethi PD HPTLC High performance thin layer chromatography, First edition, CBS Publisher
and Distributers.
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 LayerChromatography
HPTLC), 1993;25:61-71.
https://youtu.be/dNueDQ0Fn9U?si=-3naUSRD3dSUqr8a
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