Presented by:

NILESH S.JAWALKAR
Guided by:
S.R.KALE
Pune University
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Introduction
Principle of HPTLC
Difference between HPTLC & TLC
Features of HPTLC
Instrumentation
Steps involved in HPTLC
Rf value
Factor affecting HPTLC
References


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Chromatography: method used for separation
of the multi-component mixture.

HPTLC-High Performance Thin Layer
Chromatography.
It is a sophisticated & automated form of
TLC.

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Same theoretical principle of TLC.
Separation may result due to
adsorption phenomenon.
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HPTLC TLC
Particle size 4-8m 5-20m
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Sorbent layer 100m 250m
Efficiency High Less
Separations 3-5cm 10-15cm
Analysis time greatly reduced slower
Development
chamber
Less amount of
mobile phase
More amount

Simple & rapid
Several analysts work simultaneously
Lower separation time
Less cost
Low maintenance cost
Low mobile phase consumption per sample
No interference from previous analysis
Visual detection possible

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Sample Preparation
Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
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Normal phase chromatography: non polar solvent
Reversed phase chromatography: polar solvent

Selection of chromatography layer
Depends on nature of material to be separated
Commonly used(silica gel, alumina)
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It is purification step
Mainly methanol is used
Essential for quantitative evaluation
Stability testing

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Also called Chamber Saturation
Low polarity mob. Phase:- no need
High polar mob. Phase:- desirable
For reverse phase saturate chamber with polar
solvent
Sample application
1-5 l
Linomat IV Applicator
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Linomat lV applicator
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Ascending development
Descending development
Horizontal development
Problem encountered
Tailing
Diffusion
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Generally, detection by iodine vapour
Alternatively by
Visual detection at 254nm UV-Light
Detection at 365nm UV-Light
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Use white light
Registered absorption spectra
Lamp & detector are placed

450m above surface of HPTLC
plate
45 angle bet. Lamp & detector
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Rf = Zs
Zo

Where,
Rf = Retardation factor
Zs = distance of centre of spot from starting line(mm)
Zo = distance of solvent front from starting line(mm)

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Type of stationary phase
Mobile phase
Layer thickness
Temperature
Mode of development
Amount of sample
Dipping zone
others
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Kasture A.V A text book of
Pharmaceutical Analysis (Instrumental
methods), 14
th
edition, page no.28-30
Elke Hahn Deinstrop Applied TLC, 2
nd

edition
Site www.Google.com (Google images)
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