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HPTLC vs TLC: Key Differences & Features

This document presents information about high performance thin layer chromatography (HPTLC). It discusses the principles and process of HPTLC, highlighting key differences between HPTLC and traditional thin layer chromatography (TLC). HPTLC provides higher efficiency separations using a thinner stationary phase layer over a shorter development time compared to TLC. The document outlines the basic steps of an HPTLC procedure including sample preparation, chromatography development, detection and documentation of results. It also discusses factors that can affect HPTLC separations and provides references for additional information.

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420 views18 pages

HPTLC vs TLC: Key Differences & Features

This document presents information about high performance thin layer chromatography (HPTLC). It discusses the principles and process of HPTLC, highlighting key differences between HPTLC and traditional thin layer chromatography (TLC). HPTLC provides higher efficiency separations using a thinner stationary phase layer over a shorter development time compared to TLC. The document outlines the basic steps of an HPTLC procedure including sample preparation, chromatography development, detection and documentation of results. It also discusses factors that can affect HPTLC separations and provides references for additional information.

Uploaded by

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Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd

Presented by:

NILESH [Link]
Guided by:
[Link]
Pune University
1
Introduction
Principle of HPTLC
Difference between HPTLC & TLC
Features of HPTLC
Instrumentation
Steps involved in HPTLC
Rf value
Factor affecting HPTLC
References


2
Chromatography: method used for separation
of the multi-component mixture.

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

3
Same theoretical principle of TLC.
Separation may result due to
adsorption phenomenon.
4
HPTLC TLC
Particle size 4-8m 5-20m
5
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

6
Sample Preparation
Selection of
chromatography layer
Pre-washing
Pre-conditioning
Application of sample
Chromatography development
Detection of spots
Scanning & documentation
7
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)
8
It is purification step
Mainly methanol is used
Essential for quantitative evaluation
Stability testing

9
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
10
Linomat lV applicator
11
Ascending development
Descending development
Horizontal development
Problem encountered
Tailing
Diffusion
12
Generally, detection by iodine vapour
Alternatively by
Visual detection at 254nm UV-Light
Detection at 365nm UV-Light
13
Use white light
Registered absorption spectra
Lamp & detector are placed

450m above surface of HPTLC
plate
45 angle bet. Lamp & detector
14
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)

15
Type of stationary phase
Mobile phase
Layer thickness
Temperature
Mode of development
Amount of sample
Dipping zone
others
16
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 [Link] (Google images)
17

You
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