3. Introduction
Chromatography –
Chromatography is an important biological technique that enables
the separation , Identification and purification of the components of
a mixture for qualitative and quantitative analysis .
The Russian botanist Mikhail tswett coined the term
chromatography in 1906 .
The first analytical use of chromatography for the analysis of fatty
acids mixtures
4. Classification of chromatography
According to
Principle
• Adsorption
• Partition
• Ion exchange
• Size exclusion
• Affinity
According to
polarity of phases
• Normal phase
• Reverse phase
According to
Mobile phase
• Liquid
chromatography
• Liquid –Liquid
• Liquid – Solid
• Gas
chromatography
• GSC
• GLC
• Supercritical fluid
chromatography
According to
geometry
• Planner
chromatography
• Paper
• TLC
• HPTLC
• Column
chromatography
• Column
• HPLC
• UPLC
5. High Performance Thin Layer Chromatography
HPTLC ( High Performance Thin Layer Chromatography ) is the
automated , sophisticated form and improved method of TLC .
It is a powerful analytical method equally suitable for qualitative and
quantitative analytical tasks .
It is also known as planner or flat bed chromatography .
HPTLC is very popular for many reasons such as -
Visual chromatogram
Multiple sample handling
Enables the most complicated separation ,
6. Principle
Same theoretical principle pf TLC ( Adsorption chromatography ) i.e. the
principle of separation is adsorption .
Mobile phase flow by capillary action effect .
And component move according to their affinities towards the adsorbent.
The component with higher affinity towards adsorbent travels slowly.
And the component with lesser affinity towards the stationary phases
travels faster .
Thus the component are separated on a chromatographic plate according to
their affinity and separation also based on their solubility in mobile phase .
9. Steps involved in HPTLC
Stationary phase :
Silica gel GF- 254 (SO2 + CaSO4 + zinc silicate )
Particle size in 7μm .
Thickness of plate 100μ .
Decreased particle size , increased surface area , so increasing
efficiency increasing resolution & decreasing analysis time .
10. Sample preparation
It is important to prepare proper sample for successful separation .
Sample and reference substances should be dissolved in the same
solvent to ensure comparable distribution at starting zones .
Solvents used are : Methanol , Chloroform : Methanol
(1:1) , Ethyl acetate : methanol (1:1) etc.
11. Sample application
Usual concentration range is 0.1-1μg/l .
Above this causes poor separation .
Volume recommended for HPTLC is 0.5 – 5μl.
The size of sample spot must be not large than 1mm in diameter .
The problem of overloading can be overcome by applying the
sample as band .
The major criteria is that it should not damage the surface while
applying sample .
12. Selection of mobile phase
Chemical properties of analytes &sorbent layer should be
considered while selection of mobile phase .
The less amount of mobile phase is required then TLC .
Due to small amount of mobile phase , it prevents errors in result
which is occur due to mobile phase .
13. Pre-conditioning (chamber saturation)
Chamber is saturated by lining with filter paper for 30 min . Prior to
development of chromatograph .
For low polarity mobile phase there is no need of saturation .
However saturation is needed for highly polar mobile phase .
Chamber saturation influence separation profile .
14. Chromatographic development & drying
Plates are spotted with sample and air dried & placed in the
developing chambers.
The different methods used for development of chromatographs are
like : Ascending , Descending , Horizontal
After development , remove the plate and and mobile phase is
removed from the plate to avoid contamination of lab atmosphere .
Dry in vacuum desiccators with protection from heat and light .
15. Detection and visualization
Detection under UV light is first choice .
Fluorescent compounds can be seen at 254 nm (short wavelength )
or at 336 nm (long wavelength ).
Spots of non fluorescent compounds can be seen by using
fluorescent stationary phase e.g silica gel Gf .
Non UV absorbing compounds are visualized by dipping the plates
in 0.1% iodine solution .
16. Scanning & documentation
The scanner converts band into peak and peak area which is use to
determine the concentration of substance on spot / band .
This is measured by instrument and recorded .
This is result is also get by hard copy or we can get print form of our
results
17. Example of sennoside
Determination of sennoside (Rf = 0.35 ,0.25 , 0.46 ,0.61 )
Mobile phase – 2 propanol : ethyl acetate : water : formic acid
(8.5 : 9.5 :6 : 1=25M )
Test( 10 μg/ml ) standard (10μg/ml ) in methanol
19. Factors affecting HPTLC :
Types of stationary phase
Types of mobile phase
Layer thickness
Temperature
Mode of development
Amount of sample
Dipping zone
20. Applications of HPTLC
Pharmaceutical industry : Quality control , purity test .
Food analysis : QC , stability testing
Clinical applications : Metabolism studies , drug screening etc.
Biomedical analysis : Separation of gangliosides
Forensic : Poisoning investigation
Environment analysis : pesticides in drinking water etc.
Analysis of drug in blood etc.
21. Reference
HPTLC –Quantitaive analysis of pharmaceutical formulation by
P.D. Sethi
http://images.google.co.in/images?q=hptlc+plates&ie=ISO-8859-
1&hl=en
www.camag.com
Pharmaceutical Analysis vol-II by Dr.A.V.Kastur Dr. K.R .Mahadik
Nirali Publishers page no.28-30 .