High Performance Thin Layer Chromatograph (General topic covered)

1. High Performance Thin Layer Chromatography
Prepared by-
Kartik Tiwari
M. Pharm
HYGIA INSTITUTE OF PHARMACEUTICAL
EDUCATION AND RESEARCH, LUCKNOW

2. Content
 Introduction
 Chromatography
 Classification
 High Performance Thin Layer Chromatography
 Principle
 Instrumentation
 Steps involved in HPTLC
 Factors affecting
 Application
 Reference

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 .

7. Instrumentation
Fig.1: Linomat 5 Fig.2: Chromatogram immersion

8. Instrumentation
Fig.3: Visualizer Fig.4: HPTLC Scanner

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

18. Fig.5: Sennoside TLC picture

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 .