High Performance Thin Layer Chromatography.pptx

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

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 ( SO 2 + CaSO 4 + zinc silicate ) Particle size in 7 μ m . Thickness of plate 100 μ . 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 0.1-1 μ g/l . Above this causes poor separation . Volume recommended for HPTLC is 0.5 – 5 μ l. 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 μ g/ml ) standard (10 μ g/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 .

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