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A SeminaronHPTLC : 

A SeminaronHPTLC Presented by: Surjeet Thakur Guided by: Mr. Sumit Bansal Shoolini University 1

Contents : 

Contents Introduction Principle of HPTLC Difference between HPTLC & TLC Features of HPTLC Instrumentation Steps involved in HPTLC Rf value Factor affecting HPTLC References 2

Introduction : 

Introduction 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

Principle : 

Principle Same theoretical principle of TLC. Separation may result due to adsorption phenomenon. 4

Difference between HPTLC & TLC : 

Difference between HPTLC & TLC HPTLC TLC Particle size 4-8µm 5-20µm 5 Sorbent layer 100µm 250µm Efficiency High Less Separations 3-5cm 10-15cm Analysis time greatly reduced slower Development chamber Less amount of mobile phase More amount

Features of HPTLC : 

Features of HPTLC 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

Steps Involving in HPTLC : 

Steps Involving in HPTLC Sample Preparation Selection of chromatography layer Pre-washing Pre-conditioning Application of sample Chromatography development Detection of spots Scanning & documentation 7

Sample preparation : 

Sample preparation 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

Pre-washing : 

Pre-washing It is purification step Mainly methanol is used Essential for quantitative evaluation Stability testing 9

Pre-conditioning : 

Pre-conditioning 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 µl Linomat IV Applicator 10

Slide 11: 

Linomat lV applicator 11

Chromatographic Development : 

Chromatographic Development Ascending development Descending development Horizontal development Problem encountered Tailing Diffusion 12

Detection of Spot (Evaluation) : 

Detection of Spot (Evaluation) Generally, detection by iodine vapour Alternatively by Visual detection at 254nm UV-Light Detection at 365nm UV-Light 13

Diode-Array Detector : 

Diode-Array Detector Use white light Registered absorption spectra Lamp & detector are placed 450µm above surface of HPTLC plate 45º angle bet. Lamp & detector 14

Rf value (Retardation factor) : 

Rf value (Retardation factor) 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

Factor affecting HPTLC : 

Factor affecting HPTLC Type of stationary phase Mobile phase Layer thickness Temperature Mode of development Amount of sample Dipping zone others 16

References: : 

References: “Kasture A.V” A text book of Pharmaceutical Analysis (Instrumental methods), 14th edition, page no.28-30 “Elke Hahn Deinstrop” Applied TLC, 2nd edition Site –www.Google.com (Google images) 17

“Thank : 

“Thank You” 18

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