hptlc

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HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY : 

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

CHROMATOGRAPHY : 

CHROMATOGRAPHY Chromatography is a physical process of separation in which the components to be separated are distributed between 2 immiscible phases ­ a stationary phase which has a large surface area and mobile phase which is in constant motion through the stationary phase.

Introduction of HPTLC : 

Introduction of HPTLC HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way. HPTLC takes place in high­speed capillary flow range of the mobile phase. There are three main steps HPTLC procedure, they are 1] Sample to analyzed to chromatogram layer, volume precision and exact position are achieved by use of suitable instrument. 2] Solvent (mobile phase) migrates the planned distance in layer (stationary phase) by capillary action. In this process sample separated into it’s components. 3] Separation tracks are scanned in densitometer with light beams in visible or uv region

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Fig. Hptlc instrumentation

Selection of HPTLC plates : 

Selection of HPTLC plates Previously hand made plates is used in TLC for both qualitative and quantitative work. Certain drawbacks with that is non­uniform layer, formation of thick layer, paved for advent of precoated plates. Nowadays precoated plates are available in different format and thickness by various manufactures. Precaoted plates can be used for both qualitative and quantitative work in HPTLC, they are GLASS PLATES POLY ESTER/POLYETHYLYNE ALUMINIUM PLATES

Glass Plates: : 

Glass Plates: Offers superior flat and smooth surface. - fragile - high weight - higher production cost Polyester/polyethylene plates: Thickness of plate is 0.2mm. - It can be produced in roll forms. - Unbreakable. - Less packing material is required. Development of plate cann’t be above temperature 1200 c loses its shape.

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Aluminium plates: - Thickness of plate is 0.1mm. - It can be produced in roll forms. - Unbreakable. - Less packaging material is required.

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SORBENTS USED IN HPTLC PLATES: sorbents which are used in convential TLC are also used in HPTLC with or without modification. - silica gel 65F - highly purified silicagel 60 - aluminium oxide - cellulose microcrystalline - silica gel - reversed stationary phase

Layer thickness : 

Layer thickness The layer thickness in HPTLC is around 100-200cm,in conventional it is 250mm. Layer prewashing: - Ascending method - Dipping method - Continuous method

ACTIVATION OF PRECOATED PLATES : 

ACTIVATION OF PRECOATED PLATES The plates are activated by placing in an oven at 110­1200 C for 30 min, this step will removes water that has been physically absorbed on surface at solvent layer. Freshly opened box of HPTLC plates usually does not require activation. Activation at higher temp and for longer time is avoided which leads to very active layer and there is risk of sample being decomposed.

Solvents used in HPTLC : 

Solvents used in HPTLC - Methanol (commonly used) - Chloroform:methanol:ammonia(90:10:1) - Chloroform:methanol(1:1) - Methylene chloride:methanol(1:1) - Ammonia(1%)solution

Application of sample and standard : 

Application of sample and standard Usual concentration range is 0.1-1µg / µl,above this causes poor separation. Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe on TLC plates as bands. Band wise application - better separation - high response to densitometer.

Chromatographic development and drying : 

Chromatographic development and drying After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere. Dry in vacuum desiccator - avoid hair drier because essential oil components may evaporate.

Detection and visualization : 

Detection and visualization Detection under UV light is first choice - non destructive. Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length). Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF.

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Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution. When individual component does not respond to UV - derivatisation required for detection .

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HPTLC 100µm High due to smaller particle size generated 3 - 5 cm Shorter migration distance and the analysis time is greatly reduced Wide choice of stationary phases like silica gel for normal phase and C8 , C18 for reversed phase modes New type that require less amount of mobile phase Auto sampler Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer TLC 250µm Less 10 - 15 cm Slower Silica gel , Alumina & Kiesulguhr More amount Manual spotting Not possible

APPLICATIONS : 

APPLICATIONS Pharmaceutical Researches Bio­medical Analysis Clinical Analysis Environmental Analysis Food Industry Therapeutic drug monitoring to determine concentration of drug and it’s metabolite in blood, urine etc Analysis of environmental pollutions levels Quantitative determination of prostaglandin’s and thromboxanes in plasma Determination of mercury in water Analysis of nitrosoamines in food and body fluids Determination of sorbic acid in wine Characterization of hazards in industrial waste

REFERENCES : 

REFERENCES Principles of instrumental analysis skoog, Holler, Nieman Instrumental methods of analysis Willard ,Merrit, Dean Pharmaceutical analysis Munson Sharma J.friedB.Handbook of TLC

THANK YOU : 

THANK YOU

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