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HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY Presention by Guided by Yogesh A. Shirsath Prof.Arun M.Kashid Sinhgad Institute of Pharmacy Narhe , Pune . 31/12/2010 1


Contents Introduction. Principle of HPTLC. Difference between HPLC, TLC & HPTLC. Steps involved in HPTLC. Material used for plates. Mobile phase. Sample application. HPTLC Plate development. 3/28/2011 2


Introduction Chromatography : Separation of the multi-component mixture. HPTLC - High Performance Thin Layer Chromatography. Sophisticated & automated form of TLC. History In l973 Halpaap introduced first. ‘‘nano-TLC’’ plates (Merck manufacturer). In 1977 the first major HPTLC publication appeared, simply called ‘‘HPTLC – high” . 3/28/2011 3


Principle Separation may result due to adsorption or partition or by both phenomenon depending upon the nature of adsorbents used on plates and solvents system used for development. 3/28/2011 4

Difference between HPTLC & TLC:

Difference between HPTLC & TLC HPTLC TLC Particle size 4-8 µm 5-20 µm Sorbent layer thickness 100 µm 250 µm Efficiency High Less Separations 3-5 cm 10-15cm Analysis time Faster Slower Development chamber 3/28/2011 5 Less amount of mobile phase More amount of mob. phase


HPTLC TLC Sample spotting Auto sampler Manual spotting Scanning. Visible/ Fluores- scanner scan Not possible the entire chromatogram (Densitometer) 3/28/2011 6


DIFFERENCE BETWEEN HPTLC & HPLC HPTLC HPLC Simultaneous processing simultaneous process of sample & standard of sample & std. not under same condition possible Extreme flexibility for Limited flexibility various steps Technically, it is simple Skilled & well-trained to learn & operate personnel are needed 3/28/2011 7


HPTLC HPLC Several analyst either Only one analyst can of the same lab. or can work at a given different lab can time simultaneously work on the system 3/28/2011 8 Sample preparations the critical Sample preparations is simple Less maintenance cost High maintenance cost

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3/28/2011 9 STEPS IN HPTLC Sample & Standard Preparation Selection of Chromatographic Layer Application of Sample & Standard Chromatographic Development Detection of Spot Scanning & Documentation of Chromatoplate Layer Pre-wash

Plates Materials:

Plates Materials Cellulose Cellulose with starch as binder Cellulose (Microcrystalline) fluorescent indicator Acetylated Cellulose + CaSo 4 Silica gel Silica gel with Starch 3/28/2011 10

Support Materials:

Support Materials 3/28/2011 11 Materials Advantage Disadvantage Glass 1.Ressistant to heat and chemicals 2.Easy to handle and offers superior flat surface for work 1. Fragility 2.Relatively High wt 3.Costs more for additional packaging Polyester sheets (0.2 mm thick) 1.More economical as produced even in roll forms 2.Unbreakable 3.Less packing material 4.Can be cut and eluted thus eliminates dust from scrapping 1.Charring reactions if temperature exceeds 120 o c as the plates are dimensionally unstable beyond this temperature Aluminum Sheets(0.1mm) 1.Increasesed temperature resistance 1.Eluents containing high concentration of mineral acids or ammonia can attack chemically on aluminum

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3/28/2011 12 Some of the sorbents used in HPTLC: No Examples Applications 1. Silica gel 60F (Unmodified ) Analysis of 80% of drugs. 2. Alluminium oxide Basic substances ,alkaloids and steroids 3. Cellulose (microcrystalline ) Amino acids ,peptides ,sugars and other liable compounds which cannot be chromatographed on the active layers of silica gel. 4. Silica gel chemically modified a) Amino group ( NH2) b ) CN COOH ,Phenols ,Nucleotides Pharmaceutical preservations.

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13 Plate Size – 20X20cm 5X7.5 cm 5X10 cm 10X20cm. Pre-washing of pre-coated Plates Sorbent layer absorb water vapour & other impurities from atmosphere Pre-washing method- Ascending Dipping Continuous 3/28/2011

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3/28/2011 14 3/28/2011 14 Solvents used for pre-washing Methanol Chloroform: Methanol ( 1:1 ) Chloroform: Methanol: Ammonia (90:10:1 ) Methylene chloride: Methanol ( 1:1 ) Ammonia solution (1%)

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3/28/2011 15 After washing Dry for sufficient time for removal washing liquids. Avoid hot/cold air dryer. Use desiccators for storing. Activation of pre-coated plates Fresh open box of HPTLC plate not require activation, Other activated by placing 110 o -120 o c for 30 min. Dry in vertical position.

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3/28/2011 16 Mobile phase Solvent composition expressed in v/v. Mobile phase should be of high graded. Chemical properties , analytes and sorbent layer factors should be considered while selection of mobile phase. If possible mobile phase containing more than 3 or 4 components should be avoided.

Mobile phase(cont.):

Mobile phase(cont.) 3/28/2011 17 Prevents contamination of solvents. Multi-component of mobile phase once used is not recommended for re-use. Chemical reaction avoided between SP & MP. e.g. Acetic acid, Ammonia.

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3/28/2011 18 Sample Preparation For normal chromatography: Solvent should be non-polar and volatile. For reversed chromatography: Polar solvent is used for dissolving the sample Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones.

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3/28/2011 19 Application of sample The selection of sample application technique and device to be used depends primarily on: Sample volume No. of samples to be applied Required precision

Application of sample (cont.) :

Application of sample (cont.) 3/28/2011 20 Micro syringes are preferred if automatic application devices are not available. Volume recommended for HPTLC-0.5-5 μ l. Sample spotting should not be excess or not low. Problem from overloading can be overcome by applying the sample as band.

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HPTLC SAMPLE APPLICATION SPOT BAND Non – uniform Distribution Diffusion Enlarged Low volume Scan problems Not uniform sensation. Uniform Distribution Compact Better resolved Larger volume No overload Better sensation Better precision. No problem Uniform sensation. VS 3/28/2011 21

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3/28/2011 23 Some applicators used for application of sample a) Capillary tubes. Samples applied in the form of spots. Volume of 0.1-0.2μl b) Micro bulb pipettes.

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3/28/2011 24 c) Micro syringes. Sample can apply either as spot or band Volume- 1μl. d)Automatic sample applicator . Sample can apply either as spot or band.

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3/28/2011 25 Pre-conditioning (Chamber Saturation) Time required for the saturation depends on the mobile phase. If unsaturated chamber used for development, the solvent evaporates from the plate mainly at the solvent front and it results in increased R f values.

H P T L C Development:

H P T L C Development Vertical Development. Vario method development. Horizontal development. Automatic Multiple Development (AMD)/( Gradient ). 3/28/2011 26

Twin Trough Chambers:

Twin Trough Chambers 3/28/2011 27

Vario Chamber Development :

Vario Chamber Development VARIO CHROMATOGRAM 3/28/2011 28

Horizontal Development:

Horizontal Development 3/28/2011 29 HPTLC plate is developed from both opposing sides towards the middle. Plate sizes 10x10cm and 20x10cm

Automatic Multiple Development(AMD):

Automatic Multiple Development(AMD ) 3/28/2011 30

Gradient H P T L C :

Gradient H P T L C High resolution -polymers. Trace analysis -pesticide etc residues Multipolar samples -lipids, natural products Closely related compounds -isomers, org. Synthesis Where To Use 3/28/2011 31

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Post Chromatography Steps 1) Visualization. Photo documentation. Densitometry measurements. 3/28/2011 32

Visualisation :

Visualisation UV Cabinet - 3 3/28/2011 33

Densitometry measurements:

Densitometry measurements 3/28/2011 34 Measures visible, UV absorbance or Fluorescence.

Photo-documentation With Digital Camera:

Photo-documentation With Digital Camera 3/28/2011 35


Photo-documentation VISUAL CONFIRMATION/ VISUAL DIFFERENTIATION 254 + 366 NM, UV, VISIBLE 3/28/2011 36

Reversed Phase HPTLC(RPHPTLC):

Reversed Phase HPTLC(RPHPTLC) 3/28/2011 37 Stationary phases are modified hydrophobic substances Liquid paraffin Hydrocarbons like Alkyl chlorosilanes . Mobile phase- Methanol Acetonitrile Dioxane

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3/28/2011 38 Factors Influencing Separation And Resolution Of Spots Type of stationary phase Layer thickness Binder in layer Mobile phase Solvent purity Size of developing chamber Sample volume to be spotted Size of initial spot Solvent level in chamber

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3/28/2011 39 Applications of HPTLC Pharmaceutical industry : Quality control, content uniformity, identity/purity check. Food Analysis: Quality control , additives , pesticides ,stability testing , Clinical Applications: Metabolism studies , drug screening ,stability testing etc Industrial Applications: Process development and optimization, In-process check ,validation etc. Forensic : Poisoning investigations


References 3/28/2011 40 1.Sethi P D. HPTLC High Performance Thin Layer Chromatography, First edition(1996),CBS Publishers and Distributers, 4-30 2.Sherma J. Fried B. Handbook of thin-layer chromatography, Second edition(1996), vol-89, Marcel. Dekker, New York, 1104 3.Beckett A H. Stenlake J B. Practical pharmaceutical chemistry,fourth edition(2004),part-two, First edition, CBS Publishers and Distributors New delhi,124

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3/28/2011 41 4.Meyyanathan S N, Basic Principles of HPTLC , Pharmainfo.net,2003 5.http:// www.camag.com /products/application/ disposa ble.html 6.http:// www.bcon-nstruments.nl /products/ hptlc.html

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