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By-Saurabh Rawat Guided by MR. Uttam Singh Baghel

Chromatography: method used for separation of the multi-component mixture. HPTLC-High Performance Thin Layer Chromatography. It is a sophisticated & automated form of TLC. It is also known as planar chromatography or Flatbed chromatography.

Same as TLC. The principle of separation is adsorption. One or more compounds are spotted on a thin layer of adsorbent coated on chromatographic plate. The mobile phase solvent flows through because of capillary action The components move according to their affinities towards the adsorbent. The component with more affinity towards the stationary phase travel slower and lesser affinity towards stationary phase travel faster. Thus the components are separated.

Steps Involving in HPTLC

Sample Preparation Selection of chromatography layer Pre-washing Pre-conditioning Application of sample Chromatography development Detection of spots Scanning & documentation

Selection of chromatography layer

Depends on nature of material to be separated. Commonly used(silica gel, alumina)

Materials Glass Advantage 1.Ressistant to heat and chemicals 2.Easy to handle and offers superior flat surface for work Disadvantage 1. Fragility 2.Relatively High wt 3.Costs more for additional packaging 1.It react if temperature exceeds 120c as the plates are dimensionally unstable beyond this temperature

Polyester sheets (0.2 mm 1.More economical as thick) produced even in roll forms 2.Unbreakable 3.Less packing material 4.Spots can be cut and eluted thus eliminates dust from scrapping Aluminum Sheets(0.1mm) 1.Increasesed resistance

temperature 1.Eluents containing high concentration of mineral acids or ammonia can attack chemically on aluminum

Some of the sorbents used in HPTLC:

No Examples 1. 2. 3. Silica gel 60F Alluminium oxide Cellulose (microcrystalline ) Applications 80% of analysis is done on this layer. Basic substances ,alkaloids and steroids Amino acids ,peptides ,sugars and other liable compounds which cannot be chromatographed on the active layers of silica gel.


Silica gel chemically modified COOH ,Phenols ,Nucleotides a) Amino group ( NH2) Pharmaceutical preservations. b ) CN

Some of the binders used: Gypsum (G) Starch (S) Layer containing fluorescent indicator (F) Plate size: 20X20cm 10X20cm 5X10 cm 5X7.5 cm Good cut edges of sheets is important to obtain constant Rf values.

Pre coated plates:

The plates with different support materials and sorbent layers with different format and thickness are used. Plates with sorbent thickness of 100-250 m are used.

Pre washing of pre coated plates

The main purpose of the pre-washing is to remove impurities which include water vapours and other volatile substances from the atmosphere when they get exposed in the lab environment. Silica gel 60F is most widely used sorbent. The major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using Methanol : water in the ratio of 9:1.This is the major advantage of the step of pre-washing.

Solvents used for pre-washing

1.Methanol 2.Chloroform: methanol ( 1:1 ) 3.Choloroform: Methanol: Ammonia (90:10:1 ) 4.Methylene chloride: Methanol ( 1:1 ) 5.Ammonia solution (1%)

Activation of plates:
Freshly opened box of HPTLC plates doesn t need activation. Plates exposed to high humidity or kept in hand for long time require activation. Plates are placed in oven at 110o-120oc for 30 min prior to the sample application. Activation at higher temperature for longer period is avoided as it may lead to very active layers and risk of the samples being decomposed.

Sample Preparation:
Proper sample preparation is require for proper separation. For normal chromatography: Solvent should be nonpolar. For reversed chromatography: Polar solvent is used for dissolving the sample

Application of sample:
The selection of sample application technique and device to be used depends on: Sample volume No. of samples to be applied Some applicators used for spotting are: a) Capillary tubes b) Micro bulb pipettes c) Micro syringes, d)Automatic sample applicator.

The major criteria is that they shouldn t damage the surface while applying sample. The sample should be completely transferred to the layer. Micro syringes are preferred if automatic application devices are not available. Volume recommended for HPTLC-0.5-5 l to keep the starting zone down to minimum of 0.5-1 mm in concentration range of 0.1- g/ml Sample spotting should not be excess or not low. Problem from overloading can be overcome by applying the sample as band.

Advantages of application of sample as band are

Better separation because of rectangular area. Response of densiometer is higher in case of band than that observed from an equal amount/equal volume of sample applied as a spot.

Automatic applicators used:

1) CAMAG Nanomat: Samples applied in the form of spots. The volume is controlled by disposable platinum iridium of glass capillary which has volume of 0.1-0.2 l.

2) CAMAG Linomat
Automated sample application device. Sample is loaded in micro syringe (Hamilton Syringe) 1 l capacity. Sample can apply either as spot or band by programming the instrument with parameters like spotting volume ,band length etc.

3) CAMAG automatic TLC sampler III : Applies sample as spot or bands automatically from the rack of sample vials.

Mobile phase
Mobile phase should be of high graded. Mobile phase selection is depends upon the chemical property of analytes and the sorbent layer. Use of mobile phase containing more than three or four components should normally be avoided as it is often difficult to get reproducible ratios of different components.

Mobile phase optimization is necessary while performing HPTLC. Various components of MP should be measured separately and then placed in mixing vessel. This prevents contamination of solvents and also error arising from volumes expansion or contraction on mixing. Trough chambers are used in which smaller volumes of MP usually 10-15 ml is required.

Different components of MP are mixed first in mixing vessel and then transferred to developing chambers. Chambers containing multi component MP are not generally used for re-use for any future development , due to differential evaporation and adsorption by layer and also once the chamber is opened , solvents evaporate disproportionally depending on their volatilities.

Pre-conditioning : (Chamber Saturation)

Chamber saturation has a pronounced influence on the separation profile. Time required for the saturation depends on the mobile phase. If plates are introduced into the unsaturated chamber ,during the course of development , the solvent evaporates from the plate mainly at the solvent front and it results in increased Rf values.

The different methods used for development of chambers are like-Ascending , descending .2dimentional, horizontal , multiple overrun , gradient ,radial ,anti-radial ,multimodal ,forced flow planar chromatography. Plates are spotted with sample and air dried and placed in the developing chambers. After the development plate is removed from chamber and mobile phase is removed under fume cup-board to avoid contamination of laboratory atmosphere. The plates should be always laid horizontally because when mobile phase evaporates the separated components will migrate evenly to the surface where it can be easily detected

Drying :
Drying of chromatogram should be done in vacuum desiccators with protection from heat and light. If hand dryer is used there may be chances of getting contamination of plates ,evaporation of essential volatile oils if any present in the spot or compounds sensitive to oxygen may get destroyed due to the rise in temperature.

Detection of spot
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

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 Documentation E - Merck introduced plates with imprinted identification code - supplier name. Item number, batch number and individual plate number - Avoid manipulation of data at any stage - coding automatically get recorded during photo documentation

Factors influencing separation and resolution of spots:

Type of stationary phase Type of pre-coated plates 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 Greater the difference between two spots and smaller the initial spot diameter of sample and better will be the resolution

Differences between TLC and HPTLC:

Parameter Chromatographic plate used Sorbent layer thickness Particle size range Pre-washing of the plate Application of sample Shape Spot size Sample volume Application of larger volume No. of samples/plate (20X20) Optimum development distance Development time Reproducibility of results TLC Hand made /pre-coated 250 micro meter 5-20 m Not followed Manual/Semi automatic Spot 2-4mm 1-10 l HPTLC Pre-coated 100-200micro meter 4-8 m Must Semi automatic/Automatic Spot/Band 0.5-1mm 0.2-5 l

Spotting which leads to over Can be applied as bands loading 15-20 10-15 cm Depends on mobile phase Difficult 40-50 5-7 cm 40% Less than TLC Reproducible

Applications of HPTLC:
Pharmaceutical industry: Quality control, content uniformity, uniformity test, 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

HPTLC- Quantitative analysis of pharmaceutical Formulations by P.D. Sethi Kasture A.V A text book of Pharmaceutical Analysis (Instrumental methods), 14th edition, page no.28-30 Watson David G.Pharmaceutical analysis pp 290292. e=ISO-8859-1&hl=en &lr=&ie=ISO-8859-1&q=linomat

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