(HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY)

By-Saurabh RawatGuided by –MR. Uttam Singh Baghel

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.It is also known as planar chromatography or Flat-bed chromatography.

PRINCIPLE• 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 PreparationSelection of

chromatography layer

Pre-washing

Pre-conditioning

Application of sample

Chromatography development

Detection of spots

Scanning & documentation

Selection of chromatography layerSelection of chromatography layer

Depends on nature of material to be separated.

Commonly used(silica gel, alumina)

SupportsMaterials Advantage Disadvantage

Glass 1.Ressistant to heat and chemicals2.Easy to handle and offers superior flat surface for work

1. Fragility2.Relatively High wt3.Costs more for additional packaging

Polyester sheets (0.2 mm thick)

1.More economical as produced even in roll forms2.Unbreakable3.Less packing material4.Spots can be cut and eluted thus eliminates dust from scrapping

1.It react if temperature exceeds 120ºc 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

Some of the sorbents used in HPTLC:

No Examples Applications

1. Silica gel 60F 80% of analysis is done on this layer.

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 modifieda) Amino group ( NH2)b ) CN

COOH ,Phenols ,NucleotidesPharmaceutical preservations.

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 100-250μm 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 non-polar.

• 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-0.5-5μl to keep the starting zone down to minimum of 0.5-1 mm in concentration range of 0.1-μg/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 0.1-0.2μl.

2) CAMAG Linomat

Automated sample application device. Sample is loaded in micro syringe (Hamilton Syringe) 1μl 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.

Development • The different methods used for development of

chambers are like-Ascending , descending .2-dimentional, 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 TLC HPTLC

Chromatographic plate used Hand made /pre-coated Pre-coated

Sorbent layer thickness 250 micro meter 100-200micro meter

Particle size range 5-20 μm 4-8 μm

Pre-washing of the plate Not followed Must

Application of sample Manual/Semi automatic Semi automatic/Automatic

Shape Spot Spot/Band

Spot size 2-4mm 0.5-1mm

Sample volume 1-10 μl 0.2-5 μl

Application of larger volume Spotting which leads to over loading

Can be applied as bands

No. of samples/plate (20X20) 15-20 40-50

Optimum development distance 10-15 cm 5-7 cm

Development time Depends on mobile phase 40% Less than TLC

Reproducibility of results Difficult 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

References: • 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 290-292.• www.pharmainfo.net• http://images.google.co.in/images?q=hptlc+plates&ie=ISO-

8859-1&hl=en• http://images.google.co.in/images?

svnum=10&hl=en&lr=&ie=ISO-8859-1&q=linomat• www.camag.com• http://www.infoexpo.ch/abstract

Thank you