(HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY)

Presented by:
ABDUL RAZZAQ M.Pharma in Pharmaceutical Chemistry LUQMAN COLLEGE OF PHARMACY GULBARGA

Definition:
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:
HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way. It is also known as planar chromatography or Flat-bed chromatography.

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 mm 5-20 m Not followed Manual/Semi automatic Spot 2-4mm 1-10 l HPTLC Pre-coated 100-200mm 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

SAMPLE AND STANDARD PREPARATION

SELECTION OF CHROMATOGRAPHIC PLATES

LAYER PRE-WASHING

LAYER PRE-CONDITIONING

APPLICATION OF SAMPLE

CHROMATOGRAPIC DEVELOPMENT

DETECTION OF SPOTS

SCANNING AND DOCUMENTATION OF CHROMOPLATE USING PC CATS SOFTWARE

 Selection of HPTLC plates Hand plates were available which are made up of cellulose and other materials which are not used much now-a days.

 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 for qualitative and quantitative analysis.

Supports
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.Charring reactions if temperature exceeds 120oc 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 (Unmodified ) Alluminium oxide Cellulose (microcrystalline ) Applications 60F 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.

4.

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 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.

Some common methods involved in pre: washing: Ascending method: Dipping method: Continuous method:

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 doesnt 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 an important prerequisite for success of TLC separation. 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.

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 and degree of automation.

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 shouldnt 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. Large quantiti

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. Chemical properties ,analytes and sorbent layer factors should be considered while selection of mobile phase. 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.

Development of chambers:
1.Twin trough chamber.

2.Rectangular chambers

3. V-shaped chambers 4.Sandwitch chamber 5.Horizontal development chamber 6.Automatic development chamber

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 and Drying:

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

Simulation chamber
The simultan developing chamber is a thick walled clear glass tank with vertical grooves and a heavy ground-glass lid.

Round chamber
These cylindrical chambers are ideal for use with narrower width plates.

Nano chamber
The nano chamber is suitable for the development of 10x10cm TLC plates and features a heavy glass lid for gas-tight seal and optimum vapour saturation.

HPTLC chamber
Ideal for the development of HPTLC 5x5cm plates.

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.

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

Gradient Relative humidity Temperature Flow rate in solvent Separation distance Mode of Derivatization Greater the difference between two spots and smaller the initial spot diameter of sample and better will be the resolution

Detection and visualization

One of the characteristic feature of HPTLC is the possibility to utilize postchromatographic off line derivatization Detection are of two types: Qualitative Quantitative

 Qualitative detection; HPTLC is routinely used for qualitative analysis of raw materials , finished products ,plant extracts etc. It involves the identification of unknown sample mixture by comparing the Rf values of the sample components with the standards. Quantitation Evaluation: Quantitative of the chromatogram by HPTLC basically involves direct and indirect methods;

Densiometry;

Documentation:
1. Documentation is important because labeling every single chromatogram can avoid mistake in respect of order of application. 2. Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded. 3. TO assist the analysts and researchers E .merck has introduced HPTLC pre-coated plates with an imprinted identification codes. 4. Suppliers name, item number, batch no. , individual plate no. are imprinted near upper edge of pre-coated plates. This will not only help in traceability of analytical data, but will also avoid manipulation of data at any stage as coding will automatically get recorded using photo-documentation.

Applications of HPTLC:
Pharmaceutical industry: Quality control, content uniformity, uniformity test, identity/purity check. Food Analysis: Quality control , additives , pesticides ,stability testing ,analysis of submicron levels of aflotoxins etc 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 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