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M.M.U COLLEGE OF PHARMACY

RAMANAGARAM-571511SEMINAR ON

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

SUBMIT TOMr. NIRMAL.T.HAVANAVARPROFESSOR & HODPHARMA.CHEMISTRY

PRESENTED BY

AZIM ARSHI

1ST M.PHARMA DEPARTMENT OF PHARMACEUTICS

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INTRODUCTION 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

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.

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Principle

HPTLC takes place in high-speed capillary flow range of the mobile phase. There are three main steps HPTLC procedure

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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Sample & slandered Preparation

Layer Pre - Washing

Layer Pre- Conditioning

Application Of Sample & Standard

Chromatographic Development

Detection of spot

Scanning & documentation of chromatoplate

Selection Of Chromatographic

layer

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SELECTION OF HPTLC PLATES

Previously hand made plates is used in TLC for both qualitative

and quantitative work. Certain drawbacks with that is non-

uniform 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.

GLASS PLATES

POLY ESTER/POLYETHYLYNE

ALUMINIUM PLATES

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GLASS PLATES

GLASS PLATESResistant to heat & ChemicalEasy to handleOffers superior Flat & smooth surface

FragileHigh weightHigher production cost

Thickness of Plate 1.3 mm

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It can be produced in Roll formsUnbreakableLess packing material required

Development of plate can not be above temp. 1200 losses of it shape

POLY ESTER/POLYETHYLYNEThickness of Plate 0.2 mm

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ALUMINIUM PLATES

It can be produced in Roll formsUnbreakableLess packing material required

Eluent with high Concentration mineral acid or ammonia chemical attacks aluminum plate

Thickness of Plate 0.1 mm

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SORBENTS USED IN HPTLC PALTES

Silica gel 65F(modified)  

Highly purified Silica gel

60

Aluminium oxide 

Cellulose Microcrystalline

Slica gel  

Reversed stationary phase

 

Hybrid Plates

PARTICAL SIZE OF SORBENTSHPTLC 6m, TLC 10m.

Sorbent, which are used in conventional TLC, are also used in HPTLC with or without modification

LAYER THICKNESSThe layer of thickness in HPTLC is around 100-200 m,where as 250m in conventional TLC.

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LAYER PRE -WASHING

Ascending

method

Dipping method

Continuous

methodSolvents used for pre washing  Methanol (commonly used)

Chloroform: Methanol: Ammonia (90:10:1)

Chloroform: Methanol (1:1)

Methylene chloride: Methanol (1:1)

Ammonia solution (1%)

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ACTIVATION OF PRECOATED PLATES

The plates are activated by placing in an oven at 110-120c 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 pleads to very active layer and there is risk of sample being decomposed.

SAMPLE PREPATION

Proper sample prepation an in imp.prerequsite for success of TLC separation.Besides maximizing the yield of analyte in selected solvent, stability of analyte during extraction and analysis must be considered. Therefore choice of suitable solvent for given analysis is very imp.Solvent for dissolving sample should be non polar and volatile as far as possible, since polar solvents are likely to induces circular chromatogram at the origin.

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APPLICATION OF SAMPLE AND STANDERED SOLUTION

Sample application is one imp. And critical step for obtaining good resolution for quantification by HPTLC. Sample / std. Is applied as a sporty or band depending upon the analysis.Spot application is done by using

1) Capillary tubes2) Micro bulb pipettes3) Micro syringes4) Automatic sample applicator

compare sample / std. Application HPTLC from that of TLC

Parameter TLC HPTLC

Spotting vol 1-10l 0.1-2l

Spot diameter 3-6mm 1-2mm

Sample / std. 0.1-1g/ml 0.1-1g/ml

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CAMAG LINOMAT

Camag Linomat with spray tech. Is usually automated sample application device. The sample is loaded in micro syringe (Hamilton syring ) of 1.0 l capacity. The sample is applied either as a spot or band by programming instrument parameters like spotting volume, band length, no. of spot/ band, space between bands etc.The nozzle is placed at tip of syringe, air is coming out at high pressure atomizes sample solution into fine spray. It results in concentration and spraying of sample as a narrow band of suitable length.

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CHROMATOGRAM DEVELOPMENT

After application of sample in HPTLC

plate, chromatogram is developed by

dipping in suitable solvent system taken

in a developing chamber. The solvent

system is rises over the layer by

capillary action and separation of sample

in to different components takes place.

Selection of solvent system / mobile

phase

Chamber saturation

Type of development and developing

device.

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In a close bed tec. Such as HPLC only Linear developmentIs possible, But an open bed tec. Like HPTLC does not suffer this limitation.HPTLC can develop byAscending (linear )CircularAnti circular

LINEAR & RADIAL DEVELOPMENT

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Circular ChromatographyAnti Circular Chromatography

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DETECTION OR VISULATION OF SPOTS / BANDS

There is no difficulty in

detecting the colored

substances or colorless

substances absorbing UV-

radiations or with fluoresce

(Riboflavin)

“Derivatisation”

Detection of spots / bands are

done by

1) Destruction / Non-reverse

2) Non-destructive /

Reversible

3) Misc.methods

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EVALUATION OF SPOTS / BANDS

After detection of spots / band, upon objective of expt.

Chromatogram is used for several purposes

Qualitative Evaluation

Quantitative Evaluation

DOCUMENTATION OF CHROMATOGRAM

HPTLC plates that have been evaluated quantitatively and qualitatively, should be documented as per guidelines of GMP, GLP common methods of documentation are

Photo documentation

Video documentation

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APPLICATIONS OF HPTLC

Pharmaceutical

Researches

Bio-medical 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.

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PARAMETERS TLC HPTLC

TYPE OF CHROMATOGRAPHIC PLATE

Handmade / Precoated

Per coated

ADSORBANT LAYER

200-250 m 100-200 m

PARTICAL SIZE RANGE

5-20 m 4-8 m

APPLICATION OF SAMPLE

Manual / Semiautomatic

Semiautomatic / Automatic

SHAPE OF SAMPLE

Spot Spot / Band

SPOT SIZE 3-6mm 1-2mm

SAMPLE VOLUME 1-10 l 0.1-2l

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PARAMETERS TLC HPTLC

NO.OF SAMPLE PER PLATE

15-20 40-50

OPTIMAL DEVP.DISTANCE

10-15cm 5-7cm

DEVP. TIME Depends on Mobile Phase

40% less than TLC

QUANTITATIONS Manual / Instrument Instrumental

REPRODUCIBILITY OF RESULTS

Difficult Reproducible

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REFERENCES:-Principles of Instrumental Analysis,Skoog,Holler,Nieman.Instrumental Methods of Analysis. Willard, Merrit,Dean.Pharmaceutical Analysis. Munson.Instrumental Methods of Chemical Analysis. Gurdeep R. Chatwal, Shyam K. Anand.Sherma J, Fried B. Handbook of thin layer chromatography. 3rd ed. New York: Marcel Dekker, Inc.; 2003. p. 3-4.Sethi PD. HPTLC: Quantitative analysis of pharmaceutical formulations. 1st ed. New Delhi: CBS Publisher; 1996. p. 44-57. Peter EW. Thin layer chromatography: A modern practical approach. UK: The royal society of chemistry; 2005. p. 6-154.

http://pharamcytimes.wordpress.com/category/instrumental-analysis-studies/chromatography/ http://www.interchromforum.com/html/body_qnt_err_hptlc.htmlhttp://www.pharmainfo.net/reviews/validated-analytical-methods-determination-active-ingredients-bulk-drugs-and-pharmaceutical-http://www.selectscience.net/commNWDetails.aspx?mailID=1035

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Thankyou