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1
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