Slide 1: SEMINAR ON HPTLC ( H igh P erformance T hin L ayer C hormatography) PRESENTED BY SUBMITTED TO KANTILAL B. NARKHEDE Mr.JADHAV V.B. F.Y.M.PHARM. HOD, DEPT.OF MEDI.CHEM., DEPT.OF P’CEUTICS SND COLLEGE OF PHARMACY SND COLLEGE OF PHARMACY Slide 2: Contents: Introduction. Introduction to H.P.T.L.C. principle. Selection of HPTLC plates. Activation of pre coated plates. Sample preparation. Evaluation of spot or band. Application of HPTLC. Difference between TLC and HPTLC . Slide 3: Introduction: chromatography is a physical process of separation in which the component is to be separated are distributed between two immiscible a stationary phase with has large surface area and a mobile phase which is in constant motion through the stationary phase. Slide 4: Introduction to H.P.T.L.C H.P.T.L.C is the improved method of T.L.C which utilize the conventional technique of TLC in more optimize way. It is also known as a planer chromatography or Flat- bad chromatography . Slide 5: Principle : HPTLC take place in high speed capillary flow rang of the mobile phase, There are three main step in HPTLC 1] sample to analyzed to chromatogram layer volume precision and suitable position are achieved by use of suitable instrument. 2] solvent (mobile phase) migrates the planned distance in layer (stationary) by capillary action in this process sample separated in its components. 3] separation tracks are scanned in densitometer with light beam in visible or uv region Slide 7: Selection of HPTLC plates: Previously hand made plate is used in TLC for both qualitative and quantitative work, certain draw back with that is non uniformly layer , formation of thick layer paved for advent pre coated plates. Now a days pre coated plates are available in different format and thickness by different manufactures. these plates are used for both qualitative and quantitative purpose in HPTLC. glass plates . Polyester /polyethylene. Aluminium plates . Slide 8: GLASS PLATES: Resistance to heat Easy to handle Thickness 1.3mm Offer superior plane and smooth surface. Fragile High weight High production cost Slide 9: POLY ESTER POLYETHYLENE : Thickness of plate 0.2mm It can be produce in Roll form. Unbreakable. Less packing material required. Development of plate is not above temp. 120 0 losses of its shape . Slide 10: Aluminium plates: Thickness of plate 0.1mm It can be produce in Roll form. Unbreakable. Less packing material required. Development of plate is not above temp. 120 0 losses of its shape. Slide 11: Sorbents used in HPTLC Plates: Sorbent used in conventional TLC can be used in HPTLC with or with out modification. Silica gel 65F(modified) Highly purified silica gel60. Aluminium oxide. Microcrystalline. Silica gel G particle size of sorbent Reversed stationary phase. HPTLC 6 m Hybrid plates. TLC 10 m Layer thickness in HPTLC -100-200 m in TLC -250 m Slide 12: Layer prewashing : Ascending method . continuous method. Deeping method . solvent used for washing Methanol Chloroform: methanol:amonia(90:10:1) Chloroform: methanol(1:1) Ammonia solution (1%) Slide 13: Activation of precoated plates : The plates are activated by placing in oven at 110-120 0 c for 30 minutes, this step will remove the water that has been physical absorbed on the surface at solvent layer. Freshly open box of HPTLC plates usually not required activation. Activation at higher temperature and long time is avoided which may tends to vary active layer and sample decomposition . Slide 14: SAMPLE PREPARATION: Proper sample preparation is pre requisite for the success HPTLC separation. Beside maximizing the yield of analyte in the selected solvent ,stability of the analyte during extraction and analysis must consider. there for choice of suitable solvent for given analysis is very important . Solvent for dissolving the sample should be non polar and non volatile as far as possible since polar solvent are likely to induce circular chromatogram at the origin . Slide 15: Application of sample and standard solution. Sample application is one imp and critical step for obtaining the good resolution for quantification by HPTLC. sample/std are applied as sport or band depending upon the analysis spot application is done by using Capillary tubes. Micro bulb pipettes. Micro syringe. Automatic sample applicator. compare sample/ std applications FIG:Automatic HPTLC sampler Slide 16: Chromatogram developement: After application of sample in HPTLC plate, chromatogram is developed by dipping in suitable solvent system Taken in developing chamber. The solvent system rises over the layer by capillary action and separation of sample in different components take place. selection of solvent system Chamber saturation Type of development and developing device . Fig :DEVELOPMENT CHAMBER Slide 17: HPTLC can develop by Ascending . Descending . Circular. Anti circular . FIG: HPTLC of Ginseng. Slide 18: Detection or visulation of spot/ band: There is no difficult in detecting the colored substance. or color les substance absorbing the uv radiation or with fluoresce (Riboflavin) Detection of spots/band are done by destruction/Non reverse. Nondestructive/reversible. Misc. method. Slide 19:
Evaluation of spot or band: After detection of spot /band upon objective of experiment chromatogram is used for several purpose.
Evaluation . Quantitative Evaluation .
Application of HPTLC: Pharmaceutical research. Biomedical Analysis. Clinical Analysis.
Analysis. Food industry. Therapeutic drug monitoring to determine its concentration and metabolites in blood urine etc. Analysis of environment pollution level. Quantitative determination of prostaglandin s and thromboxanes in plasma. Determination of mercury in water. Characterization of hazard in industrial waste.
Slide 21: PARAMETER TLC HPTLC TYPES OF CHROMATOGRAPHIC PLATES HANDMADE/PRECOATED PRECOATED ABSORBENT LAYER 200-250 m 100-150m PARTICLE SIZE RANGE 5-20 m 4-8 m APPLICATION OF SAMPLE MANUAL/SEMIAUTOMATIC MANUAL/SEMIAUTOMATIC SHAPE OF SAMPLE SPOT SPOT/BAND SPOT SIZE 3-6mm 1-2mm SAMPLE VOLUME 1-10 0.1-2 DIFFERENCE BETWEEN TLC AND HPTLC . Slide 22: PARAMETER TLC HPTLC NO OF SAMPLE OER PLATE 15-20 40-45 OPTIMAL DEVELOPMENT DISTANCE 10-15cm. 5-7cm DEVELOPMENT TIME DEPEND UPON MOBILE PHASE 40 % LESS THAN TLC QUANTITATION MANUAL MANUAL /INSTRUMENTION. REPRODUBILITY OF RESULT DIFFUCULT REPRODUCIBLE. Slide 23: References. Willard H.H., m errritt L.L., Dean J,A., Settle F.A., i nstrumental methods of analysis, seventh edn ., CBS publishers and distribution pvt . Ltd.,new delhi , Chatwal G.R., a nand S.K.,Instrumental methods of chemical analysis, Himalaya publishing house, d elhi , Mahajan S.S., i nstrumental methods of analysis, First edition, popular prakashan , New delhi , 274-280. : *Thank you*