“ “ A REVIEW ON HIGH A REVIEW ON HIGH PERFORMANCE THIN LAYER PERFORMANCE THIN LAYER CHROMATOGRAPHY” CHROMATOGRAPHY” PRESENTING BY, PRESENTING BY, Ms.SONALI DIWATE B. Ms.SONALI DIWATE B. GUIDED BY, GUIDED BY, Mrs.TAMBE V.S. Mrs.TAMBE V.S. MODERN C.O.P.,MOSHI, PUNE-412105 MODERN C.O.P.,MOSHI, PUNE-412105 1
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““A REVIEW ON HIGH PERFORMANCE A REVIEW ON HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY”THIN LAYER CHROMATOGRAPHY”
PRESENTING BY,PRESENTING BY,Ms.SONALI DIWATE B.Ms.SONALI DIWATE B.
GUIDED BY,GUIDED BY,Mrs.TAMBE V.S. Mrs.TAMBE V.S.
MODERN C.O.P.,MOSHI, PUNE-412105 MODERN C.O.P.,MOSHI, PUNE-412105
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AIMAIMTo Review and Study the High Performance Thin Layer To Review and Study the High Performance Thin Layer ChromatographyChromatography
OBJECTIVEOBJECTIVE
1. TO COLLECT THE INFORMATION ABOUT CHROMATOGRAPHIC TECHNIQUES.1. TO COLLECT THE INFORMATION ABOUT CHROMATOGRAPHIC TECHNIQUES.2. TO UNDERSTAND THE PRINCPLE INVOLVED IN HPTLC2. TO UNDERSTAND THE PRINCPLE INVOLVED IN HPTLC3. TO UNDERSTAND THE FEATURES OF HPTLC .3. TO UNDERSTAND THE FEATURES OF HPTLC .4. TO STUDY THE STEPS INVOLVED IN HPTLC.4. TO STUDY THE STEPS INVOLVED IN HPTLC.5. TO UNDERSTAND THE INSTRUMENTATION OF HPTLC.5. TO UNDERSTAND THE INSTRUMENTATION OF HPTLC.6. TO STUDY THE DIFFERENCE IN BETWEEN HPTLC & TLC.6. TO STUDY THE DIFFERENCE IN BETWEEN HPTLC & TLC.7. TO ANALYSE THE VALIDATION PARAMETERS. 7. TO ANALYSE THE VALIDATION PARAMETERS. 8. TO EXPLAIN APPLICATIONS OF HPTLC.8. TO EXPLAIN APPLICATIONS OF HPTLC.9. SUMMERIZATION OF THE INFORMATION.9. SUMMERIZATION OF THE INFORMATION.
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CONTENTS…..CONTENTS…..
1. AIM AND OBJECTIVE1. AIM AND OBJECTIVE2.INTRODUCTION TO CHROMATOGRAPHY & HPTLC2.INTRODUCTION TO CHROMATOGRAPHY & HPTLC3.FEATURES OF HPTLC3.FEATURES OF HPTLC4. STEPS INVOLVED IN HPTLC4. STEPS INVOLVED IN HPTLC5. INSTRUMENTATION5. INSTRUMENTATION6. DIFFERENCE BETWEEN TLC & HPTLC6. DIFFERENCE BETWEEN TLC & HPTLC7. VALIDATION PARAMETERS7. VALIDATION PARAMETERS8. APPLICATIONS OF HPTLC8. APPLICATIONS OF HPTLC9. CONCLUSION9. CONCLUSION10. BIBLIOGRAPHY10. BIBLIOGRAPHY
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INTRODUCTION TO HPTLCINTRODUCTION TO HPTLC[2]
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THE WORD CHROMATOGRAPHY DERIVED FROM GREEK WORD CHROMA MEANS COLOUR & GRAPHEIN MEANING WRITE.
HPTLC STANDS FOR HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY. IT IS THE SCIENCE OF SEPERATION USED EITHER FOR IDENTIFICATION OR
QUANTITATION OF CHEMICAL SUBSTANCES. THIS SEPARATION TECHNIQUE BASED ON THE DIFFERENT INTERACTIONS OF
COMPOUNDS WITH TWO PHASES,A MOBILE PHASE & A STATIONARY PHASE,AS THE COMPOUNDS TRAVEL THROUGH A SUPPORTING MEDIUM.
COMPONENTS –
MOBILE PHASE:A SOLVENT THAT FLOWS THROUGH THE SUPPORTING MEDIUM STATIONARY PHASE:A LAYER OR COATING ON THE SUPPORTING MEDIUM THAT
INTERACTS WITH THE ANALYTS SUPPORTING MEDIUM:A SOLID SURFACE ON WHICH THE STATIONARY PHASE IS
BOUND OR COATED.
HISTORY OF ANTIDEPRESSANTSHISTORY OF ANTIDEPRESSANTS
SIMULTANEOUS PROCESSING OF SAMPLE & STANDARD.
BETTER ANALYTICAL PRECISION & ACCURACY
SEVERAL ANALYSTS WORK SIMULTANEOUSLY
LOWER ANALYSIS TIME & LESS COST PER ANALYSIS
LOW MAINTENANCE COST
SIMPLE SAMPLE PREPARATION
NO PRIOR TREATMENT FOR SOLVENT LIKE FILTRATION & DEGASSING
VISUAL DETECTION IS POSSIBLE.6
FEATURES OF HPTLCFEATURES OF HPTLC[2]
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1. SELECTION OF HPTLC PLATES2. SELECTION OF CHROMATOGRAPHIC LAYER3.SAMPLE & STANDARD PREPARATION4.LAYER PREWASHING5.SAMPLE APPLICATION6.DEVELOPMENT7.DETECTION OF SPOTS8.SCANNING & DOCUMENTATION
SCHEMATIC PROCEDURE FOR HPTLC[4]
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HAND MADE PLATES:THE AMOUNT OF EACH SORBENT INDICATED IS SUFFICIENT TOCOAT 5 PLATES OF 20*20EACH.EG.CELLULOSE,CELLULOSE WITH STARCH,SILICA GEL WITH STARCHPRECOATED COATS:IT IS AVAILABLE WITH SUPPORT MATERIAL LIKEGLASS, POLYESTER OR
ALUMINIUM.SELECTION OF PLATE DEPENDS UPON- ADSORBANT MATERIAL USED FOR COATING ANALYTE TO BE SEPERATED THICKNESS OF PLATE
SELECTION OF HPTLC PLATES
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SELECTION OF CHROMATOGRAPHIC LAYER
PRECOATED PLATES OF DIFFERENT MATERIALS OR DIFFERENT SORBENTS ARE AVAILABLE
SILICA GEL GF : IT IS USED FOR BASIC SUBSTANCE LIKE ALKALOIDS,ALUMINIUM HYDROXIDE &
USED IN 80 % OF ANALYSIS.RP2,RP8, RP18:IT IS USED FOR NONPOLAR SUBSTANCES LIKE FATTY ACIDS, CAROTENOIDS,CHOLESTEROL.HYBRID PLATES RPWF254S: PRESERVATIVES,BARBITURATES
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SAMPLE & STANDARD PREPARATION
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LAYER PREWASHING
ACTIVATION
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SAMPLE APPLICATION
USUAL CONCENTRATION RANGE IS 0.1µg/µl ABOVE THIS CAUSES POOR SEPARATION EG.LONOMAT IV = IN THIS NOTROGEN GAS SPRAYS
SAMPLE & STANDARD FROM SYRING ON TLC PLATES AS A BAND
BAND WISE APPLICATION GIVES BETTER SEPARATION & HIGH RESPONSE TO DENSITOMETER
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SELECTION OF MOBILE PHASE
PRECONDITIONING
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CHROMATOGRAPHIC DEVELOPMENT
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DETECTION OF SPOTS
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SCANNING & DOCUMENTATION
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CHROMATOGRAM IMMERSION DEVICE
HPTLC SPRAYER
TLC SPRAY CABINET
TLC PLATE HEATER
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For proper execution of the dipping technique, the chromatogram must be immersed and withdrawn at a controlled uniform speed. By maintaining a well defined vertical speed and immersion time, derivatization conditions can be standardized and «tide marks», which can interfere with densitometric evaluation, are avoided.Key features• Uniform vertical speed, freely selectable between 30 mm/s and 50 mm/s.• Immersion time selectable between 1 and 8 seconds and indefi nitely.• The device can be set to accommodate 10 cm and 20 cm plate height.• Battery operated, independent of power supply.
CHROMATOGRAM IMMERSION DEVICE
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The TLC sprayer consists of a charger and a pump unit with two kinds of spray heads.• spray head type A is for spray solutions of normal viscosity, e.g. lower alcohol solutions.• spray head type B is for liquids of higher viscosity, e.g. sulfuric acid reagents.Key features• Easy to use, with electro-pneumatic spray function.• Formation of fine aerosol with particles of 0.3 to 10 μm.• Homogenous distribution with low reagent consumption.
HPTLC SPRAYER
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The TLC spray cabinet ensures the complete removal of reagent mist while spraying tlc plates. There is no deflection of the spray mist before it reaches the chromatogram, an effect often occurring in a normal laboratory fume hood. The TLC spray cabinet is made of PVC. The blower, a radial fan driven by a motor outside of the fume duct, produces airflow of 130 cubic feet (3.7 cubic meter) per minute. The bottom of the spray cabinet has a built in tray, which is removable for easy cleaning.Dimensions: 470 x 490 x 490 mm (width x depth x height).
TLC SPRAY CABINET
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TLC PLATE HEATER
The TLC plate heater III is designed for heating TLC plates to a given temperature, while ensuring homogenous heating across the plate. The TLC plate heater III has a CERAN® heating surface which is resistant to all common reagents and is easily cleaned. The 20 x 20 cm heating surface has a grid to facilitate correct positioning of the TLC plate. programmed and actual temperature are digitally displayed. The temperature is selectable between 25 and 200 °c. The plate heater is protected from overheating.
The TLC plate heater III is designed for heating TLC plates to a given temperature, while ensuring homogenous heating across the plate. The TLC plate heater III has a CERAN® heating surface which is resistant to all common reagents and is easily cleaned. The 20 x 20 cm heating surface has a grid to facilitate correct positioning of the TLC plate. programmed and actual temperature are digitally displayed. The temperature is selectable between 25 and 200 °c. The plate heater is protected from overheating.
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DIFFERENCE BETWEEN HPTLC & TLC[1]
PARAMETERS HPTLC TLC
LAYER OF SORBENT
100 MICROMETER 250 MICROMETER
EFFICIENCY HIGH DUE TO SMALLER PARTICLE SIZE LESS
SEPARATIONS 3-5 CM 10-15CM
ANALYSIS TIME SHORTER MIGRATION DISTANCE & ANALYSIS TIME IS GREATLY REDUCED
SLOWER
SOLID SUPPORT WIDE CHOICE OF STATIONARY PHASE LIKE SILICA GEL FOR NORMAL PHASE & C8,C18 FOR REVERSED MODES.
SILICA GEL,ALUMINA KIESULGUHR
SAMPLE SPOTTING AUTOMATIC MANNUAL
SCANNING USE OF UV/VISIBLE/FLURESCENCE SCANS THE ENTIRE CHROMATOGRAMQUALITATIVELY & QUANTITATIVELYIT IS ADVANCED TYPE OF DENSITOMETER
NOT POSSIBLE
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BIOCHEMICAL RESEARCH
CLINICAL RESEARCH
COSMETICS ANALYSIS
ENVIRONMENTAL ANALYSIS
FOOD ANALYSIS
NATURAL PRODUCTS PLANT INGREDIENTS
TOXICOLOGY & FORENSIC ANALYSIS
DOPING ANALYSIS27
APPLICATIONS[5]
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CONCLUSIONCONCLUSION
1. CHROMATOGRAPHY IS A SCIENCE OF SEPERATION USED EITHER FOR IDENTIFICATION OR QUANTITATION OF CHEMICAL SUBSTANCES.
2. VARIOUS MODES OF CHROMATOGRAPHIC TECHNIQUES WERE DEVELOPED BASED ON INITIAL DISCOVERY BY MICHAEL T.SWETT.
3. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY IS MOST ADVANCED TECHNIQUE NOW A DAYS.
4. IT IS THE MODERN SEPARATION TECHNIQUE WHICH IS ACCEPTRD AS AN EXTREAMLY FLESIBLE,RELIABLE & COST EFFICIENT METHOD.
5.BY THIS TECHNIQUE,ACCURATE SEPARATION OF SAMPLE TAKES PLACE.6.IT CAN BE CONSIDERED AS A TIME MACHINE THAT CAN SPEED UP OUR
WORK & SO MANY THINGS AT A TIME USUALLY NOT POSSIBLE WITH OTHER ANALYTICAL TECHNIQUE.
7.HPTLC CAN SIMULTANEOUSLY HANDLE SEVERAL SAMPLES EVEN OF DIFFERENT NATURE & COPOSITION SUPPORTING SEVERAL ANALYTE OF A GIVEN TIME.
2. Dr. Harish Chandra Andola, “High Performance Thin Layer Chromatography (HPTLC): A Modern Analytical tool for Biological Analysis”, Nature and Science , 2010;8(10)
3.3. CAMAG, 2010-2011. BASIC EQUIPMENT FOR MODERN THIN LAYER CHROMATOGRAPHY. CAMAG, 2010-2011. BASIC EQUIPMENT FOR MODERN THIN LAYER CHROMATOGRAPHY. SWITZERLAND: CAMAG.AVAILABLE FROM: SWITZERLAND: CAMAG.AVAILABLE FROM: CAMAG.COM/DOWNLOADS/FREE/BROCHURES/CAMAG-BASIC-EQUIPMENT-08.PDFCAMAG.COM/DOWNLOADS/FREE/BROCHURES/CAMAG-BASIC-EQUIPMENT-08.PDF
5.5. PATEL, R.B. AND PATEL, M.R. AND PATEL, B.G. (2011) EXPERIMENTAL ASPECTS AND PATEL, R.B. AND PATEL, M.R. AND PATEL, B.G. (2011) EXPERIMENTAL ASPECTS AND IMPLEMENTATION OF HPTLC. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER, PP. 41- IMPLEMENTATION OF HPTLC. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER, PP. 41- 54.54.
6.6. SHRIVASTAVA, M.M. (2011) AN OVERVIEW OF HPTLC: A MODERN ANALYTICAL TECHNIQUE SHRIVASTAVA, M.M. (2011) AN OVERVIEW OF HPTLC: A MODERN ANALYTICAL TECHNIQUE WITH EXCELLENT POTENTIAL FOR AUTOMATION, OPTIMIZATION, HYPHENATION, AND WITH EXCELLENT POTENTIAL FOR AUTOMATION, OPTIMIZATION, HYPHENATION, AND MULTIDIMENSIONAL APPLICATIONS. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER, MULTIDIMENSIONAL APPLICATIONS. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER, PP. 3- 24.PP. 3- 24.