HPTLC : SEPERATION TECHNIQUE

““A REVIEW ON HIGH PERFORMANCEA 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-412105MODERN C.O.P.,MOSHI, PUNE-412105
1

AIMAIM
To Review and Study the High Performance Thin LayerTo 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 HPTLC
3. 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.
2

CONTENTS…..CONTENTS…..
1. AIM AND OBJECTIVE1. AIM AND OBJECTIVE
2.INTRODUCTION TO CHROMATOGRAPHY & HPTLC2.INTRODUCTION TO CHROMATOGRAPHY & HPTLC
3.FEATURES OF HPTLC3.FEATURES OF HPTLC
4. STEPS INVOLVED IN HPTLC4. STEPS INVOLVED IN HPTLC
5. INSTRUMENTATION5. INSTRUMENTATION
6. DIFFERENCE BETWEEN TLC & HPTLC6. DIFFERENCE BETWEEN TLC & HPTLC
7. VALIDATION PARAMETERS7. VALIDATION PARAMETERS
8. APPLICATIONS OF HPTLC8. APPLICATIONS OF HPTLC
9. CONCLUSION9. CONCLUSION
10. BIBLIOGRAPHY10. BIBLIOGRAPHY
3

INTRODUCTION TO HPTLCINTRODUCTION TO HPTLC[2]
5
 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]

7
1. SELECTION OF HPTLC PLATES
2. SELECTION OF CHROMATOGRAPHIC LAYER
3.SAMPLE & STANDARD PREPARATION
4.LAYER PREWASHING
5.SAMPLE APPLICATION
6.DEVELOPMENT
7.DETECTION OF SPOTS
8.SCANNING & DOCUMENTATION

SCHEMATIC PROCEDURE FOR HPTLC[4]

9
HAND MADE PLATES:
THE AMOUNT OF EACH SORBENT INDICATED IS SUFFICIENT TO
COAT 5 PLATES OF 20*20EACH.
EG.CELLULOSE,CELLULOSE WITH STARCH,SILICA GEL WITH STARCH
PRECOATED 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

10
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:

11
SAMPLE & STANDARD PREPARATION

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
14

CHROMATOGRAPHIC DEVELOPMENT
17

 CHROMATOGRAM IMMERSION DEVICE
 HPTLC SPRAYER
 TLC SPRAY CABINET
 TLC PLATE HEATER
22

22
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
23

23
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
24

24
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
25

25
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.
26

26
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 CHROMATOGRAM
QUALITATIVELY & QUANTITATIVELY
IT IS ADVANCED TYPE OF DENSITOMETER
NOT POSSIBLE
27

 BIOCHEMICAL RESEARCH
 CLINICAL RESEARCH
 COSMETICS ANALYSIS
 ENVIRONMENTAL ANALYSIS
 FOOD ANALYSIS
 NATURAL PRODUCTS PLANT INGREDIENTS
 TOXICOLOGY & FORENSIC ANALYSIS
 DOPING ANALYSIS 27
APPLICATIONS[5]
28

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.
31

BIBLIOGRAPHYBIBLIOGRAPHY
1.1. P.D.SETHI, “HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-QUANTITATIVE ANALYSIS OFP.D.SETHI, “HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-QUANTITATIVE ANALYSIS OF
PHARMACEUTICAL FORMULATION”,CBS PUBLISHERS & DISTRIBUTORS,1PHARMACEUTICAL FORMULATION”,CBS PUBLISHERS & DISTRIBUTORS,1STST
EDITION,2001,P.G.141-142EDITION,2001,P.G.141-142
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
4.4. http://www.pharmainfo.net/files/images/stories/article_images/INTRODUCTION_ANALYTIChttp://www.pharmainfo.net/files/images/stories/article_images/INTRODUCTION_ANALYTIC
ALMETHODFig10.JPGALMETHODFig10.JPG
5.5. PATEL, R.B. AND PATEL, M.R. AND PATEL, B.G. (2011) EXPERIMENTAL ASPECTS ANDPATEL, 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 TECHNIQUESHRIVASTAVA, M.M. (2011) AN OVERVIEW OF HPTLC: A MODERN ANALYTICAL TECHNIQUE
WITH EXCELLENT POTENTIAL FOR AUTOMATION, OPTIMIZATION, HYPHENATION, ANDWITH 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.
32

Thank
You…..--------------------------------------------------------------------------------
Thank
You…..--------------------------------------------------------------------------------

HPTLC : SEPERATION TECHNIQUE

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HPTLC : SEPERATION TECHNIQUE