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HPTLC PROCEDURE FOR HERBAL

CHARACTERISATION
SAMPLE / STD. EXTRACTION

HPTLC SEPARATION (METHOD DEVELOPMENT)

SCAN UV ( MWL ) (METHOD DEVELOPMENT)

IN – SITU UV SPECTRA FOR FP

SCAN FLUOR 366 nm FOR FP

SCAN UV ( MWL ) AT  MAX

FOR FP + QKF
OF FRACTIONS

VIDEO– 254 + 366 + VIS FOR FP

POST – CHROM. DERIVATIZATION FOR (QKF AND OR FP)

SCAN – VIS (+F) VIDEO – VIS (+F)

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STANDARDIZATION
STANDARDIZATION OF
OF CRUDE
CRUDE DRUG
DRUG EXTRACTS
EXTRACTS

C H A R A C T E R I S A T I O N DATA OF

TYPICAL HPTLC FINGERPRINT

 FINGERPRINT

 QUANTITATIVE ANALYSIS

e.g. ADHATODA VASAKA

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QUANTIFICATION OF KNOWN

FRACTIONS BY HPTLC
e.g. ADHATODA VASAKA
METHOD DEVELOPMENT
 MATCHING OF SPECTRA
 SPECTRA COMPARISON PURITY
 MULTIWAVELENGTH CALIBRATION
 LINEARITY

FINGERPRINT
 UV SPECTRUM OF - ALL PEAKS
 MULTIWAVELENGTH SCAN
( 205, 260 & 385 nm )
  FLUORESCENCE SCAN (366 nm)
  DIGITAL PHOTODOC.
( 254nm + 366nm & VISIBLE )
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STANDARDIZATION
STANDARDIZATION OF
OF CRUDE
CRUDE DRUG
DRUG EXTRACTS
EXTRACTS

CHROMATOGRAPHY MET

HOD DEVELOP MENT

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METHOD
METHOD DEVELOPMENT
DEVELOPMENT

MULTIWAVE MEASUREMENT AT 20 nm INTERVAL FOR

SCANNING. WAVELENGTH 230nm SEEMS TO GIVE
MAXIMUM NUMBER OF PEAKS.
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METHOD
METHOD DEVELOPMENT
DEVELOPMENT

MULTIWAVELENGTH SCAN ( AT 5 nm INTERVAL ) 230

nm ± 20 nm. FOR FINETUNING. THIS MEASUREMENT
CONFIRMS THAT DATA OF SCAN AT 230 nm IS THE
BEST FOR FURTHER STUDIES.
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SPECTRUM
SPECTRUM MESURMENT
MESURMENT

SPECTRUM OF MAJOR PEAKS DETECTED AT230 nm. THE  max

OF VARIOUS PEAKS ARE THE WAVELENGTH CHOSEN FOR
ACTUAL MULTIWAVELENGTH ANALYSIS e.g. PEAK AT Rf 0.51
HAS  max OF 290 nm. SO 290 nm IS TO BE CHOSEN AS ONE OF
THE MWLs.
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MULTIWAVELENGTH
MULTIWAVELENGTH ANALYSIS
ANALYSIS

MULTIWAVE MEASUREMENT FOR ACTUAL ANALYSIS. BY CHOOSING

THE  max OF EACH PEAK, IT IS ENSURED THAT
ALL IMPORTANT PEAKS ARE DETECTED AT THEIR BEST
WAVELENGTH. IN A GIVEN CHROM. MWL MEASUREMENT
MAY BE DONE USUALLY BETWEEN 3 TO 6 WAVELENGTHS.
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FLUORESCENCE
FLUORESCENCE SCAN
SCAN

FLUORESCENCE SCAN FOR FINGERPRINT. GIVES UNIQUE

INFORMATION, BASED ON NATIVE FLUORESCENCE.
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DIGITAL PHOTODOCUMENTATION

Photodoc 254 nm
Photodoc 366 nm

Photodoc Visible
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QUANTITATIVE

HERBAL ANALYSIS

e.g. VASICINE FROM

ADHATODA VASAKA

 SCAN OF ALL TRACKS ( 290 nm )

 SPECTRUM MATCH OF
VASICINE
STD. PEAK VS SAMPLE
 ANALYSIS METHOD / REPORT
 CALIBRATION CURVE
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SPECTRUM
SPECTRUM FOR
FOR IDENTITY
IDENTITY

MATCHING OF SPECTRA OF KNOWN FRACTIONS WITH THE

CORRESPONDING FRACTION IN UNKNOWN TO ENSURE THAT
THEY ARE THE SAME SUBSTANCES.
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SAMPLE
SAMPLE FOR
FOR PURITY
PURITY CHECK
CHECK

PURITY CHECK OF A FRACTION IS DONE BY RECORDING ITS

ABS. SPECTRA AT THREE DIFFERENT PLACES i.e. PEAK MAX
PLUS BOTH THE FLANKS. COMPUTER COMPARES THE THREE
AND GIVES CORREATION FACTORS. THIS TEST CHECKS FOR
HOMOGENITY OF THE FRACTION.
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QUANTITATIVE
QUANTITATIVE ANALYSIS
ANALYSIS

MULTILEVEL CALIBRATION USING KNOWN CONCENTRATIONS OF

VASICINE, IS NECESSARY TO QUANTIFY IT FROM THE SAMPLES.
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CALIBRATION
CALIBRATION CURVE
CURVE