HPTLC PROCEDURE FOR HERBAL

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• HPTLC PROCEDURE FOR HERBAL
• CHARACTERISATION

SAMPLE / STD. EXTRACTION
(METHOD DEVELOPMENT)
HPTLC SEPARATION
SCAN UV ( MWL )
(METHOD DEVELOPMENT)
IN SITU UV SPECTRA
FOR FP
FOR FP
SCAN FLUOR 366 nm
SCAN UV ( MWL ) AT ? MAX OF FRACTIONS
FOR FP QKF
VIDEO 254 366 VIS
FOR FP
POST CHROM. DERIVATIZATION
FOR (QKF AND OR FP)
SCAN VIS (F)
VIDEO VIS (F)
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CAMAG - HPTLC STANDARDIZATION OF CRUDE DRUG
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
• M E T H O D D E V E L O P M E N T
• MATCHING OF SPECTRA
• SPECTRA COMPARISON PURITY
• MULTIWAVELENGTH CALIBRATION
• LINEARITY
• F I N G E R P R I N T
• UV SPECTRUM OF - ALL PEAKS
• MULTIWAVELENGTH SCAN
• ( 205, 260 385 nm )
•  FLUORESCENCE SCAN (366 nm)
•  DIGITAL PHOTODOC.
• ( 254nm 366nm VISIBLE )
  

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CAMAG - HPTLC STANDARDIZATION OF CRUDE DRUG
EXTRACTS
C H R O M A T O G R A P H Y M E T H
O D D E V E L O P M E N T
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METHOD DEVELOPMENT

• MULTIWAVE MEASUREMENT AT 20 nm INTERVAL FOR
  SCANNING. WAVELENGTH 230nm SEEMS TO GIVE MAXIMUM
  NUMBER OF PEAKS.

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METHOD 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 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 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 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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Q U A N T I T A T I V E H E R B A L A N A L Y
S I S

• 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 FOR 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 FOR PURITY 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 ANALYSIS

• MULTILEVEL CALIBRATION USING KNOWN CONCENTRATIONS
  OF VASICINE, IS NECESSARY TO QUANTIFY IT FROM THE
  SAMPLES.

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CALIBRATION CURVE