ANALYSIS OF HERBAL DRUGS USING TLC AND HPTLC Presented By: DHRUTI AVLANI B.Pharm, 3 rd year, 6 th sem Registration No.: 122770210011 Roll No.: 27701912011 NSHM Knowledge Campus, Kolkata – Group of Institutions
ANALYSIS OF HERBAL DRUGS USING
TLC AND HPTLC
Presented By: DHRUTI AVLANI
B.Pharm, 3rd year, 6th semRegistration No.: 122770210011Roll No.: 27701912011NSHM Knowledge Campus, Kolkata – Group of Institutions
INTRODUCTION
BOTANICAL ANALYSIS• Macroscopic• Microscopic
PHYSICAL ANALYSIS• Moisture Content• Extractive Value• Ash ValueCHEMICAL ANALYSIS
• Chromatographic StudiesMICROBIOLOGICAL ANALYSIS
• Microbial Contamination
The quality control of herbal crude drugs and their bio-constituents is of paramount importance. Drug evaluation means conformation of its identity and determination of its quality and purity and detection of adulterants. The different techniques involved in the standardization of crude drugs are as follows:
THIN LAYER CHROMATOGRAPHY (TLC)
TLC is a method of separation of a mixture of components into individual components by using finely divided adsorbent solid spread over a plate and liquid as a mobile phase.Therefore, the principle of separation is absorption.
COMPONENTS OF TLC :-1) TLC plates2) TLC chamber3) Mobile phase4) Filter paper5) Detecting or visualising agents
Components of TLC
ADVANTAGES Simple method Any type of compound can be analysed Rapid technique and not time consuming Detection is easy
METHOD OF TLC
1Prepar-
-ation of the devel-
-oping chamber
2 Prepar--ation of the TLC plate and sample applica-
tion
3Column develo--pment
4Visualiza-
-tion of the spots (UV-VIS Spectroh-otometer)
5- Determination of the Retention Factor (Rf)(Rf) = Distance travelled by solute Distance travelled by solvent
Rf of A = y/z ; Rf of B = x/z
Normal Value = 0 – 1
Ideal Value = 0.3 – 0.8
APPLICATION Separation of carbohydrates, alkaloids, glycosides, etc. Identification of drugs.
METHOD OF TLC (continued)
Thin Layer Chromatogram
HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC)
HPTLC is a sophisticated and automated form of TLC with a number of enhancements made to the basic method of TLC to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements. The principle of separation is absorption.
ADVANTAGES Promotes high separation efficiencies of zones due to higher number of theoretical plates. Shorter developing time Enormous flexibility
COMPONENTS OF HPTLC1) HPTLC PLATES
Silica Gel 60 GF-254 The smaller particle size and the thinner layer (200 µm or 100 µm) ultimately result in highly increased sensitivity and faster analysis
HPTLC PLATES
2) HPTLC CHAMBERLow solvent Consumption Better saturation of the chamber for better chromatogram Lesser development/analysis time Example of solvent system used – Chloroform:Methanol (1:1) HPTLC
CHAMBERS
COMPONENTS OF HPTLC (continued)3) SAMPLE APPLICATOR – Linomat 5
Samples are sprayed onto the plates in the form of bands with nitrogen or compressed air (1-5µl) Applicator – Pt-Iridium capillary winCATS Planar Chromatography Manager - software Sample Applicator
4) SCANNING DENSITOMETER – SCANNER 4 The scanner features three light sources, a deuterium lamp, a tungsten lamp and a mercury lamp. The scanner enables densitometric determination of individual spots along with spectrum of each spot.
ScanningDensitometer
5) TLC VISUALISER The system provides illumination with direct and/or transmitted white light as well as with direct UV 254nm and UV 366nm light. Integrated camera The process is conveniently controlled by the TLC software.
TLC Visualiser
At 254 nm
TLC chromatogram of standard herbal extract
At 366 nm
COMPONENTS OF HPTLC (continued)
Twin Trough
Chamber
FLOW DIAGRAM FOR HPTLCSample and Standard Preparation Selection of
Chromatographic layer
Layer pre-washing
Layer pre-conditioningApplication of sample
and standard
Linomat 5 Applicator
Chromatographic development
Detection of spots, scanning and documentation
Scanner 4
VisualizerChromatogram
Separation of carbohydrates, alkaloids, glycosides, etc. in herbal drugs. Finger printing analysis - identification of bio-actives of claimed herbs, batch to batch consistency of product and possible contaminants/adulterants. Determines the presence of adulterants
Chromatogram
APPLICATION OF HPTLC
COMPARISON BETWEEN TLC AND HPTLC
PARAMETER TLC HPTLCType of
chromatographic platesHand made/ Pre-coated Pre-coated
Solvent Layer 200-250nm 100-150nmParticle Size 5-20µm 4-8µm
Application of sample Manual/ Semiautomatic AutomaticShape of the sample Spot Band
Sample Volume 1-10µl 0.2-5µlNo. of samples per plate 15-20 40-45
Spot Size 3-6mm 1-2mmDevelopment Time Depends on the mobile
phase40% less than TLC
Reproducibility of results
Difficult Easy
CONCLUSION
Various chromatographic techniques can be employed for the qualitative and quantitative analysis of the drug.It is observed that HPTLC is the most widely used Chromatography instrument as it consumes less solvent for developing in less time. Reproducibility of results is also easy.
REFERENCE1) Ravi Shankar, S., 2010, Chromatography in Text Book of
Pharmaceutical Analysis, Fourth edition, Rx Publications, Tirunelveli, India.
2) Ganti Sravani, “High Performance Thin Layer Chromatography”. http://www.slideshare.net/sravaniganti1/hptlc-30991038?next_slideshow=1 (accessed as on February 7, 2015).
3) J.Raja Sudheer, “High Performance Thin Layer Chromatography. http://www.authorstream.com/Presentation/sudheerraj99-1489826-hptlc/ (accessed as on February 7, 2015)
THANK YOU!