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)

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