Dhruti Avlani - TLC & HPTLC

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.

1. 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

2. INTRODUCTION 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:

3. 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 :- TLC plates TLC chamber Mobile phase Filter paper 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

4. METHOD OF TLC

5. 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

6. 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

7. COMPONENTS OF HPTLC 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 CHAMBER Low 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

8. COMPONENTS OF HPTLC (continued) 3) SAMPLE APPLICATOR – Linomat 5 S amples 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. Scanning Densitometer

9. 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)

10. Twin Trough Chamber FLOW DIAGRAM FOR HPTLC Sample and Standard Preparation Selection of Chromatographic layer Layer pre-washing Layer pre-conditioning Application of sample and standard Linomat 5 Applicator Chromatographic development Detection of spots, scanning and documentation Scanner 4 Visualizer Chromatogram

11. 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

12. COMPARISON BETWEEN TLC AND HPTLC PARAMETER TLC HPTLC Type of chromatographic plates Hand made/ Pre-coated Pre-coated Solvent Layer 200-250nm 100-150nm Particle Size 5-20µm 4-8µm Application of sample Manual/ Semiautomatic Automatic Shape of the sample Spot Band Sample Volume 1-10µl 0.2-5µl No. of samples per plate 15-20 40-45 Spot Size 3-6mm 1-2mm Development Time Depends on the mobile phase 40% less than TLC Reproducibility of results Difficult Easy

13. 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 f or developing in less time. Reproducibility of results is also easy.

14. REFERENCE Ravi Shankar, S., 2010, Chromatography in Text Book of Pharmaceutical Analysis, Fourth edition, Rx Publications, Tirunelveli , India . Ganti Sravani , “High Performance Thin Layer Chromatography”. http://www.slideshare.net/sravaniganti1/hptlc-30991038?next_slideshow=1 (accessed as on February 7, 2015). 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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