HPTLC (High performance thin layer chromatography) VAISHNAVI U.S.D.N M.Pharmacy(Pharmaceutical Chemistry) 170213884006 UNDER THE GUIDENCE: CEEMA MATHEW,M.pharmacy Asst.Professor

INDEX INTRODUCTION ADVANTAGES OF HPTLC OVER HPLC & TLC FEATURES OF HPTLC STEPS INVOLVED IN HPTLC DIFFERENCES BETWEEN TLC AND HPTLC

INTRODUCTION

CHROMATOGRAPHY is a broad range of physical methods used to separate and or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed.  

High performance thin layer chromatography (HPTLC) is an enhanced form of thin layer chromatography (TLC). A number of enhancements can be made to the basic method of thin layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurements.

Principle of separation is ADSORPTION Automation is useful to overcome the uncertainty in droplet size and position when the sample is applied to the TLC plate by hand. Principle of separation is ADSORPTION

Advantages of HPTLC over HPLC Sample and standard, both can be used at a time. Evaluation time is less. Use of internal standard is not necessary. Simultaneously 2 or 3 persons can work but not in case of HPLC. Solvent degasing and removal of impurities is not necessary.

Advantages of HPTLC over TLC Visual chromatogram simplicity Multiple sample handling Low running and maintenance costs and disposable layer etc Detection by TLC scanner Spotting by applicator Highly precised

FEATURES OF HPTLC

Simultaneous processing of sample and standard - better analytical precision and accuracy less need for Internal Standard Several analysts work simultaneously Lower analysis time and less cost per analysis Low maintenance cost Simple sample preparation - handle samples of divergent nature No prior treatment for solvents like filtration and degassing Low mobile phase consumption per sample No interference from previous analysis - fresh stationary and mobile phases for each analysis - no contamination Visual detection possible - open system Non UV absorbing compounds detected by post-chromatographic derivatization

STEPS INVOLVED IN HPTLC

· 80% of analysis - silica gel GF Selection of chromatographic layer · Precoated plates - different support materials - different Sorbents available · 80% of analysis - silica gel GF · Basic substances, alkaloids and steroids - Aluminum oxide · Amino acids, dipeptides, sugars and alkaloids - cellulose . Non-polar substances, fatty acids, carotenoids, cholesterol - RP2, RP8 and RP18 · Preservatives, barbiturates, analgesic and phenothiazines- Hybrid plates-RPWF254s

To avoid interference from impurities and watervapours Sample and Standard Preparation To avoid interference from impurities and watervapours Solvents used are Methanol, Chloroform: Methanol (1:1) Ethyl acetate: Methanol (1:1) Chloroform: Methanol: Ammonia (90:10:1) Methylene chloride : Methanol (1:1) 1% Ammonia or 1% Acetic acid Dry the plates and store in dust free atmosphere

Layer pre-washing & pre-conditioning If any impurities are present, those are need to be removed hence pre washing is done. Conc.HCl : Methanol(1:9) Plate is dipped in the soln, allowed for the solvent to pass throughout the plate. Hence impurities are washed out

Activation of pre-coated plates Freshly open box of plates do not require activation Plates exposed to high humidity or kept on hand for long time to be activated By placing in an oven at 110-120ºc for 30’ prior to spotting (pre conditioning) Aluminum sheets should be kept in between two glass plates and placing in oven at 110-120ºc for 15 minutes.

· Above this causes poor separation Application of sample and standard · Usual concentration range is 0.1-1µg / µl · Above this causes poor separation · Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe on TLC plates as bands

Selection of Mobile phase Can be explained by STAHL’S TRIANGLE Hydrophilic Non-Polar E Polar Lipophilic M S Active Inactive EG:If E- hydrophilic then M- polar & S- inactive E- ELUENT S- SORBANT M- MIXTURE

Trial and error - one’s own experience and Literature Normal phase - Stationary phase is polar - Mobile phase is non polar - Non-polar compounds eluted first because of lower affinity with stationary phase - Polar compounds retained because of higher affinity with the stationary phase Reversed phase - Stationary phase is non polar - Mobile phase is polar - Polar compounds eluted first because of lower affinity with stationary phase - Non-Polar compounds retained because of higher affinity with the stationary phase

Chromatographic development and drying After development, remove the plate and mobile phase is removed from the plate to avoid contamination of lab atmosphere Dry in vacuum desiccator

Chromatographic Develpment 1. TWIN TROUGH CHAMBER

Detection and visualization · Detection under UV light is first choice - non destructive · Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length) · Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF · Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution · When individual component does not respond to UV - derivatisation required for detection

APPLICATIONS Pharmaceutical research Bio medical analysis Clinical analysis Environment analysis food industry Quantification of herbal extracts Therapeutic drug monitoring to determine its concentration and metabolite in blood urine Analysis of environment pollution level Characterization of hazards in industrial waste Quantitative determination of prostaglandins & thromboxanes in plasma.

DIFFERENCES BETWEEN TLC AND HPTLC

PARAMETER TLC HPTLC Type of chromatographic plates Hand made/pre coated Pre coated Absorbant layer 200-250mm 100-150mm Particle size 5-20 micrometer 4-8 micrometer Application of sample Manual/semi automatic Semi automatic Shape of sample spot band

Spot size 3-6mm 1-2mm Sample volume 1-10 microlit 0.1-2microlit No of sample per plate 15-20 40-45 Optimal development distance 10-15cm 5-7cm Development time Depend upon mobile phase 40% less than TLC Quantization Manual Manual/ instrumentation Reproducibility of result difficult Reproducible

REFERENCES Ajaz Ahmad, M Mujeeb, Bibhu Prasad Panda (2010) An HPTLC Method for the Simultaneous Analysis of Compactin and Citrinin in Penicillium citrinum Fermentation Broth. Journal of Planar Chromatography-modern TLC 23 (4), 282–285 1994 Rebecca Carrier and Julie Bordonaro - Intro to Biochemical Engineering Term Project, merged with the 1997 project by Kevin Yip   Puri, A., Ahmad, A. and Panda, B. P. (2010), Development of an HPTLC-based diagnostic method for invasive aspergillosis. Biomed. Chromatogr., 24: 887–892. doi: 10.1002/bmc.1382 Wikipedia Pharmainfo.dot