HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)
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The document discusses High Performance Thin Layer Chromatography (HPTLC) as an advanced method of analyzing chemical substances, detailing its principles, applications, and advantages over traditional Thin Layer Chromatography (TLC). It outlines key parameters such as chromatographic plate types, sample application methods, mobile phase selection, and post-chromatographic techniques. Additionally, it highlights specific applications in pharmaceuticals, food analysis, clinical settings, and forensic investigations.
HPTLC- Principle, Instrumentation and Software (Abhishek Gupta)
- 1. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY Abhishek Gupta Young Scientist Fellow Pharmacognosy & Ethnopharmacology Division CSIR-NBRI Lucknow
- 2. Thin layer chromatography (TLC) • Is a method for identifying substances and testing the purity of compounds • TLC is a useful technique because it is relatively quick and requires small quantities of material
- 3. • Separations in TLC involve distributing a mixture of two or more substances between a stationary phase and a mobile phase. • The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a plate. • The mobile phase: is a developing liquid which travels up the stationary phase, carrying the samples with it. • Components of the samples will separate on the stationary phase according to how much they adsorb on the stationary phase versus how much they dissolve in the mobile phase.
- 5. HPTLC • HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way • It is also known as planar chromatography or Flat- bed chromatography Principle: • Chromatography is a physical process of separation in which the components to be separated are distributed between two immiscible i.e. the principle of separation is adsorption
- 6. • The mobile phase solvent flows through because of capillary action. The components move according to their affinities towards the adsorbent • The component with more affinity towards the stationary phase travels slower. The component with lesser affinity towards the stationary phase travels faster • Thus the components are separated on a chromatographic plate
- 7. Differences between TLC and HPTLC Parameter TLC HPTLC Chromatographic plate used Hand made /pre-coated Pre-coated Sorbent layer thickness 250 mm 100-200mm Particle size range 5-20 μm 4-8 μm Pre-washing of the plate Not followed Must Application of sample Manual/Semi automatic Semi automatic/Automatic Shape Spot Spot/Band Spot size 2-4mm 0.5-1mm Sample volume 1-10 μl 0.2-5 μl Application of larger volume Spotting which leads to over loading Can be applied as bands No. of samples/plate (20X20) 15-20 40-50 Optimum development distance 10-15 cm 5-7 cm Development time Depends on mobile phase 40% Less than TLC Reproducibility of results Difficult Reproducible
- 9. Selection of HPTLC plates • Hand plates were available which are made up of cellulose and other materials which are not used much • Precoated plates The plates with different support materials and sorbent layers with different format and thickness are used. Plates with sorbent thickness of 100-250μm are used for qualitative and quantitative analysis.
- 10. Supports Materials Advantage Disadvantage Glass 1.Ressistant to heat and chemicals 2.Easy to handle and offers superior flat surface for work 1. Fragility 2.Relatively High wt 3.Costs more for additional packaging Polyester sheets (0.2 mm thick) 1.More economical as produced even in roll forms 2.Unbreakable 3.Less packing material 4.Spots can be cut and eluted thus eliminates dust from scrapping Charring reactions if temperature exceeds 120oc as the plates are dimensionally unstable beyond this temperature Aluminum Sheets(0.1mm) 1.Increasesed temperature resistance Eluents containing high concentration of mineral acids or ammonia can attack chemically on aluminum
- 11. Some of the sorbents used in HPTLC No Examples Applications 1. Silica gel 60F (Unmodified ) 80% of analysis is done on this layer. 2. Aluminum oxide Basic substances ,alkaloids and steroids 3. Cellulose (microcrystalline ) Amino acids, peptides, sugars and other liable compounds which cannot be chromatographed on the active layers of silica gel. 4. Silica gel chemically modified a) Amino group ( NH2) b ) CN COOH ,Phenols ,Nucleotides Pharmaceutical preservations.
- 12. Binders • Gypsum (G) • Starch (S) • Layer containing fluorescent indicator (F)
- 13. Plate size • 20X20cm • 10X20cm • 5X10 cm • 5X7.5 cm • Good cut edges of sheets is important to obtain constant Rf values.
- 14. Pre washing of pre coated plates To remove impurities which include water vapours and other volatile substances Silica gel 60F is most widely used sorbent. The major disadvantage of this sorbent is that it contain iron as impurity which is removed by using Methanol : water in the ratio of 9:1.This is the major advantage of the step of pre-washing
- 15. Solvents used for pre-washing 1. Methanol 2. Chloroform: methanol ( 1:1 ) 3. Choloroform: Methanol: Ammonia (90:10:1) 4. Methylene chloride: Methanol ( 1:1 ) 5. Ammonia solution (1%)
- 16. Activation of plates • Freshly opened box of HPTLC plates doesn’t need activation • Plates exposed to high humidity or kept in hand for long time require activation • Plates are placed in oven at 110o -120o c for 30 min prior to the sample application • Activation at higher temperature for longer period is avoided as it may lead to very active layers and risk of the samples being decomposed
- 17. Sample Preparation • Proper sample preparation is an important pre- requisite for success of TLC separation. • For normal chromatography: Solvent should be non-polar and volatile. • For reversed chromatography: Polar solvent is used for dissolving the sample • Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones.
- 18. Application of sample The selection of sample application technique and device to be used depends primarily on: • Sample volume • No. of samples to be applied • Required precision and degree of automation
- 19. Some applicators used for spotting are: a) Capillary tubes b) Micro bulb pipettes c) Micro syringes, d) Automatic sample applicator. – The major criteria is that they shouldn’t damage the surface while applying sample.
- 20. • The sample should be completely transferred to the layer. • Micro syringes are preferred if automatic application devices are not available. • Volume recommended for HPTLC-0.5-5μl • Sample spotting should not be excess or not low • Problem from overloading can be overcome by applying the sample as band.
- 21. Advantages of application of sample as band • Better separation because of rectangular area. • Response of densiometer is higher • Large quantity
- 22. Automatic applicators 1. CAMAG Nanomat: Samples applied in the form of spots. The volume is controlled by disposable platinum iridium of glass capillary which has volume of 0.1-0.2μl 2. CAMAG Linomat: Automated sample application device. Sample is loaded in micro syringe (Hamilton Syringe) 1μl capacity. Sample can apply either as spot or band by programming the instrument with parameters like spotting volume ,band length etc 3. CAMAG automatic TLC sampler III: Applies sample as spot or bands automatically from the rack of sample vials
- 23. Mobile phase • Mobile phase should be of high grade • Chemical properties ,analytes and sorbent layer factors should be considered while selection of mobile phase • Use of mobile phase containing more than three or four components should normally be avoided as it is often difficult to get reproducible ratios of different components • Mobile phase optimization is necessary while performing HPTLC • Various components of MP should be measured separately and then placed in mixing vessel. This prevents contamination of solvents and also error arising from volumes expansion or contraction on mixing
- 24. • Trough chambers are used in which smaller volumes of MP usually 10-15 ml is required • Chambers containing multi component MP are not generally used for re-use for any future development , due to differential evaporation and adsorption by layer • Once the chamber is opened , solvents evaporate disproportionally depending on their volatilities.
- 25. Polarity of Solvents Water Acetic Acid Ethyleneglycol Methanol Ethanol Isopropanol Acetonitrile Nitromethane Aniline Dimethylsulfoxide Ethylacetate Acetone Dicholoroethane Tetrahydrofuran Chloroform Diethylether Benzene Toluene Carbontetrachloride Cyclohexane Petroleum ether Non-polar Polar
- 26. Development of chambers 1.Twin trough chamber 2.Rectangular chambers
- 27. Simulation chamber The simultion developing chamber is a thick walled clear glass tank with vertical grooves and a heavy ground-glass lid. Round chamber These cylindrical chambers are ideal for use with narrower width plates. Nano chamber The nano chamber is suitable for the development of 10x10cm TLC plates and features a heavy glass lid for gas-tight seal and optimum vapour saturation. HPTLC chamber Ideal for the development of HPTLC 5x5cm plates.
- 28. Pre-conditioning (Chamber Saturation) • Chamber saturation has a pronounced influence on the separation profile • Time required for the saturation depends on the mobile phase • If plates are introduced into the unsaturated chamber,during the course of development , the solvent evaporates from the plate mainly at the solvent front and it results in increased Rf values
- 29. Development and Drying • Ascending, descending, horizontal • Plates are spotted with sample and air dried and placed in the developing chambers • After the development plate is removed from chamber and mobile phase is removed under fume cup-board to avoid contamination of laboratory atmosphere • The plates should be always laid horizontally because when mobile phase evaporates the separated components will migrate evenly to the surface where it can be easily detected
- 30. Drying • Drying of chromatogram should be done in vacuum desiccators with protection from heat and light • If hand dryer is used there may be chances of getting contamination of plates, evaporation of essential volatile oils if any present in the spot or compounds sensitive to oxygen may get destroyed due to the rise in temperature
- 31. Factors influencing separation and resolution of spots • Type of stationary phase • Type of pre-coated plates • Layer thickness • Binder in layer • Mobile phase • Solvent purity • Size of developing chamber • Sample volume to be spotted
- 32. • Size of initial spot • Solvent level in chamber • Relative humidity • Temperature • Mode of Derivatization Greater the difference between two spots and smaller the initial spot diameter of sample and better will be the resolution
- 33. 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 One of the characteristic feature of HPTLC is the possibility to utilize post-chromatographic off line derivatization Detection are of two types: • Qualitative • Quantitative
- 34. • Qualitative detection: HPTLC is routinely used for qualitative analysis of raw materials, finished products, plant extracts etc. It involves the identification of unknown sample mixture by comparing the Rf values of the sample components with the standards. • Quantitative Evaluation: Quantitative evaluation of the chromatogram by HPTLC basically involves estimation of chemical compounds present in the sample
- 35. Densiometry •Separation of Herbal sample and reference substances, there is a broad array of other detection modes available. •Detection is a very flexible and independent step. Multiple detection is possible without repeating the chromatography. •Allows measuring the absorption and/or fluorescence of underivatized or derivative substances at wavelengths between 200 and 800 nm. Up to 31 wavelengths can be evaluated and spectra of any peak can be recorded. •Following integration densitometric data can be quantitatively evaluated.
- 36. Documentation • Labeling every single chromatogram can avoid mistake in respect of order of application • Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded • Suppliers name, item number, batch no., individual plate no. are imprinted near upper edge of pre-coated plates. Helps in traceability of analytical data • Avoids manipulation of data at any stage as coding will automatically get recorded using photo-documentation
- 37. POST CHROMATOGRAPHIC DERIVATIZATION • Chromatogram Immersion Device. • TLC Sprayer. • Reagent Spray. • TLC Spray Cabinet. • TLC Plate Heater
- 38. Detecting Agents • p-Anisaldehyde – sulfuric acid: phenols, sugars, steroids, and terpenes • Aluminium chloride: flavonoids • Antimony (III) chloride: vitamins A & D, carotenoids, steroids, sapogenins, steroid glycosides, terpenes • Bromthymol blue: lipids and phospholipids • Diphenylamine: glycosides, glycolipids • Dragendorff reagent: nitrogen compounds, alkaloids, antiarrhythmic drugs, surfactants • Gentian Violet – Bromine: lipids • Iodine vapor: Relatively unspecific universal reagent for many organic compounds
- 39. Applications of HPTLC • Pharmaceutical industry: Quality control, content uniformity, uniformity test, identity/purity check. • Food Analysis: Quality control , additives , pesticides ,stability testing ,analysis of sub- micron levels of aflotoxins etc • Clinical Applications: Metabolism studies , drug screening ,stability testing etc • Industrial Applications; Process development and optimization, In-process check ,validation etc. • Forensic : Poisoning investigations
- 40. HPTLC of Centella asiatica
- 41. Mobile phase: Toluene: Ethyl acetate: Formic acid (5:5:1) for Asiatic acid n-Butanol: Ethyl acetate: Water (5:1:4) for Asiaticoside & Madecassoside
- 42. HPTLC of Centella asiatica
- 43. HPTLC of Jatropha curcas
- 44. HPTLC of Jatropha curcas L.
- 45. HPTLC of Salvadora persica L. Fig. 2 HPTLC profiles of S. persica stem (SS), twig (ST) & Std. Ferulic acid Fig. 3 Densitometric chromatogram of S. persica stem, twig and ferulic acid (A: Extract of twig; B: extract of stem; C: Standard Ferulic acid)
- 46. Figure 4: HPTLC profile of C. forskohlii samples with standard forskolin A: Cf-Salem; B: Cf-Bhuwali; C: Cf- Bhadrapur; D: Cf-Jabalpur; E: Cf-Banglore F1, F2, F2, F3, F4, F5, F6: Standard forskolin levels Figure 2: Calibration curve of standard forskolin-Conc. vs AU HPTLC of Coleus forskohlii
- 47. Mobile Phase: Toluene: Ethylacetate: Methanol (9:3:0.5) Rf: 0.41 HPTLC of Coleus forskohlii Figure 3: Comparative HPTLC chromatogram of forskolin in samples and standard A: Cf-Banglore; B: Cf-Jabalpur; C: Cf-Bhadrapur; D: Cf-Bhuwali; E: Cf-Salem; F: Std. Forskolin Figure 1: HPTLC chromatograms of different accessions of C. forskohlii A: Cf-Salem; B: Cf- Bhadrapur; C: Cf-Jabalpur; D: Cf-Banglore; E: Cf-Bhuwali
- 48. Mobile Phase: T:Eac:F (5:4:1) Rf: 0.61 HPTLC of Urtica dioica L.
- 49. HPTLC of amino acids in Luffa cylindrica L.
- 67. Conclusion 1. CHROMATOGRAPHY IS A SCIENCE OF SEPERATION USED EITHER FOR IDENTIFICATION OR QUANTITATION OF CHEMICAL SUBSTANCES 2. VARIOUS MODES OF CHROMATOGRAPHIC TECHNIQUES WERE DEVELOPED BASED ON INITIAL DISCOVERY BY MICHAEL T.SWETT 3. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY IS MOST ADVANCED TECHNIQUE NOW A DAYS 4. IT IS THE MODERN SEPARATION TECHNIQUE WHICH IS ACCEPTRD AS AN EXTREMLY FESIBLE,RELIABLE & COST EFFICIENT METHOD 5. BY THIS TECHNIQUE ACCURATE SEPARATION OF SAMPLE TAKES PLACE 6. IT CAN SPEED UP OUR WORK & SO MANY THINGS AT A TIME USUALLY NOT POSSIBLE WITH OTHER ANALYTICAL TECHNIQUE 7. HPTLC CAN SIMULTANEOUSLY HANDLE SEVERAL SAMPLES EVEN OF DIFFERENT NATURE & COMPOSITION SUPPORTING SEVERAL ANALYTE AT A GIVEN TIME
- 68. References: • HPTLC- Quantitative analysis of pharmaceutical Formulations by P.D. Sethi • Dr. Harish Chandra Andola, “High Performance Thin Layer Chromatography (HPTLC): A Modern Analytical tool for Biological Analysis”, Nature and Science , 2010;8(10) • CAMAG, 2010-2011. BASIC EQUIPMENT FOR MODERN THIN LAYER CHROMATOGRAPHY. SWITZERLAND: CAMAG.AVAILABLE FROM: CAMAG.COM/DOWNLOADS/FREE/BROCHURES/CAMAG-BASIC-EQUIPMENT-08.PDF • http://www.pharmainfo.net/files/images/stories/article_images/INTRODUCTION_ANALYTICALME THODFig10.JPG • PATEL, R.B. AND PATEL, M.R. AND PATEL, B.G. (2011) EXPERIMENTAL ASPECTS AND IMPLEMENTATION OF HPTLC. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER, PP. 41- 54. • SHRIVASTAVA, M.M. (2011) AN OVERVIEW OF HPTLC: A MODERN ANALYTICAL TECHNIQUE WITH EXCELLENT POTENTIAL FOR AUTOMATION, OPTIMIZATION, HYPHENATION, AND MULTIDIMENSIONAL APPLICATIONS. IN: SHRIVASTAVA, M.M. HPTLC. NEW YORK: SPRINGER, PP. 3- 24. • http://www.infoexpo.ch/abstract