High Performance Thin Layer Chromatography

Hptlc|Principle|comparison with tlc,hptlc|Steps|Instrumentation|Factors|Applications

1. High-Performance Thin-layer Chromatography (HPTLC) Dept. Of Pharmaceutical Sciences & Technology BIRLA INSTITUTE OF TECHNOLOGY, MESRA Presented by- PROTTAY DUTTA(MPH/10055/20) Facilitated to- Dr. B.N.SINHA Professor Dept. Of Pharmaceutical Sciences & Technology BIT Mesra, Ranchi

2. CONTENTS Introduction Principle Comparison Of TLC , HPLC, HPTLC Steps involved Instrumentation factors influencing separation and resolution of spots Applications References

3. INTRODUCTION High-performance thin-layer chromatography  ( HPTLC ) is an enhanced form of thin-layer chromatography (TLC). It is also known as planar or flat bed chromatography. HPTLC is a powerful analytical method equally suitable for qualitative and quantitative analytical tasks.

4. HPTLC is popular for many reasons such as :- visual chromatogram, Multiple sample handling, Enables the most complicated separation Detection limit in nano gram range with UV-absorption detection and in picogram range with fluorometric detection Large number of theoretical plates in minimum area of plates. Analysis time greatly reduced in HPTLC due to short migration distant. Simplicity high efficiency due to small particle size Fast and inexpensive

5. Principle HPTLC have similar approach and employ the same physical principles of TLC (Adsorption chromatography) i.e. the principle of separation is adsorption. 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, component with lesser affinity towards the stationary phase travels faster.

6. Parameter for comparison TLC HPLC HPTLC AVERAGE PARTICLE SIZE 19-15µm 3-5µm 5-7µm PARTICLE SIZE DISTRIBUTION wide Normal Normal LAYER THICKNESS 250µm Not applicable 100-200µm NUMBER OF SAMPLES Max 12 One sample at a time 36-72 on a 20cm plate SEPARATION DISTANCE 100-150µm Not applicable 30-70mm RUNNING TIME 30-200 minutes 10-30 minutes or longer 3-10 minutes SOLVENT CONSUMPTION 50ml 1ml/minute For a typical run time of 30minutes , 30ml of solvent is required. 5-10ml DETECTION LIMIT (ABSORBANCE MODE) 100-1000ng ng/ml to mg/ml 10-100ng DETECTION LIMIT (FLOURESCENCE MODE ) 1-100ng ng /ml 0.1-10ng

7. Parameter for comparison TLC HPLC HPTLC Usage - Column life of 1000-2000 samples Single use plates Sample application - Auto-sampler Manual/ Auto-sampler Elution Technique - Isocratic / gradient Isocratic / gradient Development - Single dimension only possible 2 dimensional development possible which can separate a complex mixture . Dimensions - Column length – 5-30cm Diameter- 4.6mm Plate size of 5x10cm,10x10cm & 20x20cm. Separation Type - Normal / Reversed Phase Normal / Reversed Phase Material - Silica gel / NH 2 Diol / CN Silica gel 60 F254 / NH 2 Diol / CN , Cellulose

8. STEPS INVOLVED IN HPTLC

9. STATIONARY PHASE: HPTLC can be regarded as the most advanced form of modern TLC. It uses HPTLC plates featuring small particles with a narrow size distribution. As a result, homogenous layers with a smooth surface can be obtained. HPTLC uses smaller plates (10 × 10 or 10 × 20 cm) with significantly decreased development distance (typically 6 cm) and analysis time (7–20 min). HPTLC plates provide improved resolution, higher detection sensitivity, and improved in situ quantification and are used for industrial pharmaceutical densitometric quantitative analysis. Normal phase adsorption TLC on silica gel with a less polar mobile phase, such as chloroform– methanol, has been used for more than 90% of reported analysis of pharmaceuticals and drugs. Lipophilic C-18, C-8, C-2; phenyl chemically-modified silica gel phases; and hydrocarbon- impregnated silica gel plates developed with a more polar aqueous mobile phase, such as methanol–water or dioxane–water, are used for reversed-phase TLC.

10. Other precoated layers that are used include aluminum oxide, magnesium silicate, magnesium oxide, polyamide, cellulose, kieselguhr, ion exchangers, and polar modified silica gel layers that contain bonded amino, cyano, diol, and thiol groups. Optical isomer separations that are carried out on a chiral layer produced from C-18 modified silica gel impregnated with a Cu (II) salt and an optically active enantiomerically pure hydroxyproline derivative, on a silica layer impregnated with a chiral selector such as brucine, on molecularly imprinted polymers of a-agonists, or on cellulose with mobile phases having added chiral selectors such as cyclodextrins have been reported mostly for amino acids and their derivatives. Mixtures of sorbents have been used to prepare layers with special selectivity properties. HPTLC plates need to be stored under appropriate conditions. Before use, plates should be inspected under white and UV light to detect damage and impurities in the adsorbent. It is advisable to prewash the plates to improve the reproducibility and robustness of the results.

11. SILICA BASED NON-SILICA BASED

12. MOBILE PHASE: The selection of mobile phase is based on adsorbent material used as stationary phase and physical and chemical properties of analyte. General mobile-phase systems that are used based on their diverse selectivity properties are diethyl ether, methylene chloride, and chloroform combined individually or together with hexane as the strength-adjusting solvent for normal-phase TLC and methanol, acetonitrile, and tetrahydrofuran mixed with water for strength adjustment in reversed-phase TLC . Accurate volumetric measurements of the components of the mobile phase must be performed separately and precisely in adequate volumetric glassware and shaken to ensure proper mixing of the content. Volumes smaller than 1 ml are measured with a suitable micropipette. Volumes up to 20 ml are measured with a graduated volumetric pipette of suitable size. Volumes larger than 20 ml are measured with a graduated cylinder of appropriate size. To minimize volume errors, developing solvents are prepared in a volume that is sufficient for one working day.

13. Generally used Mobile phase in detection of some chemical compounds

14. LAYER PREWASHING: Plates are generally handled only at the upper edge to avoid contamination. Usually plates are used without pretreatment unless chromatography produces impurity fronts due to contamination of the plate. For reproducibility studies and quantitative analysis, layers are often prewashed using 20 ml methanol (generally, methanol is used as a prewashing solvent; however, a mixture of methanol and ethyl acetate or even mobile phase of the method may also be used) per trough in a 20 × 10 cm twin-trough chamber ( TTC ). Up to two 20 × 10 cm or four 10 × 10 cm plates can be developed back-to-back in each trough of the TTC . Remove the plate and dry it for 20 min in a clean drying oven at 120°C. Equilibrate plate with laboratory atmosphere (temperature, relative humidity) in a suitable container providing protection from dust and fumes.

15. PREPARATION OF PLATE: Precoated layers: TLC plates can be made in any lab with suitable apparatus. However such layers do not adhere well to the glass support. Precoated plates that use small quantities of very high molecular weight polymer as binder overcomes most limitations of a home-made layer. Precoated layers are reasonably abrasion resistant, very uniform in layer thickness, reproducible, preactivated, and ready to use. They are available with glass or aluminum or polyester support. Aluminum foil plates are less expensive to buy, cheaper, can be cut, and therefore easy to carry around or transport or mail. Glass plates are the best for highest quality of results. Most often, layers containing a fluorescent indicator F 254 are used. This enables the visualization of samples in a UV cabinet very simply, instantly, and in a nondestructive manner. Commonly used size of plates in TLC is 20×20 cm and in HPTLC 20 × 10 cm or 10 × 10 cm is widespread.

16. SAMPLE PREPARATION AND APPLICATION: Sample preparation It’s important to prepare proper sample for successful separation. Sample and reference substances should be dissolved in the same solvent to ensure comparable distribution at starting zones. It needs a high concentrated solution, as very less amount of sample need to be applied. Sample application In Thin-Layer Chromatography manual sample application with capillaries is usually performed for simple analyses. Sample volumes of 0.5 to 5 μL can be applied as spots onto conventional layers without intermediate drying. HPTLC layers take up to 1 μL per spot. More demanding qualitative, quantitative, and preparative analyses or separations are made possible only by instruments for band wise application of samples using the spray-on technique. Particularly HPTLC takes full advantage of the gain in separation power and reproducibility available by precise positioning and volume dosage.

17. AUTOMATIC TLC SAMPLER  Automatic sample application is a key factor for productivity of the HPTLC laboratory. The requirements for an instrument serving this purpose, i.e. precision, robustness during routine use and convenient handling are fully met by the Automatic TLC Sampler 4. The ATS 4 offers fully automatic sample application for qualitative and quantitative analyses as well as for preparative separations. I t is best suited for routine use and high sample throughput in mass analysis. Samples are either applied as spots through contact transfer (0.1–5 μl)or as bands or rectangles (0.5 to > 50 μl) using the spray-on technique. Starting zones sprayed on as narrow bands offer the best separation attainable with a given chromatographic system.

18. Key features: • Fully automatic sample application, suitable for routine. • Application of spots, bands, or rectangles. • Data input and monitoring through WINCATS. • Application of solutions onto any planar medium. • Application of sample volumes between 0.1 and 5 μl by contact transfer. • spray-on application of sample volumes between 0.5 and > 50 μl. Application in the form of rectangles allows precise application of large volumes without damaging the layer. Prior to chromatography, these rectangles are focused into narrow bands with a solvent of high elution strength. The ATS 4 allows “ over spotting”, i.e. a sequential application from differentials onto the same position. This technique can be used e.g. in pre chromatographic derivatization, spiking, etc.

19. DEVELOPMENT OF CHROMATOGRAM: Thin-layer chromatography differs from all other chromatographic techniques in the fact that in addition to stationary and mobile phases, a gas phase is present. This gas phase can significantly influence the result of the separation. Processes in the Developing Chamber The “ classical” way of developing a chromatogram is to place the plate in a chamber, which contains a sufficient amount of developing solvent. The lower end of the plate should be immersed several millimeters. Driven by capillary action the developing solvent moves up the layer until the desired running distance is reached and chromatography is stopped. The following considerations primarily concern silica gel as stationary phase and developments, which can be described as adsorption chromatography.

20. Provided the chamber is closed, four partially competing processes occur: 1. Between the components of the developing solvent and their vapor, an equilibrium will be established eventually . This equilibrium is called chamber saturation. Depending on the vapor pressure of the individual components the composition of the gas phase can differ significantly from that of the developing solvent. 2 While still dry, the stationary phase adsorbs molecules from the gas phase. This process, adsorptive saturation, is also approaching an equilibrium in which the polar components will be withdrawn from the gas phase and loaded onto the surface of the stationary phase . 3 Simultaneously the part of the layer which is already wetted with mobile phase interacts with the gas phase. Thereby especially the less polar components of the liquid are released into in the gas phase . Unlike this process is not as much governed by vapor pressure as by adsorption forces. 4 During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts.

21. DERIVATIZATION: Post chromatographic Derivatization It is an inherent advantage of Thin-Layer Chromatography that fractions remain stored on the plate and can be derivatized after chromatography. By derivatization substances that do not respond to visible or UV light can be rendered detectable. In many cases, substances or classes of substances can be identified by specific reagents. 1. Changing non-absorbing substances into detectable derivatives 2. Improving the detectability (lowering detection limits) 3. Detecting all sample components 4. Selectively detecting certain substances 5. Inducing fluorescence QUANTIFICATION Most modern HPTLC quantitative analysis are performed in situ by measuring the zones of samples and standards using a chromatogram spectrophotometer usually called a densitometer or scanner with a fixed sample light beam in the form of a rectangular slit. Generally, quantitative evaluation is performed with the TLC Scanner 3 using WINCATS software. It can scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode; scanning speed is selectable up to 100 mm/s.

22. DOCUMENTATION Each developed plate is documented using digital documentation system under UV light at 254 nm, UV light at 366 nm, and white light. If a type of light does not produce usable information, that fact must be documented. If a plate is derivatized, images are taken prior and after derivatization. Factors affecting HPTLC Types of stationary phase. Mobile phase Layer thickness Temperature Mode of development Amount of sample Dipping zone

23. Isolation of Drug from excipients We can understand this by taking an example – Determination of Paracetamol, Pseudoephedrine and Loratidine in Tablets. A sensitive, accurate and selective high performance thin layer chromatography (HPTLC) method was developed and validated for the simultaneous determination of paracetamol (PAR), its toxic impurity 4-aminophenol (4-AP), pseudoephedrine HCl (PSH) and loratidine (LOR).  After the completion of the process we obtain the following graphs :-

24. HPTLC chromatogram of (A) blank plasma at 254 nm, (B) blank plasma at 208 nm, (C) a mixture of 4 μ g/band PAR, 0.8 μ g/band LOR and 2 μ g/band pseudoephedrine in spiked human plasma at 254 nm and (D) a mixture of 4 μ g/band PAR, 0.8 μ g/band LOR and 2 μ g/band pseudoephedrine in spiked human plasma at 208 nm

25. 1. Pharmaceutical applications  Quality control  Content Uniformity Test (CUT)  Identity- and purity checks  Stability tests, etc. 3. Cosmetics  Identity of raw material  Preservatives, colouring materials, etc.  Screening for illegal substances, etc. 6. Industrial applications  Process development and optimization  Process monitoring  Cleaning validation, etc. 2. Clinical applications  Lipids  Metabolism studies  Drug screening  Doping control, etc. 4. Herbal medicines and botanical dietary supplements  Identification  Stability tests  Detection of adulteration  Assay of marker compounds, etc. 5.Food and feed stuff  Quality control  Additives ( e.g. vitamins)  Pesticides  Stability tests (expiration), etc. 7. Forensics  Detection of document forgery  Investigation of poisoning  Dyestuff analyses, etc. Applications of HPTLC

26. Foods usually originate as botanical products and therefore are naturally variable as well as complex. HPTLC can confirm identities of complex mixtures as well as detect adulteration. HPTLC is the first method of analysis because it is simple, risk free, fast, economical and analyses 100-120 samples a day, without producing much waste.  HPTLC-FOOD ANALYSIS

27. HPTLC- HERBAL APPLICATIONS HPTLC Fingerprint is a technique adopted by the US & European Pharmacopoeias recently for the purpose of identification of “botanical materials”, all of which are very complex in nature. HPTLC Fingerprint is the representation of the phytochemical composition of a plant extract or formulation, in the form of a conventional image i.e. a photograph. Fingerprint can also be used to monitor batch to batch consistency and stability studies of herbal medicines, dietary supplements etc. HPTLC-FORENSIC ANALYSIS Forensic analysis is the multi-disciplinary application of scientific knowledge and sophisticated instruments for investigating crime related materials and biological samples. A frequent but challenging aspect of forensic toxicology is the identification of unknown poisonous substances in lethal intoxication cases. HPTLC offers identification as well as qualitative and quantitative analysis for toxic substances, CAMAG HPTLC offers rapid identification of such toxins for antidote administration.

28. High Performance Thin Layer Chromatography (HPTLC) is a valuable tool to check purity, impurity of any non-volatile organic industrial materials such as dyes, surfactants, pesticides, perfumery compounds, intermediates etc. It is far simpler, cheaper and easier to understand than other similar methods of analysis. Chemical reactions can be studied very quickly e.g. within 2 hours. Complex mixtures like biological samples, reaction mixtures, fermentation broth can be easily chromatographed without much sample preparation. HPTLC- DYES AND INTERMEDIATES ANALYSIS Dyes and intermediates are non-volatile organic substances and so best suited for HPTLC analysis. HPTLC analysis is very low cost and results are “visible”. Colored substances are therefore particularly ideal for HPTLC analysis. HPTLC enables comparison of different samples or with standard, analysis of competition samples apart from purity and impurity determination. All kinds of optical brightner, intermediates, dyes, banned amines can be analyzed by HPTLC. HPTLC-SPECIALITY CHEMICALS ANALYSIS

29. HPTLC-BIOTECHNOLOGY APPLICATIONS HPTLC is a most versatile analytical technique which offers a great separation power using precise sample application, software-controlled chromatographic steps, chromatogram development and scanning and photo documentation. Separated samples can be visually checked on the plate is an unique aspect of HPTLC. Biotechnology industry is considered as one of the most research-intensive sectors in the world. Therefore, shorter analysis time, low sample analysis cost per sample, minimal contamination possibilities and reliable accurate results are essential which are provided by HPTLC. HPTLC can analyse different samples simultaneously with zero risk of cross contamination. HPTLC also offers the advantage of evaluating a plate by any specific process using different detection modes (UV, fluorescence, etc.) By coupling HPTLC with a MS or other suitable methods such as NMR, FTIR, ESI, MALDI, one can identify and or confirm the chemical structures of analytes under study.

30. REFERENCES :-   CAMAG, 2010-2011. Instrumental thin layer chromatography. Switzerland: Camag . Available from: camag.com/downloads/free/brochures/CAMAG_TLC10-11_E.pdf 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. https://academic.oup.com/chromsci/article/54/4/647/2754774 https://www.pharmatutor.org/articles/high-performance-thin-layer-chromatography- hptlc-instrumentation-overview A Textbook of Pharmaceutical Analysis By Dr. S . Ravi Shankar

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