hptlc

a presentation on HPTLC

1. HPTLC Presented by SREYA S 1st Semester M Pharm Dept. of pharmacology

2. Introduction Chromatography is a physical process of separation in which the component is to be separated are distributed between two immiscible a stationary phase with has large surface area and a mobile phase which is in constant motion through the stationary phase. In 1973,Halpaap introduced first “ Nano TLC plates’’ In 1977,the first major HPTLC publication is “HPTLC-high”

3. Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel,  aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase. 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 .

4. Principle Same physical principal of TLC, ie the principle of separation is Mobile phase containing the dissolved solutes passes over the surface of stationary surface The components move according their affinities towards the adsorbent. Component with more affinity towards stationary phase travel slower and less affinity travels faster. Adsorption may not always be the principle ,separation may be achieved by partition , ion exchange and molecular exclusion. Rf = Distance travelled by solute

5. INSTRUMENTATION Sample applicators Development chamber Scanner Visualizer

6. STEPS INVOLVED SAMPLE AND STANDARD PREPARATION SELECTION OF CHROMATOGRAPHIC PLATES LAYER PRE-WASHING LAYER PRE-CONDITIONING APPLICATION OF SAMPLE CHROMATOGRAPIC DEVELOPMENT DETECTION OF SPOTS SCANNING AND DOCUMENTATION

7. SELECTION OF HPTLC PLATES Hand plates were available which are made up of cellulose and other materials which are not used much now a days Pre coated 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.

8. Supports Materials Advantage Disadvantage Glass 1.Resistant 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 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 Eluent containing high conc entration of mineral acids or ammonia can attack chemically on aluminum

9. SORBENTS USED IN HPTLC Silica gel 60F, it analyses 80% of drugs. Aluminium oxide, it analyses the basic substances and steroids. Cellulose. Silica gel chemically modified in amino group and CN. Some of the binders used : • Gypsum (G) • Starch (S) • Layer containing fluorescent indicator(F)

10. PLATE SIZE : 20X20cm 10X20cm 5X10 cm 5X7.5 cm Good cut edges of sheets is important to obtain constant Rf values.

11. Pre washing of pre coated plates The main purpose of the pre-washing is to remove impurities which include water vapour and other volatile substances from the atmosphere when they get exposed in the lab environment. Silica gel 60F is most widely used sorbent. The major disadvantage of this sorbent is that it contain iron as impurity. This iron is removed by using Methanol : water in the ratio of 9:1. This is the major advantage of the step of pre-washing.

12. Some common methods involved in pre-washing Ascending method : In this technique the chromatographic plates are run blank ( i . e. before application of the sample with suitable solvent / mobile phase.) The solvent/mobile phase carries the impurities to the top of the plate. It takes longer time but cleaning effect is superior. The disadvantage of this technique is active dirt gets accumulated at the solvent front

13. Dipping method : In this technique, the chromatographic plate is dipped in a suitable solvent for specified period of time, removed from the chamber and finally dried. Dipping method is quicker and yields uniform layer but cleaning effect is often not as good as ascending method Continuous method : In this technique, the plate to be washed is placed in chamber having an entrance and exit slits. The solvent is made to flow continuously through the chamber that carries the impurities from the plate.

14. SOLVENTS USED FOR PRE-WASHING Methanol • Chloroform: methanol ( 1:1 ) • Choloroform : Methanol: Ammonia (90:10:1 ) • Methylene chloride: Methanol ( 1:1 ) • Ammonia solution (1%) 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-120oc 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.

15. SAMPLE PREPARATION Proper sample preparation is an important pre-requisite for success of TLC separation. For normal chromatography: Solvent should be nonpolar 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 .

16. 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. It is the most critical step for obtaining good resolution for quantification by HPTLC. 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

17. • The major criteria is that they shouldn’t damage the surface while applying sample 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 to keep the starting zone down to minimum of 0.5-1 mm in concentration range of 0.1-μg/ml Sample spotting should not be excess or not low. Problem from overloading can be overcome by applying the sample as band.

18. MOBILE PHASE Mobile phase should be of high graded. 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. Trough chambers are used in which smaller volumes of MP usually 10-15 ml is required.

19. Different components of MP are mixed first in mixing vessel and then transferred to developing chambers. Chambers containing multi component MP are not generally used for re-use for any future development , due to differential evaporation and adsorption by layer and also once the chamber is opened, solvents evaporate disproportionally depending on their volatilities Solvent composition expressed in v/v.

20. DEVELOPMENT CHAMBERS .Twin trough chamber: Low solvent consumption Reproducible pre-equilibration of the plate with solvent vapour is possible by placing it in the empty trough opposite that containing the solvent Sandwitch chamber : In this method, a sandwich is created by covering the TLC layer with a glass plate. The TLC layer and cover plate should be slightly separated using spacers such that only the bottom-most zone (about 2 cm in width) remains uncovered. The cover plate should not be dipped into the solvent. The sandwich design can be used in any type of TLC development chamber. Round chamber: These cylindrical chambers are ideal for use with narrower width plates.

21. Horizontal development chamber: n this method, a sandwich chamber is used for horizontal development of TLC plates. TLC development can be performed from one or both sides of the plate. Simulation chamber :The simultan 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.

22. AUTOMATIC APPLICATORS USED 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 0.1-0.2μ 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. 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 areintroduced in to theunsaturated chamber , during the course of development , the solvent evaporates from the plate mainly at the solvent front and it results in increased Rf values. If tank is saturated prior to the development, solvent vapour soon get uniformly distributed through out the chamber. As soon as the plate is kept in such a saturated chamber ,it soon gets pre-loaded with solvent vapors thus less solvent is required to travel a particular distance, resulting in lower Rf values. But in some cases depending on their polarity saturation and non-saturation of chambers are required

24. Eg : Pre-equilibrium is often recommended in case of solvent with high polarity. Development in a non-saturation or partially saturated atmosphere is recommended in solvents used in a composition leadingto phase separation such as mixture of n- butanol , water and glacial acetic acid. Preloading of dry layer with solvent vapors should be avoided for low polar molecules .

25. DEVELOPMENT AND DRYING The different methods used for development of chambers are like-Ascending , descending , 2-dimentional , horizontal , multiple over run , gradient , radial , anti-radial , multimodal , forced flow planar chromatography. 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

26. 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 .

27. 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 Size of initial spot Solvent level in chamber

28. Gradient Relative humidity Temperature Flow rate in solvent Separation distance Mode of Derivatization Greater the difference between two spots and smaller the initial spot diameter of sample and better will be the resolution

29. DETECTION AND VISUALIZATION Detection are of two types: Qualitative Quantitative 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. • Quantitation Evaluation : Quantitation of the chromatogram by HPTLC basically involves direct and indirect methods

30. Indirect method; It involves removal of analyte from the plate followed by quantitation . Eg ; Scrapping and elution which is followed by analysis of eluant by convenient methods like 1) Spectrophotometry 2) Flourimetry 3) Colourimetry Collection of samples from scrapping will results in the loss of sample, so vaccum devices and elution chamber are used.

31. Photo-documentation With Digital Camera

32. DOCUMENTATION 1.Documentation is important because labeling every single chromatogram can avoid mistake in respect of order of application. 2.Type of plate, chamber system, composition of mobile phase, running time and detection method should be recorded. 3.To assist the analysts and researchers E.Merck has introduced HPTLC pre-coated plates with an imprinted identification codes. 4.Suppliers name, item number, batch no. , individual plate no. are imprinted near upper edge of pre-coated plates. This will not only help in traceability of analytical

33. 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

34. UHPLC UHPLC, Ultra-High-Performance Liquid Chromatography is similar to HPLC, in that it is a technique used to separate different constituents of a compound.  Used predominately to identify, quantify and separate components of a mixture by using high pressure to push solvents through the column. In UHPLC, particle sizes less than 2um can be used, providing better separation than HPLC where particle size is limited to 5um. These smaller particles require higher pump pressures (100MPa vs.40 MPa , making this technique very efficient with fast analysis and higher resolution

35. DIFFERENCES BETWEEN HPLC AND UHPLC Particle sizes – In HPLC particle sizes of the stationary phase are typically in the order of 3-5 µm, whilst UHPLC is characterised by particles of 2 µm or less. Column dimensions – As with particle sizes there is a corresponding reduction in column dimensions with UHPLC. A typical HPLC column has an internal diameter of 4.6 mm and a length of 250 mm, whilst a UHPLC column has internal diameters of 2.1 mm or less and is much shorter, 100 mm for example. Flow rates – UHPLC runs at much lower flow rates than HPLC, for example 0.2 – 0.7 ml/min against 1-2 ml/min respectively.

36. Backpressure – With the smaller particles and reduced column diameter then this manifests itself in to higher backpressures in UHPLC compared to HPLC. HPLC instruments typically operate at maximum pressures of 400-600 bar, whilst UHPLC instruments can operate at up to 1500 bar. Detection parameters – Narrow peaks are produced with UHPLC, requiring a detector that can keep pace and provide the required number of data points per peak for detection. Most modern  detectors , though, are capable of detection speeds of up to 250 Hz, which is sufficient for both HPLC and UHPLC.

37. TECHNICAL SPECIFICATION   Pump pressure – up to 100 MPa Pump flow rate – 0.05 to 8.0 mL /min Injection volume – 1 to 100 uL Column compartment temperature – 5 to 110 °C Column particle size – less than 2 um Detection UV/VIS, 190 to 800 nm Fluorescence

38. STRENGTH   Smaller particle size Faster analysis Higher resolution   LIMITATIONS   Cannot separate inorganic ions or polysaccharides Lack of an ideal universal detector Aggressive non-polar solvents are not compatible with the instrument  

39. IDEAL USE Biochemistry for the analysis of constituents of a compound. Separation and identification of amino acids, nucleic acids, proteins, hydrocarbons, pesticides, carbohydrates, antibiotics, steroids and countless other inorganic substances. Quantitation of analytes present Sample purity determination Quality assurance and control