HPTLC_planer chromatography Seperation Technique

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1. HPTLC : SEPERATION TECHNIQUE Priyanka Singh Sientist B Kerry ingredients India Pvt ltd

2. HPTLC A science of separation ; used for both identification and quantification. This separation technique is based on the different interactions of compounds with two phases, A mobile phase A stationary phase, as the compounds travel through a supporting medium.

3. Theoretical Considerations

4. some of the major parameters that influence chromatographic system efficiency with special reference to HPTLC are - Separation Efficiency Partition Coefficient Retardation Factor Capacity Factor Spot Capacity Plate Height Resolution Chromatographic Seperation influencer

5. Here, the flow of the mobile phase is not controlled as in the column methods; but mobile phase runs by capillary forces and the position of the mobile phase at time t is given by Zf is the distance moved by the mobile phase k is the velocity constant . Velocity constant is dependent on – Saturation level of vapor phase in contact with the stationary phase. properties of the mobile and stationary phases by the equation Ko is the permeability constant dp is the average particle diameter γ is the surface tension of the mobile phase, η is the viscosity of the mobile phase θ is the contact angle. Separation Efficiency

6. Partition Coefficient The ratio of the equilibrium concentration of an analyte in the stationary phase divided by its equilibrium concentration in the mobile phase is described by the distribution constant Ka and is represented by the equation Ka=CS/CM CS - concentration of the analyte in the stationary phase CM - concentration in the mobile phase Thus a solute with large Ka has a great affinity for stationary phase and will travel slowly .

7. Retardation Factor Retardation factor ( Rf ) is defined as the amount of separation due to the solvent The position of any solute spot in TLC is characterized by its retention/retardation factor Rf . A fundamental qualitative value and is expressed as

8. Capacity Factor The capacity factor of a substance is defined as “the ratio of its retention time in the stationary phase to that in the mobile phase.” K= ts /tm Ideally, the retention factor for an analyte is between 1 and 5. Thus,higher the capacity factor, the longer the retention time.

9. Spot Capacity The separation number or spot capacity is defined as the maximum number of substances , which are completely separated between Rf =0 and Rf =1, provided that the separation conditions are isocratic. A typical capillary controlled HPTLC has a separation number of 10–20

10. Plate Height M easurement of the efficiency of a chromatographic system is the plate number. It is proportional to the migration length of the mobile phase ( Zf ), If Zs /Ws ratio is constant, an increase in Zf results in an increase of N and better separation . N can also be calculated by the equation :

11. HETP value where H is the so-called HETP value (i.e., height equivalent of a theoretical plate). The quantity H, or simply the plate height, measures the efficiency of a given chromatographic system per unit length of the migration distance, Zf , of the mobile phase.

12. Resolution The separation between two spots is measured by the quantity Rs, and is called resolution. Resolution of two adjacent chromatographic spots 1 and 2 is defined as being equal to the difference between the Rf of both the spots divided by the mean spot widths. Rs =Rf2-Rf1/0:5 (w1+w2)

13. Steps of HPTLC

14. Selection of chromatographic layer Precoated plates of different sorbents - Silica gel 60F : More than 80% of analysis is done on these plates. Aluminum oxide : Basic substances, alkaloids, steroids. Microcrystalline cellulose : Amino acids, sugars, antibiotics. RP-2, RP-8 and RP-18 : chemically modified silica gel plates - analysis of fatty acids, carotenoids , steroids, and cholesterol etc.

15. Plate handling The active layer of the plate is quite delicate – Take special care to avoid any damage or contamination. Use plates without any pre-treatment “out of the box” for most qualitative analysis . Freshly open box of plate do not require activation. Plates exposed to high humidity or kept on hand for long time to be activated. Prior to spotting Aluminium Sheets should be kept in between two glass plates and place in oven at 110 – 120 ◦C for 15 minutes.

16. Prewashing of plate To avoid any possible interference, impurities and water vapors clear the plates before chromatography . This process is called as prewashing. Silica gel adsorbs water vapor upon storage and handling which affects the Activity of the plate Rf value of the analyte Selectivity of the separation Use standardized cleaning procedure for validated analytical method (stability test, quantification), for reproducible results. Low signal-to-noise ratio Straight base line Improvement of LOD

17. Prewashing technique Prewashing is done by various technique like Ascending dipping - Ascending technique takes longer time but cleaning effect is superior to other. The dirt get accumulated at the rear end of the plate. Continuous mode technique. 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. Dry the plate for 20 min in a oven at 120C and store in dust free atmosphere.

18. Sample application The sample preparation is not as demanding as for other chromatographic techniques. Solvent for dissolving sample should be nonpolar and volatile (i.e., methanol, ethanol, or chloroform). Apply the sample as a sharp band using the spray-on technique – for better Resolution and high response to densitometer. Usual concentration range is 0.1–1 mg/ml; above this causes poor separation. Defined Plate layout - for reproducible results and comparability of data from plate to plate.

19. Layout for an HPTLC plate with position of samples (orange) and labels. (a) x-position of first track - 15 mm (b) - min. 10 mm (c) Band length - 8 mm (d) Distance from lower edge of plate for use in TTC - 8 mm (e) Solvent run - 62 mm (f ) Solvent front - 70 mm Minimum space between bands/spots - 2 mm Maximum diameter of application spot - 5 mm Maximum number of tracks on a 10 × 10 cm plate 7 Maximum number of tracks on a 20 × 10 cm plate 16

20. Selection of mobile phase Based on one’s own experience and literature. Poor grade of solvent - decreases resolution, Rf reproducibility and spot definition. A good solvent system is one that does not put anything on the solvent front, but moves all components of the mixture off the baseline. Between Rf 0.15 and 0.85 range. If normal stationary phase is polar and mobile phase selected is nonpolar , then nonpolar compounds are eluted first because of lower affinity with stationary phase and polar compounds retained because of higher affinity with the stationary phase and vice versa. Taking into consideration, sorbent layer mobile phase and the chemical properties of the analyte should be chosen.

21. Preconditioning (Chamber saturation) Chamber saturation has a pronounced influence on the separation profile. Time required for the saturation depends on the mobile phase. Unsaturated chamber- solvent evaporates from the plate mainly at the solvent front; result in increased Rf . Saturated chamber- plate gets preloaded with solvent vapors hence less solvent is required to travel a particular distance, resulting lower Rf . But in some cases depending on their polarity saturation and non-saturation of chambers are required. Filter paper lining for 30 min prior to development in saturation chamber leads to uniform distribution of solvent vapours and less solvent require for the sample to travel.

22. Chamber saturation Place a correctly sized piece of filter paper in the rear trough of TTC and carefully pour prepared mobile phase into chamber so that the filter paper is thoroughly wetted and adheres to rear wall of TTC. Tilt TTC to the side (about 45) so that the solvent volume in both troughs equalizes. Set chamber on bench, replace the lid and let chamber equilibrate for 20 min.

23. Chromatographic development and drying Plates are spotted with sample and air dried Mark the desired developing distance (70 mm from lower edge of plate) with a pencil on the right edge of the plate. Slide off the lid to the side and place the plate into the front trough in such a way that the layer and filter paper should face each other and the back of the plate is resting against front wall of TTC. Replace the lid and develop plate to the mark. Remove plate from chamber and dry it (vertically in the direction of chromatography) for 5 min in a stream of cold air. After each development, remaining mobile phase and filter paper are discarded. Prior to being prepared for the next run, the chamber is dried and, if necessary, also cleaned.

24. HPTLC differs from all other chromatographic techniques in the fact that in addition to a stationary and mobile phase a gas phase is present which can influence the separation. For adsorption chromatography on silica gel, four partially competing processes occur in the closed developing chamber . Chamber saturation & Chromatogram development Saturation pre-conditioning Evaporation Formation of secondary fronts

25. Chamber saturation & Chromatogram development Contd …. Saturation - Between the components of the developing solvent and their vapour phase pre-conditioning - While still dry, the stationary phase adsorbs molecules from the gas phase (adsorptive saturation). This way particularly the polar components will be withdrawn from the gas phase Evaporation - Simultaneously , the part of the layer which is already wetted with mobile phase interacts with the gas phase and, especially, the less polar components of the liquid are given off in the gas phase. Formation of secondary fronts - During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts.

26. Detection Detection under UV light is the first choice as it is nondestructive. 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 plate is derivatized , take images prior and after derivatization .

27. Derivatization Derivatization Derivatization is a procedural technique that modifies functionality of an analyte’s to enable chromatographic separations. Derivatization can be performed by immersing the plates plates within a suitable reagent, f or better reproducibility Heating To induce or optimize the derivatization reaction, it may be necessary to heat the plates. Use plate heater for uniform heating.

28. Quantification Q uantitative evaluation is performed with the TLC Scanner 3 using Vison CATS 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. 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 with a fixed sample light beam in the form of a rectangular slit. Slit Size used is 6× 0.45 mm for 8mm band length of applied sample /std track. The resolution of compounds to be separated on the chromo-plate is followed by measuring the optical density of the separated spots directly on the plate. Densitometry is a simplest way of quantifying sample and standard directly applied on the plate. Concentration of analyte in the sample is calculated by considering the sample initially taken and dilution factors.

29. Qualitative Evaluation with a TLC Scanner For qualitative evaluation of samples with complex compositions, a “fingerprint” method is usually performed using a TLC scanner C reate a spectrum for each spot on the plate. Results are then compared to standard spectra to identify the substances in the sample.

30. A Comparison evaluation of HPLC and HPTLC Criteria HPTLC System HPLC System Validation Relatively simple Relatively simple Documentation Meets all the requirements Meets all the requirements Photo documentation Possible Not possible Sample preparation Very simple and fast; dissolve; centrifuge and supernatant for application Expensive, time consuming, complex, extraction, filtration is essential prior to chromatography Number of CUT’S (content uniformity test) system handled at a time Up to 5 tests of 17 samples each Maximum of 1 sample and 1 test at a time Chromatography time of each CUT (content uniformity test) 45-60 minutes 4-6 minutes Urgent Samples Start analysis any time on receipt of sample, but finish 60 minutes. 1-2 hours start up time and then analysis time Analysis requiring post chromatographic derivatization Simple, additional 10-15 minutes requires after chromatographic separation. Complicated, additional 1-3 hours may be required after chromatography.