HPTLC

Introduction, instrumentation, working , difference between HPTLC and TLC,Hyphenation,

1. High-Performance Thin Layer Chromatography (HPTLC) Presented by:- Jasmine Kaur M.Pharm (I st year)

2. High-Performance Thin Layer Chromatography (HPTLC) A sophisticated and automated form of TLC Useful in qualitative a nd quantitative analysis of natural products The principle of separation is adsorption (same as that of TLC) In HPTLC, the pre-coated plates are used and the particle size of stationary phase is 5-6μm in diameter There is a wide choice of stationary phases like silica gel for normal phase and C18, C8 , etc., for reverse phase mode

3. Provides a higher efficiency than TLC because adsorbents used are small and uniform in size. A very less amount of sample is spotted on the plate so the sample prepared should be highly concentrated. The size of the sample spot should not be more than 1 mm in diameter. The samples are spotted by various techniques and commonly used method is by semiautomatic linomet V apparatus.

4. New types of development chambers are used in HPTLC that require less amount of solvents for developing. A linear development technique is commonly used. The plate is placed vertically in development chambers containing solvent, and chromatogram can be developed from the sides. In HPTLC, UV/VIS/fluorescence scanner is used; therefore, it scans the entire chromatogram qualitatively and quantitatively. The scanner is an advanced type of densitometer . HPTLC is used for the standardization of herbal extracts and other formulations . By using this technique, the analytical profiles of alkaloids, cardenolides , anthracene glycosides, flavonoids , lipids, steroidal compounds, etc., have been developed.

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. This is how components are separated on the chromatographic plate.

6. Instrumentation The HPTLC instrumentation involves mainly Sample applicators, Development chamber, Scanner and Visualizer . Sample applicators : Camag Nanomat (Manual sample applicator) Sample to be spotted is filled in glass capillaries, and precise volume of sample is applied on HPTLC plates in form of spots.

7. 2. Camag Linomat (Semi-automatic applicator) Sample is applied by spraying, by this solvent for sample gets evaporated. This leads to concentration of sample as narrow band leading to higher resolution for qualitative and quantitative analysis. 3. Camag Automatic TLC sampler-4 (ATS) Sample is applied as spot through contact transfer or as a band by sprayon technique. Optional heated nozzle is also there for aqueous samples.

8. Development Chamber : Camag Twin Trough Chamber (TTC) Bottom of chambers is divided into 2 troughs allowing control over gas phase saturation, humidity and pH. They are economical, requires minimum solvent (10-20 ml). 2. Camag Horizontal development chamber It is designed for chromatogram development from both sides of a glass plate. It is rapid and cost effective. About 72 samples can be run simultaneously.

9. 1.HPTLC plate (layer facing down) 2. glass plate for sandwich configuration 3. reservoir for developing solvent 4. glass strip 5. cover plate 6. conditioning tray

10. 3. Camag automatic development chamber 2 (ADC-2) Here there is isocratic development of HPTLC plates. Steps like preconditioning of layer, chamber saturation, development distance and final drying can be preset and automatically monitored independent of environmental effects. 4. Camag Automatic Multiple Development System-2 (AMD-2) Gradient elution with 5 solvents. Plates are repeatedly developed in same direction . After every run the solvent is completely removed from chamber and the layer is dried under vacuum. It is controlled by system manager software. Samples that are complex or have different polarity fractions or a large number of fractions are best resolved by AMD.

11. Camag TLC scanner-4 The most advanced automatic densitometric evaluation of TLC/HPTLC plates can be done by scanning in UV, visible and fluorescence modes of qualitative analysis. High quality chromatograms with well resolved sharp bands can be scanned. For identification and purity check in-situ UV-visible spectra with range of 190-900nm can be performed.

12. Camag TLC Visualizer The Visualizer captures TLC images of best quality with the use of 12- bit industrial camera. It is used mostly in finger-printing and correct identification as well as to detect adulteration. Camag Smart Digi : UV modified cabinet with camera Inspection of plates can be done at every step of HPTLC using UV (254 nm) or fluorescence (366 nm) in a non-destructive manner inside the UV cabinet. There is provision for attachment of consumer digital camera for recording images with this UV cabinet.

13. Camag automatic plate coater Provides coating of glass plates of 20x 20 cm or 20x 10 cm size. Forms layer of adsorbent very uniform. Camag Solvent Front monitor (smart ALERT) With smart ALERT the chromatographer does not 'forget' the TLC plate in the developing chamber anymore: The desired developing distance is set by the user. A light beam and a sensor monitors the selected distance. As soon as the front has reached this position, visual and acoustic signals alert the operator.

14. 1. Camag immersion device The device used for immersion of plates, which provide uniform and quick spreading of the reagent on the plate leading to improved detection. There are no tide marks and it gives better results than spraying. 2. Camag auto reagent sprayer Gives uniform and consistent fine spray. Small pump and rechargeable batteries are present. Used for normal and high viscosity solutions 3. Camag Plate heater It is designed for uniform heating of TLC pre-coated plate to a set temperature. Useful for in-situ derivatisation or for plate activation. Derivatisation

15. The cabinet is made of PVC. The blower, a radial fan driven by a motor outside of the fume duct, produces an airflow of 130 cubic feet (3.7 cubic meter) per minute. It ensures the complete removal of reagent mist while spraying TLC plates. 4. CAMAG TLC Spray Cabinet 5. CAMAG Derivatizer It sets a new standard of reproducibility and convenience in reagent transfer onto TLC/HPTLC plates by employing a unique “micro droplet” spraying technology (patent pending). The device is suitable for all common reagents.

16. The process steps of HPTLC are identical to classical TLC. The main difference between them is in the characteristics of the separation plate. HPTLC plates are based on optimized silica gel 60 with a significantly smaller particle size than used for classical TLC. This allows a higher packing density and a smoother surface. Hence, sample diffusion is reduced, resulting in compact bands or spots. Furthermore, the smaller particle size and thinner layer significantly increase detection sensitivity and analysis speed. HPTLC is a modern adaptation of TLC with better and advanced separation efficiency and detection limits. DIFFERENCE BETWEEN HPTLC AND TLC

17. CAMAG is the world leader in instrumental Thin-Layer Chromatography (TLC/HPTLC ) + Since 1961, CAMAG is dedicated to the development and manufacturing of instruments, software and consumables for all steps of the TLC/HPTLC procedure. + Their products are truly Swiss made and have an excellent reputation throughout the world . + CAMAG has its headquarters in Switzerland and daughter companies in Germany and in the United States. In these countries CAMAG sells its instruments directly to end users, in other markets - presently in over 70 countries

18. TLC/HPTLC Thin-Layer Chromatography They develop, manufacture and market sophisticated instruments and products as well as software for all steps of instrumental Thin-Layer Chromatography TLC/HPTLC :- Sample application Chromatogram development Derivatisation Densitometric evaluation UV Detection Documentation DBS Dried Blood Spot Sampling CAMAG Switzerland has developed a system for the automated extraction of Dried Blood Spot (DBS) cards that can be coupled directly to LC-MS systems. Dried blood spot (DBS )  analysis has been known for more than five decades. It is an easy way of collecting, shipping and storing blood samples. In recent years the usage of dried blood spots (DBS) has gained increasing importance since this method shows strong advantages compared to the conventional collection and analysis of blood or plasma samples.  ALOX Aluminium Oxide CAMAG has manufactured Aluminium Oxide for 50 years. The product, widely used in the pharmaceutical and chemical industries in purification processes is of excellent quality due to the fact that CAMAG has well-trained employees, has a reliable supplier of the raw material, and a standardized manufacturing process. SERVICES The company CAMAG has been certified according to the following standard(s): ISO 9001   PRODUCTS PRODUCED

19. ALOX Aluminum Oxide CAMAG has manufactured Aluminum Oxide for 50 years. It is widely used in the pharmaceutical and chemical industries in purification processes and chromatography. It is of excellent quality due to the fact that CAMAG has well-trained employees, has a reliable supplier of the raw material, and a standardized manufacturing process.

20. It was established in 2006 to meet the varied requirements in labs, research institutes and educational fraternity with precision products at competitive prices. Some of their range of instruments include spectro, laboratory & scientific analytical instruments like UV Cabinet, HPTLC Documentation Systems, TLC Applicator, HPTLC scanners.

21. WORKING Sample and Standard preparation Selection of chromatographic layers Plates Pre-washing Conditioning Sample Application Pre- conditioning Mobile phase Chromatographic Development Detection and Spot Scanning and Documentation

22. SAMPLE AND STANDARD PREPARATION It need a high concentrated solution as very less amount of sample need to be applied. Normal phase chromatography :- we use silica gel pre-coated plates. So, we use non-polar solvent for dissolving sample. Reversed phase chromatography :- we usually use polar solvents for dissolving the sample. Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Chloroform: Methanol: Ammonia (90:!0:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid 

23. SELECTION OF CHROMATOGRAPHIC LAYERS Stationary Phase - Precoated plates with different support materials (Sorbents) available:- Silica gel GF - 80% of analysis is done Aluminum oxide - Basic substances, alkaloids and steroids Cellulose - Amino acids, dipeptides , sugars and alkaloids RP2, RP8 and RP18 - polar substances, fatty acids, carotenoids, cholesterol Hybrid plates-RPWF254s - Preservatives, barbiturates, analgesic and phenothiazines

24. PLATES Precoated plates and hand made plates are used, nowadays hand made plates are not used much . 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. Different Plate sizes are available 20X20cm 10X20cm 5X10 cm 5X7.5 cm Good cut edges of sheets is important to obtain constant Rf values.

25. PRE- WASHING Pre washing is for pre coated plates The main purpose of the pre-washing is to remove impurities which include water vapours 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. Some common methods involved in pre-washing :-Ascending method ,Dipping method, Continuous method (multiple overrun). Solvents used for pre-washing :-Methanol Chloroform: methanol ( 1:1 ) ,Chloroform: Methanol: Ammonia (90:10:1 )

26. CONDITIONING Also called chamber saturation Unsaturated chamber causes high Rf values Unsaturated chamber is responsible for causing promblems like tailing and edge effect. Sample Application usual concentration of applied samples 0.1 to 1 µg / µl for qualitative Analysis and quantity may vary in quantitation based on UV absorption 1 to 5 µl for spot and 10 µL for band application MANUAL , SEMI-AUTOMATIC , AUTOMATIC APPLICATION Manual with calibrated capillaries Semi and auto-application through applicators Applicators use spray on or touch and deliver technique for application.  SAMPLE APPLICATOR

27. Selection of mobile phase •Trial and error •one’s own experience and Literature •3 - 4 component mobile phase should be avoided •Multi component mobile phase once used not recommended for further use. •Twin trough chambers are used only because 10 -15 ml of mobile phase is required MOBILE PHASE Normal phase - Stationary phase is polar and mobile phase is non polar hence non-polar compounds are eluted first because of lower affinity with stationary phase and polar compounds retained because of higher affinity with the stationary phase. Reversed phase - Stationary phase is non polar mobile phase is polar hence polar compounds are eluted first because of lower affinity with stationary phase and non-polar compounds are retained because of higher affinity with the stationary phase .

28. DEVELOPING CHAMBER 1.Twin trough 2.Automatic developing chamber 1.TWIN TROUGH CHAMBER :- Low solvent consumption: 20 mL of solvent is sufficient for the development of a 20x20cm plate. This not only saves solvent , but also reduces the waste disposal problem . CHROMATOGRAPHIC DEVELOPMENT 2. AUTOMATIC TROUGH CHAMBER :- In the ADC this step is fully automatic and independent of environmental effects. The activity and pre-conditioning of the layer , chamber saturation, developing distance and final drying can be pre-set and automatically monitored by ADC . 

29. DETECTION AND SPOT 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 , dicyloamine etc can be detected by dipping the plates in 0.1% iodine solution. Detection can be done by :- Spraying , dipping , and derivatization through gas phase Quantification •Sample and standard should be chromatographed on same plate after development chromatogram is scanned. •Concentration of analyte in the sample is calculated by considering the sample initially taken and dilution factors.

30. SCANNING The scanner is connected to a computer. The scanner features three light sources ,a deuterium lamp , a tungsten lamp and a high pressure mercury lamp. The scanning speed is selectable between 1 and 100 mm/s Densitometry measurements measures visible, UV absorbance or Fluorescence which converts the spot/band into chromatogram consisting of peaks. Sample and standard should be chromatographed on same plate after development chromatogram is scanned . Camag TLC scanner III scan the chromatogram in reflectance or in transmittance mode by absorbance or by fluorescent mode - scanning speed is selectable up to 100 mm/s - spectra recording is fast - 36 tracks with up to 100 peak windows can be evaluated .

31. DOCUMENTATION A computerized microchip and software control (win CATs) the HPTLC instrument and gives information data.

32. Journey of TLC Meinhard and Hall (1949) used a starch binder to give some firmness to the layer and described as surface chromatography . Kirchner (1951) used ascending method using silicic acid as adsorbent. Reitsema (1954) used much broader plates, was able to separate several mixtures in one run. Stahl (1956) used silica gel as stationary phase since then TLC was introduced as analytical procedure. Stahl (1965) highlighted the importance of factors affecting TLC like layer thickness , layer uniformity, pore size, particle size etc. Modifications to the silica gel began with silanization to produce reversed-phase layers. TLC was recognized as a quick, relatively inexpensive procedure for the separation of a wide range of sample mixtures. Introduction of commercial spectrodensitometric scanners that enabled the quantification of analytes directly on TLC layer.

33. In 1973 , advantage of using a smaller average particle size of the silica gel (5-6mm) in the preparation of TLC plates was recognized . Effect of particle size on development time, Rf values and plates were compared. Commerically HPTLC were first called nano - TLC plates but soon name was changed to HPTLC which added new dimension to TLC. HPTLC in 1977 was recognized as a technique which required less amount of mobile phase, precision (tenfold),and less analysis time. Later on, reversed phase HPTLC was introduced by scientist Halpaap . The era of 1980s brought improvements in spectro-densitometric scanners with full computer control. Options for peak purity and the measurement of full UV/ visible spectra for all separated components were introduced.

34. Automatic Multi- development chamber (AMD) appeared because of work of scientist Burger (1984) which has enabled a marked increased in separation and resolution of complex substances. In Image analysis video densitometry was introduced where optical scanning takes place electronically, using a computer with video digitizer, light source, monochromators , and appropriate optics to illuminate the plate and focus the image onto a charge-coupled video camera. Two-dimensional separations employs two or more coupled orthogonal separation systems represent the approach in chromatography to obtain high peak capacity for the separation of complex mixtures .

35. HPTLC- Hyphenation Combinatorial approach (hyphenation) has got significant recognition compared to a traditional one compound at a time approach. HPTLC has been successfully hyphenated with HPLC, MS, FTIR and Raman spectroscopy to give far more detailed analytical data on separated compounds. HPLC-HPTLC - Coupling is based on different modification to the spray- jet applicator . The column eluent is nebulized by mixing with nitrogen gas and sprayed as an aerosol onto the layer. The spray head is moved horizontally on one line within a defined bandwidth. HPTLC-MS - The evolution of HPTLC-MS has slow process. Dr. Luftmann developed HPTLC-MS hyphenation. The technique is divided into elution based and desorption based . Elution based – Analyte on the plate is first scraped, extracted, purified and concentrated, then transferred in the liquid phase to mass spectrometer ion source for further analysis.

36. Desorption based - Analyte is vapourized from the silica, and transferred to the mass spectrometer in the gas phase. But these days substance of interest is eluted directly from the HPTLC plates and transferred online to the mass spectrometer . Uses - 1. It has been successfully used for the identification and quantification of amino acid in peptides. 2. Fast identification of unknown impurities. 3. Problem solving technique in pharmaceutical analysis, identification of botanicals. 4. Screening for bioactive natural products in sponges, determination of ginkgolides A,B, and C.

37. HPTLC-FTIR - IR spectroscopy has a high potential for the elucidation of molecular structures and characteristic absorption bands. HPTLC- IR has been divided into direct and indirect coupling . Indirect coupling - It involves either the transfer of the substance from a TLC spot to the non- absorbing IR material ( KBr or KCl ) or insitu measurement of excised HPTLC spots when the spectra are recorded directly from the plate. Direct coupling - It is direct online HPTLC-FTIR coupling. There is ease in operation and optimization of the process. This coupled method have been in use in modern laboratories for qualitative and quantitative analysis. It is having potential applications in the fields of analysis such as drug, forensic , food, environment and biological analysis etc. HPTLC-UV - When HPTLC was coupled with UV it was only on-line coupling method available that time. Information content of UV- vis spectra is rather poor and rarely provides identification of substance .

38. For detection moreover, there is requirement of chromophore for detection in UV. HPTLC-FTIR marks over it has it makes it possible for detection and quantification of even non-UV absorbing substances on HPTLC plates. HPTLC- Laser - MALDI and APCI has been coupled and this combination has been incorporated into HPTLC- MS system. Benefits - 1. Simple transfer of analyte molecules 2. High resolution 3. Less fragmentation under atmospheric pressure Drawbacks - 1. High cost 2. Size of laser is big, not ideal for miniaturization of the whole analytical system.

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