HpTLC power

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(HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY) Presented by AACP HPTLC By Pradeep Phr.pradeep@yahoo.com

Definition: : 

Definition: Chromatography is a physical process of separation in which the components to be separated are distributed between 2 immiscible phases-a stationary phase which has a large surface area and mobile phase which is in constant motion through the stationary phase.

Introduction: : 

Introduction: 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.

Differences between TLC and HPTLC: : 

Differences between TLC and HPTLC:

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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 OF CHROMOPLATE USING PC CATS SOFTWARE

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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.

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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.

Supports : 

Supports

Some of the sorbents used in HPTLC: : 

Some of the sorbents used in HPTLC:

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Some of the binders used: Gypsum (G) Starch (S) Layer containing fluorescent indicator (F)

Plate size: : 

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

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Pre washing of 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: 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.

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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.

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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. The wanted plates should always be stored in a dust free atmosphere, under ambient conditions. Usually desiccators of suitable size are used for storage of plates.

Solvents used for pre-washing : 

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%)

Activation of plates: : 

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.

Sample Preparation: : 

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.

Application of sample: : 

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.

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1.It is the most critical step for obtaining good resolution for quantification by HPTLC. 2.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.

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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.

Advantages of application of sample as band are : 

Advantages of application of sample as band are Better separation because of rectangular area. Response of densiometer is higher in case of band than that observed from an equal amount/equal volume of sample applied as a spot. Large quantities of the sample can be handled for application, thus reducing the need of concentrating which may be damaging in case of liable substances. Position of plate for densitometric scanning is less critical as composition of the compounds is uniform in the entire area of band.

Automatic applicators used: : 

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 0.1-0.2μl.

2) CAMAG Linomat : 

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.

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Glass: Borosilicate Precision: <+/- 1% of volume Needle: Especially developed for the need of linomat III and IV

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3) CAMAG automatic TLC sampler III : Applies sample as spot or bands automatically from the rack of sample vials.

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Heated nozzle of the ATS4 (option)Raising the temperature to 60 °C reduces the time for application of an aqueous sample to about one halve. Especially in trace analysis the use of an ATS4 with heated nozzle is advantageous because large volumes usually have to be applied in order to increase detection sensitivity

Mobile phase : 

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

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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.

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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.

Development of chambers: : 

Development of chambers: 1.Twin trough chamber.

2.Rectangular chambers : 

2.Rectangular chambers

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3. V-shaped chambers 4.Sandwitch chamber 5.Horizontal development chamber 6.Automatic development chamber It gives complete control over development process , chamber saturation , developing distance and drying step as well as activity of HPTLC. Plates are controlled and guarantee the results day in and day out with documentation system called digistore-2 where images of HPTLC chromatogram can be captured within seconds

Pre-conditioning : (Chamber Saturation) : 

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.

Slide 39: 

If tank is saturated prior to the development , solvent vapors 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:

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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 leading to 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.

Development and Drying: : 

Development and Drying: The different methods used for development of chambers are like-Ascending , descending .2-dimentional, horizontal , multiple overrun , 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

Drying : : 

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.

Factors influencing separation and resolution of spots: : 

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

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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

Detection and visualization : 

Detection and visualization One of the characteristic feature of HPTLC is the possibility to utilize post-chromatographic off line derivatization Detection are of two types: Qualitative Quantitative

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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: Quantitative of the chromatogram by HPTLC basically involves direct and indirect methods;

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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.

Densiometry; : 

Densiometry;

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It is a instrumental measurement of visible , UV absorbance , fluorescence quenching directly on the layer without resorting to scrapping and elution It involves resolving of a compounds on thin layer plate , visualizing the spots and measuring the optical density of each spot / band directly on the plate. The amount of material / compound in the unknown are measured by comparing them to a standard curve from reference standard chromatographed with the same condition. Chromatographic zones remit a lower light intensity than the environment around it. Absorption spectra can be directly determined on the plate by comparison with substance free area of sorbent layer .

Slide 51: 

Measurements are usually made by reflection from the plate using single beam ,double beam or single-beam dual wavelength operation of scanning instrument. the scanner present converts the spot / band on the Layer into chromatogram consisting peaks Position of scanned peaks on recorder chart are related to Rf values of the spots on the layer and peak height or area is related to the concentration of the substance on spot. Signals which are measure represent the adsorption of transmitted or reflected light passes through the spot compared to blank portion of sorbent layer.

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Instrumentation

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It basically consists of : Light source Visible Radiation UV Radiation Wave length selection device Condensing and focusing system Photo-sensing detectors

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Theory The transmission of light in a translucent material can be described by: Io=Ia +It+Ir+Ix Where Io=Intensity of incident light Ia=Intensity of absorbed light Ix=Intensity of transmitted light Ir=Intensity of reflected light

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The densitometer work by 2 modes: 1. Transmission mode 2. Reflectance mode

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In transmission mode the ratio of It/Io is measured and converted in to absorbance values In reflectance mode the ratio Ir/Io is measured and converted in to absorbance values But it was found that transmission measurements are more sensitive than reflectance measurements.

Advantages of densitometer /Scanner : 

Advantages of densitometer /Scanner The purpose of scanner is to convert the spot /band on the layer in to densitogarm consisting of peaks similar in appearance to HPLC. The position of the scanned peaks on recorder chart is related to Rf values. Peak height/area is related to the concentration of the substance in the spot. Quantitation is faster , reliable ,accurate and reproducible Photodocumentation of HPTLC plates is possible.

Documentation: : 

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 data, but will also avoid manipulation of data at any stage as coding will automatically get recorded using photo-documentation.

Applications of HPTLC: : 

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

References: : 

References: HPTLC- Quantitative analysis of pharmaceutical Formulations by P.D. Sethi www.pharmainfo.net http://images.google.co.in/images?q=hptlc+plates&ie=ISO-8859-1&hl=en http://images.google.co.in/images?svnum=10&hl=en&lr=&ie=ISO-8859-1&q=linomat www.camag.com http://www.infoexpo.ch/abstract

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