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It is also known as planar chromatography or Flat-bed chromatography . Traditional Thin Layer Chromatography & its modern instrumental quantitative analysis version HPTLC are very popular for many reasons such as visual chromatogram, simplicity, multiple sample handling, low running and maintenance costs, disposable layer etc . INTRODUCTION


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. The component with lesser affinity towards the stationary phase travels faster. Thus the components are separated on a chromatographic plate. PRINCIPLE

Steps Involving in HPTLC:

Steps Involving in HPTLC 5 Sample Preparation Selection of chromatography layer Pre-washing Pre-conditioning Application of sample Chromatography development Detection of spots Scanning & documentation

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6 Sample and Standard Preparation Solvents used are Methanol, Chloroform: Methanol (1:1), Ethyl acetate: Methanol (1:1), Chloroform: Methanol: Ammonia (90:10:1), Methylene chloride : Methanol (1:1), 1% Ammonia or 1% Acetic acid .

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Selection of chromatographic layer Precoated plates - different support materials - different Sorbents available. 80% of analysis uses - silica gel GF · Basic substances- A lkaloids and steroids - Aluminum oxide Amino acids, dipeptides , sugars and cellulose Non-polar substances, fatty acids, carotenoids 7

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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. 8


Supports: Glass polyester sheets Aluminium sheets

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10 Some of the sorbents used in HPTLC : Silica gel 60F (Unmodified ) Alluminium oxide Cellulose (microcrystalline ) Silica gel chemically modified

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Some of the binders used Gypsum (G) Starch (S) Layer containing fluorescent indicator (F) 11

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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. 12

Plate size :

20X20cm 10X20cm 5X10 cm 5X7.5 cm Good cut edges of sheets is important to obtain constant Rf values . 13 Plate size

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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. 14

Some common methods involved in pre-washing :

Ascending method Dipping method Continuous method 15 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 . Ascending method:

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. Dipping 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. 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 . Continuous method :

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 %) 19 Solvents used for pre-washing

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 110 o -120 o c for 30 min prior to the sample application. 20 Activation of plates


Pre-conditioning Also called Chamber Saturation Unsaturated chamber causes high Rf values Sample application Usual concentration range is 0.1-1µg / µl Above this causes poor separation sample and standard application from syringe on TLC plates as bands Band wise application - better separation

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22 Some applicators used for application of sample a) Capillary tubes. Samples applied in the form of spots. Volume of 0.1-0.2μl b) Micro bulb pipettes.

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c) Micro syringes. Sample can apply either as spot or band Volume- 1μl. d)Automatic sample applicator. Sample can apply either as spot or band.


H P T L C DEVELOPMENT Vertical Development. Vario method development. Horizontal development. Automatic Multiple Development (AMD)/( Gradient ). 24

Twin Trough Chambers:

Twin Trough Chambers 25

Vario Chamber Development:

Vario Chamber Development 26 VARIO CHROMATOGRAM

Horizontal Development:

Horizontal Development 27 HPTLC plate is developed from both opposing sides towards the middle. Plate sizes 10x10cm and 20x10cm

Automatic Multiple Development(AMD):

Automatic Multiple Development(AMD) 28

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Post Chromatography Steps 1) Detection. Photo documentation. Densitometry measurements. 29

Detection :

Detection UV CABINET 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

Densitometry measurements:

Densitometry measurements 5/21/2013 31 Measures visible, UV absorbance or Fluorescence. Convert the spot/band into chromatogram consisting of peaks

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Instrumentation of HPTLC consists of following: Lamp selector Entrance lens slit Monochromator entry slit Grating Mirror Slit aperture disc Mirror Beam splitter Reference photo multiplier Measuring photo multiplier Photo diode for transmission measurements.



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Theory: According to the theory, the transmission of light in a translucent material can be described by: I0 = Ia + It + Ir + Ix Where, I0 = Intensity of incident light. Ia = Intensity of absorbed light. It = Intensity of transmitted light. Ir = Intensity of reflected light. Ix = Intensity of light lost due to scattering.

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

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Transmission mode:- In transmission mode the ratio of It/Io is measured and converted in to absorbance values Reflectance:- In reflectance mode the ratio Ir/Io is measured and converted in to absorbance values According to Goodman & Goodall transmission measurements are more sensitive than reflectance measurements

Fluorescence measurement in densitometry::

1) Measurement of direct fluorescence 2) Measurement of fluorescent quenching . 1. Direct fluorescent measurement: This method is followed if the spot exhibit fluorescence when exposed to UV light . In this two monochromators are used for selection of excitation & emission wavelength. The fluorescence is measured in reflectance mode. Fluorescence measurement in densitometry:

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2 ) Fluorescence quenching measurement: As the name indicates, it utilizes the ability of analyte to absorb & quench fluorescence light. In this technique fluorescent background is incorporated into the layer. When excited by short wavelength radiation the plate fluorescence's uniformly . If UV absorbing substance is present in the plate, a portion of the fluoresced light is absorbed & consequently quenched. This fluorescence diminution is measured as a function of amount of analyte in the spot. .

Advantages of densitometer scanner: :

The purpose of scanner is to convert the spot/band on the layer into densitogram consisting of peaks similar in appearance to HPLC. The position of the scanned peaks on the recorder chart is related to Rf values. Quantitation is faster, reliable accurate & reproducible Advantages of densitometer scanner:

Photo-documentation With Digital Camera:

Photo-documentation With Digital Camera 40


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 . 7)DOCUMENTATION:

Documentation :

video scan software for quantitative evaluation of images capture with digistore Documentation

Differences between TLC and HPTLC:

Differences between TLC and 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 Q.C. check, validation etc. Forensic : Poisoning investigations. Applications of HPTLC

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QUANTITATIVE DETERMINATION : 1 ) Biochemical research/Biotechnology- Seperation of gangliosides 2) Clinical- Inorganic & organic mercury in water & human serum. Caffeine in urine. 3) Cosmetics- Hydrocortisone & cinchocaine in lanolin ointment 4) Environmental Analysis- Pesticides in drinking water. Selenium in water. 5) Food analysis- Vitamin C in fruit juices. Aflatoxins in food stuff

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6) Pharmaceutical & chemical substance- Content uniformity test of diclofenac sodium . Vitamin B1 pharmaceutical products. 7) Natural products ,plant ingredients- Glycosides in herbal drugs. Glycyrrhizic acid in liquorice. 8) Doping analysis- Atenalol in urine. B) FINGER PRINT ANALYSIS- HPTLC finger print of Valerian. Finger print of garlic , Ashwaganda . Finger prints for identification of liquorice, ginseng .

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9) Identification and separation of phenyl thiohydantoin -amino acid. 10) analysis of drugs in blood EX : 1)separation of phenothiazine drugs like chlorpromazine, acetophenazine, perphenazine, trifluperazine and thoridazine. 11) identification of mycotoxins in admixture : EX: detection of sterigmatocystin, zearalenone, citrinin, ochrotoxinA , patulin, penicillic acid. 12) determination of polycyclic aromatic hydrocarbons in particulate sample. EX ; determination of chryesene , pyrene, fluoronthene etc.

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