THIN LAYER CHROMATOGRAPHY TYBSC Analytical Chemistry THIN LAYER CHROMATOGRAPHY Dr.Bhagure G.R.

Contents 2.2.1 Principle of Thin Layer Chromatography (TLC), Retardation factor Rf. 2.2.2 Techniques in Thin Layer Chromatography A) Stationary phase coating materials B) Preparation of Plates C) Mobile phase : Selection of Solvents D)Methods of Plate Development Ascending development, Descending Development, Two dimensional Development, Continuous Development, and Multiple Development. E) Detection methods for locating separated components: Qualitative analysis and Quantitative analysis. 2.2.3 Applications of Thin layer chromatography

THIN LAYER CHROMATOGRAPHY Principle Materials used to perform TLC Experimental technique Application Advantages Disadvantages

Principle: The sample is dissolved in in a volatile solvent Sample is applied with the help of capillary on to the base line drawn on the solid adsorbent. The plate is dipped in to the solvent working as mobile phase. As the mobile phase rises up the TLC plate by capillary action, the components dissolve in the solvent and move up the TLC plate. In adsorption separation will occur on the basis of differences in adsorption (preferential adsorption). Weekly adsorbed component will separate first and strongly later on. In partition , if the component is soluble in the mobile phase it will move with the solvent and vise versa . The movement of the solute is measured relative to that of solvent is expressed as the retardation factor.

Polarity decreases This technique manipulates POLARITY More polar substances bind strongly to the adsorbent and elute SLOWER Less polar substances bind weakly to the adsorbent and elute FASTER The strength of interactions between the adsorbent and eluting components vary approximately in this order: Salt formation > coordination > H-bonding > dipole-dipole > van der Waals Less Polar More Polar Polarity decreases

OH OH Silica Gel OH Silica Gel OH OH OH Mobile phase OH

Sample to be applied on this area Less Polar More Polar Adsorbs weakly and separate very fast Adsorbs stronger and separate very slowly Sample to be applied on this area

. distance traveled by substance . distance traveled by solvent front The Rf Value A given compound will always travel a fixed distance relative to the distance the solvent travels This ratio is called the Rf value and is calculated in the following manner: . distance traveled by substance . distance traveled by solvent front

THIN LAYER CHROMATOGRAPHY Calculation of Rf’s The Rf is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin)

Oven for activation of plate Developing chamber Mobile Phase Materials used in TLC Glass Plate Adsorbents Oven for activation of plate Developing chamber Mobile Phase A device for applying the adsorbent layer Storage facility for the prepared plate

A device for applying the adsorbent layer Materials used in TLC Glass Plate Hooper A device for applying the adsorbent layer Mobile phase Developing chamber

Alumina (Al2O3) Stationery phase Stationery phase Description Application Silica gel G Silica gel with average particle size 15µm containing ca 13% calcium sulfate binding agent Used in wide range pharmacopoeial test Silica gel G254 Silica gel G with fluorescence added Same application with Silica gel G where visualization is to be carried out under UV light. Alumina (Al2O3) Cellulose Cellulose powder of less than 30µm particle size. Identification of tetracycline's

MOBILE PHASE TLC Solvents or Solvent Systems. A single solvent or mixture of two solvents can work as mobile phase in TLC .Ex. petroleum ether, carbon tetrachloride, chloroform, ethyl acetate, hexane can used as mobile phase. The ability of mobile phase to move up is depend on the polarity itself Volatile organic solvents is preferably used as mobile phase.

MOBILE PHASE SOLVENT POLARITY INDEX Heksana Butanol 3.9 Chloroform 4.1 Butanol 3.9 Chloroform 4.1 Methanol 5.1 Ethanol Acetonitrile 5.8 Air 9.0 Water

Experimental Procedure Thin layer Chromatography

Methods used to apply adsorbent Dipping plate in slurry TLC Plate Preparation Methods used to apply adsorbent Spreading Spraying Dipping plate in slurry

TLC Plate Preparation TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. They are prepared by mixing the adsorbent, such as silica gel , with a small amount of inert binder like calcium sulphate (gypsum) and water. This mixture is spread as thick slurry on an uncreative carrier sheet, usually glass, thick aluminum foil, or plastic. The thickness of the adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes Around 0.5 – 2.0 mm for preparative TLC.

Spreading the slurry by Hooper Materials used in TLC Glass Plate Spreading the slurry by Hooper Mobile phase Developing chamber

Activation of plate Glass Plate Plate is kept for drying in oven at 100OC. This step is called as activation of plate. By doing this surface area of the adsorbent increases.

Drawing a Line and circle to apply the sample Glass Plate Circle to apply sample Mobile phase Developing chamber

Experimental Procedure TLC Chamber Preparation Cut the filter paper so that it fits in the jar, touching the bottom and reaching a height of about 1cm from the top of the jar To ensure that the filter paper will work, put it in the jar, and then place an unused TLC plate in the jar. If the above criteria are met and the plate doesn’t make any contact with the filter paper, the setup should work Remove the TLC plate, and then completely saturate the filter paper with the development solvent using a pasteur pipet. Fill the jar with development solvent to a depth no greater than 0.5cm Put the lid on the jar to preserve the saturated conditions

Application of sample Spotting the TLC Plate Dip the open end of a capillary tube into the solvent containing the compound to be eluted Touch the end of the capillary tube lightly and very briefly to the coated surface of the TLC plate Your spots should be made on the line drawn across the plate in the correct lanes and shouldn’t have a diameter much larger than the capillary tube After spotting the plate, place it in the saturated chamber and close the lid Substances should be eluted until the solvent front reaches a height of about 0.5cm from the top of the TLC plate

Materials used in TLC Glass Plate Circle to apply sample Mobile phase Developing chamber

Development of Chromatogram Chromatoplate is kept in a tank at an angle 45o The bottom the tank is nearly covered up to 1 mm by solvent. Three sides of tanks are lined with solvent saturate paper. The top of the tank is covered tightly. Solvent moves up and separation takes place in ascending way with in few minutes. Plate is removed and dried. The separated components are located by either physical or chemical method.

Methods of Plate Development Ascending development Descending Development Two dimensional Development

Application of sample and development of Chromatogram by Ascending way High Polar Less Polar

development of Chromatogram by Descending way High Polar Less Polar

Development of chromatogram by descending way Mobile Phase

Two dimensional chromatography Sample A A B B X X AC is immersed in mobile phase Solvent AB is immersed in mobile phase C C Solvent front

TLC Visualization Methods Physical Methods: Ultraviolet Light—some organic compounds illuminate or fluoresce under short-wave UV light:

Chemical methods Iodine Vapor—forms brown/ yellow complexes with organic compounds Fluorescent Indicators—compounds fluoresce when placed under UV light Silver Nitrate Spray (for Alkyl Halides)—dark spots form upon exposure to light Sulfuric Acid Spray + Heat—permanent charred spots are produced

Applications of TLC Qualitative analysis: - If the separated components are colored then identification is very easy. All the visualizing agents used in paper chromatography (Detecting agents or indicators) can be used in TLC. From Rf value qualitative analysis can be performed.

Quantitative analysis The size of the spot increases with the amount. Square root of the spot area is find out from that amount of solute can be found out. Potentiodensitometry of the plate is carried out. Flurometry or emission Spectroscopy is also used. Separated spot is removed by knife edge .Its dissolved in proper solvent and its amount is finding out by volumetric analysis.

Other Applications of TLC TLC can is applicable in the field of medicinal preparations, pharmaceutical preparations, natural product extract and related compounds. Assaying the radiochemical purity of radiopharmaceuticals. Determination of the pigments in plants. In forensic science Laboratory detection of pesticides and insectides in food, poison etc.

Other Applications of TLC:- Analyzing the dye composition of fibers in forensic study or Identifying compounds present in a given substance For Monitoring organic reactions. In clinical study to carry out qualitative and quantitative analysis of biological and metabolic samples to detect disease. Semi quantitative analysis can also performed by extracting the spot in suitable solvent and it’s determined by volumetric analysis or any other instrumental technique.

Advantages of TLC over Paper Chromatography: Separation is sharper in TLC than paper Chromatography. TLC is much more rapid The use of inorganic layer eliminates background organic effects in spectroscopic analysis. More reactive reagents like Sulphuric acid can be used. The separated spots are more distinct hence detection methods are more sensitive.

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