Thin-layer chromatography

Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures.[1] Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.[2] The mobile phase has different properties from the stationary phase. For example, with silica gel, a very polar substance, non-polar mobile phases such as heptane are used. The mobile phase may be a mixture, allowing chemists to fine-tune the bulk properties of the mobile phase.

After the experiment, the spots are visualized. Often this can be done simply by projecting ultraviolet light onto the sheet; the sheets are treated with a phosphor, and dark spots appear on the sheet where compounds absorb the light impinging on a certain area. Chemical processes can also be used to visualize spots; anisaldehyde, for example, forms colored adducts with many compounds, and sulfuric acid will char most organic compounds, leaving a dark spot on the sheet.

To quantify the results, the distance traveled by the substance being considered is divided by the total distance traveled by the mobile phase. (The mobile phase must not be allowed to reach the end of the stationary phase.) This ratio is called the retention factor or Rf. In general,a substance whose structure resembles the stationary phase will have low Rf, while one that has a similar structure to the mobile phase will have high retention factor. Retention factors are characteristic, but will change depending on the exact condition of the mobile and stationary phase. For this reason, chemists usually apply a sample of a known compound to the sheet before running the experiment.

Thin-layer chromatography can be used to monitor the progress of a reaction, identify compounds present in a given mixture, and determine the purity of a substance. Specific examples of these applications include: analyzing ceramides and fatty acids, detection of pesticides or insecticides in food and water, analyzing the dye composition of fibers in forensics, assaying the radiochemical purity of radiopharmaceuticals, or identification of medicinal plants and their constituents [3]

A number of enhancements can be made to the original method to automate the different steps, to increase the resolution achieved with TLC and to allow more accurate quantitative analysis. This method is referred to as HPTLC, or "high-performance TLC". HPTLC typically uses thinner layers of stationary phase and smaller sample volumes, thus reducing the loss of resolution due to diffusion.

1. Thin Layer Chromatography (TLC) By: AMIYA KUMAR GHOSH B.pharm,7 th sem,4 th year Roll No. : 27701912002 Registration No. : 122770210002 OF 2012-2013 NSHM Knowledge Campus-Kolkata, Group of Institutions

2. Chromatography : Chromatography is the physical separation of a mixture into its individual components . We can use chromatography to separate the components of inks and dyes , such as those found in pens, markers, clothing, and even candy shells. Chromatography can also be used to separate the colored pigments in plants or used to determine the chemical composition of many substances.

3. Gas Chromatography Used to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below. Paper Chromatography Can be used to separate the components of inks, dyes, plant compounds (chlorophyll), make-up, and many other substances Liquid Chromatography Used to identify unknown plant pigments & other compounds. Thin-Layer Chromatography Uses thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances. Examples of Chromatography

4. TLC: Thin layer chromatography (TLC) is an important technique for identification and separation of mixtures of organic compounds. In TLC, components of the mixture are partitioned between an adsorbent (the stationary phase , usually silica gel, SiO 2 ) and a solvent ( the mobile phase ) which flows through the adsorbent.

5. Why use TLC: Identification of components of a mixture (using appropriate standards) following the course of a reaction, analyzing fractions collected during purification, analyzing the purity of a compound.

6. Advantages : Easy to use Cheap Possible multiple analysis Possible recovery of the products No sample preparation required 2-dimensional analysis Drawbacks: Slow ( typically 30-60 minutes) Limited quality of the separation Limited reproducibility Evaporation of the mobile phase (composition varies during the analysis )

7. Difference between TLC and HPTLC

8. Principle : As the mobile phase rises up the TLC plate by capillary action, the components dissolve in the solvent and move up the TLC plate. Individual components move up at different rates, depending on intermolecular forces between the component and the silica gel stationary phase and the component and the mobile phase. The stationary phase is SiO 2 and is very “ polar ”. It is capable of strong dipole-dipole and H-bond donating and accepting interactions with the “ analytes ” (the components being analyzed). More polar analytes interact more strongly with the stationary phase in move very slowly up the TLC plate. By comparison, the mobile phase is relatively nonpolar and is capable of interacting with analytes by stronger London forces , as well as by dipole-dipole and H-bonding. More nonpolar analytes interact less strongly with the polar silica gel and more strongly with the less polar mobile phase and move higher up the TLC plate.

9. STATIONARY PHASE Silica is commonly used as stationary phase The separation of sample mixture will be depend on the polarity of sample . Some modified silica is also used in certain purposes. 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 GF 254 Silica gel G with fluorescence added Same application with Silica gel G where visualization is to be carried out under UV light. Cellulose Cellulose powder of less than 30µm particle size Identification of tetracyclines

10. MOBILE PHASE The ability of mobile phase to move up is depend on the polarity itself Volatile organic solvents is preferably used as as mobile phase. SOLVENT POLARITY INDEX Hexane Butanol 3.9 Chloroform 4.1 Methanol 5.1 Ethanol 5.1 Acetonitrile 5.8

11. Elution Strength of Mixed Solvents: The elution strength of the mixture is assumed to be the weighted average of the elution strengths of the components: e o net = e o A (mole % A) + e o B (mole % B) where: mole % A = (moles A) / (moles A + moles B) Thus, to determine the e o net of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the e o net of a solvent mixture prepared from 1.0 mL of ethyl acetate plus 9.0 mL of hexanes is calculated as shown below: e o net = e o EtOAc [(moles EtOAc )/(moles EtOAc+moles hexane)] + e o hexane [(moles hexane)/(moles EtOAc+moles hexane)] where: moles EtOAc = [(volume EtOAc ) (density EtOAc )] / [molecular weight of EtOAc ] thus: e o net = {0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]} {(1.0 mLEtOAc )(0.902g/mL)/88.11g/mole) + (9.0 mLhexane )(0.659g/mL)/86.18g/mole)} and e o net = 0.067

12. Materials : TLC plate ‘Developing container’ - chamber/ jar/ glass beaker Pencil Ruler Capillary pipe Solvents / mobile phase - organic solvents UV lamp

13. Procedure : 1.Developing Container Preparation: Solvent is transferred into the container with 0.5-1cm in dept from the bottom

14. 2. TLC Plate Preparation Commercialy obtained with 5cm x 20cm in size Prepare your size when neccesary Line 1 cm from the bottom with a pencil as a part should be spotted. Image Notes 1. These were made with lab grade silica gel, on glass slides, with plaster of paris as the binder 2. These were made with silica gel from dessicator packets, prepared in the same way as above but with less suspension to work with (hence the gaps near the edge)

15. 3.Spotting’ TLC plates Make sure that your sample is liquified already. stick it using capillary pipe & spott onto the line

16. 4.‘Develop the plate’ after spotting, put the plate inside the chamber in the ascendant position Make sure that the depth of solvent doesn’t touch the spots Let it develop up to the 1cm from the top of plate After that, pull out the plate from the chamber and let the solvent be vaporized

17. 5. Detection of spots The color samples are easy to be seen and no need to use UV lamp to detect them Chromatogram of 10 essential oils, Stained with vanillin reagent.

18. Detection : Compound class Derivatizing agent General Iodine vapor General Sulphuric acid (50%) Acids Bromo cresol green Aldehyde and ketone 2,4 dinitro phenyl hydrazine Amines and amino acid ninhydrin Alkaloids Mercuric nitrate Barbiturate Diphenylcarbazone Lipids Bromo thymol blue Steroids Antimony trichloride carbohydrate Aniline phthalate Chromatogram of 10 essential oils, Stained with vanillin reagent.

19. Resolution The separation between two analytes on a chromatogram can be expressed as the resolution, Rs and can be determined using the following equation: Rs = (distance between center of spots ) (average diameter of spots) In TLC, if the Rs value is greater than 1.0, the analytes are considered to be resolved.

20. Visualization Absorption of UV radiation is proportional to concentration Quantification is possible

21. Rf value: R f of component A = d A d S R f of component B = d B d S The R f value is a decimal fraction, generally only reported to two decimal places

22. THIN LAYER CHROMATOGRAPHY – R f ’s R f values can be used to aid in the identification of a substance by comparison to standards . The R f value is not a physical constant, and comparison should be made only between spots on the same sheet, run at the same time. Two substances that have the same R f value may be identical ; those with different R f values are not identical .

23. Mobile Phase Mixture Composition Number   % Ethyl Acetate   % Hexane   R f Salicylic Acid R f Acetylsalicylic Acid 1 100 0.530 0.545 2 90 10 0.620 0.600 3 70 20 0.255 0.291 4 50 30 0.945 0.291 5 30 50 0.360 0.280 6 20 70 0.270 0.250 7 10 90 0.182 0.164 8 100 Mobile Phase Mixture combinations for the separation of Salicylic Acid (1%) and Acetylsalicylic acid (1%)

24. Time (minutes) Salicylic acid R f Acetylsalicylic acid R f Reaction Mixture R f Lower Spot Reaction Mixture R f Upper Spot 15 0.385 0.346 0.385 0.481 30 0.320 0.340 0.360 0.500 45 0.264 0.321 0.321 0.491 Retardation factor ( R f ) values for TLC of salicylic acid, acetylsalicylic acid, and reaction mixture in a 50/50 ethyl acetate/hexane solvent system over time.

25. Some problem: a. The spot shape is too broad - Diameter is supposed to be < 1-2mm b. The movement of solvent - should be straight up - unproportionality in stationary phase surface will inhibit the movement of solvent c. streaking formation - caused by too concentrated sample

26. EXAMPLE : Mobile phase Stationary phase Herbs and Herbal Products 20 volumes of toluene + 45 volumes of ethyl acetate,+ 20 volumes of glacial acetic acid + 5 volumes of formic acid silica gel Amalaki 100 volumes of ethyl acetate + 11 volumes of formic acid ,+11 volumes of acetic acid + 25 volumes of water. silica gel Amra Glacial acetic acid . kieselguhr G . Arachis Oil A mixture of 1 volume of hexane and 1 volume of diethyl ether . silica gel GF254 Artemisia Toluene silica gel GF254 Clove Oil A mixture of 90 volumes of toluene and 10 volumes of ethyl acetate silica gel G Eucalyptus Oil Derivatizing agent anisaldehyde sulphuric acid reagent vanilin sulphuric acid reagent starch solution 1 volume of sulphuric acid and 0.5 volume of anisaldehyde anisaldehyde solution , anisaldehyde solution

27. Reference: R.A.Day , Jr. A.L.Undewood (1987). Analisis Kualititatif . Edisi ke Empat , ms : 474-529. David G. Watson(2005). Pharmaceutical analysis. Edisi ke-2, ms 315-331 http//orgchem.colorado.edu/ handbooksupport /TLC/TLCprocedure.html Indian pharmacopeia, volume 3, 2007 Kapp , Khail . Chapter 7, Thin Layer Chromatography, March 2, 2010 Kaine , Mary Ann. Chapter 7, Thin Layer Chromatography, March 2, 2010 . Young , Wei. Chapter 7, Thin Layer Chromatography, March 2, 2010.

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