1. Thin layer chromatography
Ridwan Islam

2. Definition:
Thin layer chromatography is a method of analysis in which the stationary phase, a finely divided solid, is spread as a thin layer on a rigid supporting plate; and the mobile phase, a liquid, is allowed to migrate across the surface of the plate by means of capillary action.

3. Theory
The mixture to be separated is dissolved in a solvent and the resulting solution is spotted onto the thin layer plate near the bottom. The mobile phase rises up the TLC plate by capillary action and the components dissolve in the solvent and move up the TLC plate.
A competition is set up between the silica gel plate and the development solvent for the spotted material. The very polar silica gel tries to hold the spot in its original place and the solvent tries to move the spot along with it as it travels up the plate. The outcome depends upon a balance among three polarities: that of the plate, the development solvent and the spot material.

4. 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 non-polar and is capable of interacting with analytes. Non-polar 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.

6. For example, if a sample consists of two components, one more polar than the other, the more polar will tend to stick more tightly to the plate and the less polar will tend to move along more freely with the solvent. Using a more polar development solvent would cause both to move along further.

7. Steps of TLC:
Description of the plate/backing support for stationary phase: As a backing support for stationary phase generally glass plates are used, although plastic sheet and aluminium foil can also be used. The size of the full plate is 20 × 20 cm.

8. Stationary phase used as thin layer in TLC plates: Mainly silica gel is used as stationary phase as thin layer on plates.
Name
Composition
Silica gel H
Silica gel without binder
Silica gel G
Silica gel + CaSO4
Silica gel GF
Silica gel + Binder + fluorescent indicator
Alumina
Al203 without binder
Al203 G
Al203 + Binder
Powdered cellulose
Cellulose without binder
Kieselguhr G
Diatomaceous earth + binder
Polyamide powder
Polyamide
Fuller’s earth
Hydrous magnesium alumina
Magnesol
Magnesium silicate
Sephadex
Cross-linked dextran 

9. To ensure that the stationary phase adheres firmly to the backing plate and does not flake off during development, binders such as calcium sulfate (gypsum), starch or carboxymethylcellulose are added to the adsorbent. Presence of binder in the adsorbent is designated with ‘G’.
Most commercially available adsorbent may be obtained with or without a chemically inert UV indicator for visualization of many compounds under UV light (254 nm or 366 nm). The presence of fluorescence indicator is designated with an ‘F’, ‘F-254’ or ‘F-366’.

10. Preparation of slurry:
TLC plates made by mixing silica gel + inert binder calcium sulphate (gypsum) + distilled water in a beaker  shake well for two minutes  slurry is prepared
Preparation of plate:
Plates are washed with soap & water (and then washed with acetone for removal of fatty materials) and dried in oven. After preparing the slurry, the TLC plates can be prepared by using any one of the following techniques:
Pouring: slurry is poured on to a glass plate which is maintained on a leveled surface. The slurry is spread uniformly on the surface of the glass plate. After setting, the plates are dried in an oven. The disadvantage is that uniformity in thickness cannot be ensured.

11. Dipping: This technique is used for small plates by dipping the two plates at a time, back to back in slurry of adsorbent. Plates are separated after removing from slurry and later dried.
Spraying: suspension of adsorbent or slurry is sprayed on a glass plate using a sprayer. The disadvantage is that the layer thickness cannot be maintained uniformly all over the plate.
Spreading: is the best technique where a TLC spreader is used. The glass plates of specific dimensions are stacked on a base plate. The slurry after preparation is poured inside the reservoir of TLC spreader. The thickness of the adsorbent layer is adjusted by using a knob in the spreader. The spreader is rolled only once on the plate.

13. Resultant plates are allowed to dry in air and further activated by heating in oven for 30 minutes at 110°C.
Activation: It is nothing but removing of moisture & other adsorbed substance from the surface of any adsorbent by heating at high temperature so that adsorbent activity is retained.
The thickness of the film of the stationary phase is very important for successful separation. It may vary according to the purpose.
mm : for analytical purpose (screening)
1-2 mm: for separation and purification (preparative TLC)
Commercial precoated TLC plates are also available.

14. Preparation & application of sample in TLC plates:
A straight line is ruled along one side of the plate about 1 inch from the edge. The sample which may range from a few micrograms to milligram dissolved in µl of a volatile solvent and are spotted usually with a capillary tube or a micro- syringe or micro-pipette. Samples may be applied as spots or as thin bands. These applied spots should be about ¾ inch apart and 1 inch from the bottom of the plate.
Pencil is always used to mark a TLC plate since the graphite carbon is inert. If organic ink is used to mark the plate, it will chromatograph just as any other organic compound and give incorrect results.

16. Development of the chromatogram
The majority of the TLC development is done by allowing the mobile phase to migrate up (ascend) the plate, which is standing in a suitable sized chamber. Developing chambers may glass, plastic or metal; glass chambers are the most common.
At first the mobile phase is taken in the development chamber and saturated completely with the mobile phase. Then the plate is inserted into the chamber keeping the origin of spot downward. Solvent level should be below the starting line of TLC, else spots will dissolve in the solvent. The solvent will gradually climb up and past the spot. Once the solvent is within 1-2 cm of the top of the TLC sheet, the TLC is removed from the developing chamber and the farthest extent of the solvent (the solvent front) is marked with a pencil. Thus a thin layer chromatogram is developed. The solvent is allowed to evaporate from the TLC sheet in the hood.

18. Detection/visualization of compounds on TLC
After the development of the chromatogram, colored compounds can easily be detected on the TLC plate.
But if the compound is colorless, it can be detected by various methods.

19. Visualization of Compounds:
If any colored spot or band is present that is marked.
Usually the compounds are not colored. Then –
 If any fluorescent or quenching compound is present, the plates can be visualized under UV lamp and marked. The UV light at 254nm (Short) and 366 nm (Long) is usually used to detect the spot or band of fluorescent compounds. Usually the commercially available precoated TLC plates itself contain a fluorescing substance (Zinc Silicate and Zinc Cadmium Sulfide, 0.5% each) which fluoresces everywhere except where a quenching compound is present on the plate. These compounds appear as black spot on the TLC plate.

20. Long wavelength (366 nm): background plate dark, spots glow
Short wavelength (254 nm): background plate green, spots dark
Under the UV light, the spots can be outlined with a pencil to mark their locations.

21. Compounds which do not contain any chromophore are not visualized under the UV light.
 The plates can be sprayed (by atomizers) with color producing reagents to detect the compounds which are not visualized by the above procedure. (Like some terpenoids which contain no chromophore in their structure).
If color develops after heating with a hair drier, the whole band is marked. Different spray reagents can be used such as:

22. A. Vanillin in sulfuric acid (1%):
It is most commonly used in the laboratory to detect organic compounds. It is prepared by dissolving 1g of vanillin in 100 ml of concentrated sulfuric acid. The TLC plate or the chromatogram is sprayed with 1% vanillin in sulfuric acid reagent and then heated for 2 minutes to develop color.
Almost all organic compounds except some alkaloids give distinctive color of bands with this reagent. So it is called general spray reagent. Example:
Terpenoids  Pink or purple color
Flavonoids  Yellow or orange color
B. Iodine:
The plates are placed in an enclosed tank containing a few crystals of iodine. The compounds particularly those containing nitrogen absorbs the iodine and the spots appear as brown areas on a faint yellow background.

24. C. 2% Ninhydrin in ethanol:
Amino acids produce mainly brown or purple color when the plate is sprayed with this reagent and heated to 120oC for 10 minutes.
D. Dragendorff’s Reagent:
It is used to detect alkaloids. The reagent is prepared by mixing equal parts (v/v) of 1.7% bismuth subnitrate dissolved in 20% acetic acid in water and 40% aqueous solution of potassium iodide. alkaloids can be detected & give orange to red color.
E. Iron II Thiocyanate Reagent:
It is used to detect peroxy compounds. The reagent is prepared by mixing 10 ml of 4% aqueous solution of ferrous sulphate with 15 ml of 3% ammonium thiocyanate in acetone. After spraying with this reagent the peroxides immediately develop a brown red color.

25. F. Aniline phthalate reagent:
It is prepared by dissolving 0.03 g of aniline and 1.6 g of phthalic acid in 100mL of butanol (saturated with water). The reagent is used to detect sugars.
G. 2,4-dinitrophenyl hydrazine in ethanol:
After spraying the plate with this reagent and heating to 120°C for 10 minutes aldehydes and ketones produce a variety of colors.

26. Preparative Thin Layer Chromatography (PTLC)
Preparative thin layer chromatography can be used for the purification of small (mg) quantities of sample. Because it allows for rapid separation of a number of components in a reaction mixture, it is especially useful for obtaining the components of natural extracts.
Procedure for running a prep TLC
Cut the plate in half by scoring with a pencil for small to moderate amounts of sample (5-25mg). For larger amounts of sample, the entire plate may be used.
Gently mark a pencil line roughly inches from one side of the plate. This is the "origin" line.

27. Prepare a relatively concentrated solution of your crude sample (1-2 mL) in a fairly low-boiling solvent (i.e. DCM or Et2O).
Using a short pipet, carefully deposit a thin line of sample across pencil line. It is very important to do this slowly and uniformly, without contacting the pipet too much against the silica as this will scrape it. The main difficulty is trying not to accidentally plunger too much at one place.
Apply remaining solution repeatedly on the original line of sample, drying in between applications. Not drying fully after an application will cause the solution to diffuse outward, broadening the band.

28. Obtain a prep TLC chamber
Obtain a prep TLC chamber. This is a large, heavy, square glass chamber with about 100 mL of eluent.
Place the plate in the chamber and seal the top with the lid or aluminum foil.
After solvent run, remove the plate and visualize under UV. If your desired band(s) are not separated enough, repeat run another elution.
Scrape off each band of interest with a spatula or razor blade.
By placing the silica gel in a pipet with a cotton plug, and washing with suitable solvent is often sufficient
After that the purity should be checked on a precoated TLC plate.
Then the eluting solvent is evaporated to obtain the pure compounds.

29. Identification of compounds by TLC: Qualitative Analysis
The components, visible as separated spots, are identified by comparing the distances they have travelled with those of the known reference materials.
At first the distance of the start line to the solvent front (a) is measured. Then the distance of center of the spot to the start line (b) is measured.

30. Then the distance moved by the individual spot is divided by the distance moved by the solvent. The resulting ratio is called Rf value. The value should be between 0.0 (spot did not moved from starting line) and 1.0 (spot moved with solvent front) and is unit less.

31. From this formula, one obtains an “observed” Rf value
From this formula, one obtains an “observed” Rf value. This calculated Rf is always (≤1).
As it has been found to be inconvenient in routine laboratory work always to write a zero and a decimal point, the Rf value is multiplied by 100, referred to as the HRf value. This is used for the qualitative description of thin-layer chromatograms.
The Rf (retardation factor) depends on the following parameters:
• solvent system
• absorbent (grain size, water content, thickness)
• amount of material spotted
• Temperature due to the fact that all those variables are difficult to keep constant, a reference compound is usually applied to the plate as well.

32. 7. Quantitative analysis
Photodensitometric analysis: Densitometer is an instrument which measures quantitatively the density of the spots. When the optical density of the spots for the standard and test solution are determined, the quantity of the substance can be calculated.
Measurement of spot area: The relationship between spot areas on the plate and concentration of the components has been investigated. A linear relationship between the logarithm of the concentration of the substance & the square root of the spot area is obtained when a plot is drawn.
UV spectrometry: After elution, the sample is diluted; the concentration is measured form UV absorbance.

33. Solvent System: Mobile Phase
If a development solvent of too high a polarity is used, all components in the mixture will move along with the solvent and no separation will be observed. If the solvent is of too low a polarity the components will not move enough and again separation will not occur. In practice, different solvents or mixtures of solvents are tried until a good separation is observed. Typically an effective solvent is one that gives Rf's in the range of
Mixtures of solvents are often used to “fine-tune” the separation of analytes. Some suggested mixtures for specific classes of organic compounds to be separated on Silica Gel are:
Ethyl Acetate: Methanol (5:1)
Methanol: Chloroform (2:3)

34. Advantages of TLC over other chromatographic techniques:
a) Simple to learn and perform, always available for use since precoated plates are usually employed without any further preparation.
b) Short development time, that means the time required to demonstrate the presence of constituents of a mixture by TLC is very short.
c) Greater sharpness of separation.
d) High sensitivity in detection.
e) Economical as the solvent consumption Is small.
f) TLC provides a chromatographic drug fingerprint and therefore suitable for determining the identity & purity of drugs.
g) TLC chromatograms can be documented and kept permanently in the record.

35. Application of Thin Layer Chromatography:
Thin-layer chromatography (TLC) is a very commonly used technique in natural chemistry for –
1. Separating sufficient amount of components of an extract (For preparative TLC),
2. Identifying the isolated compounds,
3. Determining the purity of an compound,
4. In synthetic chemistry, it is also used for following the progress of a chemical reaction,
5. TLC can also be used in quantitative analysis since the diameter of a spot is depended on the concentration of solution.

36. Special Techniques Multiple elution development
In the multiple elution development, after the TLC plate has been developed once and dried, it is returned to the chamber and redeveloped in the same direction, using the same mobile phase. The process may be repeated as many times as is necessary to ensure effective separation. This technique is useful for increasing the Rf values of compounds that move only a short distance from the origin, as they are exposed to solvent travel for a longer time period than faster eluting substances.

37. Two Dimensional TLC
Multiple Dimensional TLC

38. Problems faced in TLC