THIN LAYER CHROMATOGRAPHY
The technique of TLC was first introduced by Izmailov and Shrabier in 1938. TLC as procedure for analytical absorption chromatography was first introduced by Stahl in 1958. He was mainly responsible for bringing out standard equipment for the preparation of thin layer apparatus.
TLC is defined as a method for chromatographic analysis on thin layer of absorbent held on a glass plate. TLC is also known as open column chromatography or drop strip or spread layer or surface chromatography.
A thin layer of stationary phase is formed on a suitable flat surface such as glass or plastic plate. Since the layer is so thin, the movement of mobile phase across the layer occurs generally by simple capillary action and is rapid. As the mobile phase move across the layer from one edge to opposite edge, it transfers and analyzes the compounds placed on the thin layer. The analyte movement stops either when the mobile phase reaches the end of the layer and capillary action closed up when the plate is removed from the mobile phase i.e. solvent phase. The movement of analyte is expressed by retardation factor.
Types of TLC:
TLC is mainly of 4 types which are as follows:
a) Partition TLC: In this technique, stationary and mobile phase are in liquid. The stationary phase is liquid which is held on suitable solid such as cellulose.
b) Adsorption TLC: Stationary phase is solid and mobile phase is liquid. Stationary phase e.g. silica gel, alumina etc. is used. Mobile phase is organic solvent.
c) Reversed Phase partition TLC: Plate is coated by silica which is hydrophilic in nature and it is again coated by wax or paraffin making it hydrophobic.
d) Ion exchange TLC: A reversible exchange of ions is possible in between ions in a liquid phase and a solid insoluble substance containing ionic state (ion exchange resins).
Technique of TLC:
1. Adsorbents (coating material used in TLC):
Silica gel, alumina or aluminium powders, cellulose powder, kieselgurh (natural adsorbent) are used as adsorbent in TLC. In general silica gel and alumina are used for adsorption TLC and silica gel, cellulose and kieselguhr are used for partition TLC.
Silica gel: It is the most commonly used adsorption and slightly acidic in nature. A binding agent usually gypsum salt (CaSO4~13 %) is often added with silica gel to hold the adsorbent firmly on the plate. Silica gel is active absorbent is applicable to separation of all classes of compounds.
Alumina: They are slightly basic in nature used in separation of bases and steroids.
Kieselguhr (Diatomaceous earth): It is a natural adsorbent and is weakly absorbing than silica gel and alumina. It is an inactive type of adsorbent. It can be added to silica gel to give a less active mixed adsorbent. It has been used for separation of sugars.
Cellulose: It is used only as support for the stationary liquid phase in partition TLC in the source of as a paper of cellulose act as support in paper. Modified cellulose paper such as diethyl aminoethyl, triethyl aminoethyl and carboxy methyl cellulose are useful for separation of phospholipids, nucleolipids and pigments.
2. Preparation of plates / chromatoplates:
In TLC, the adsorbent supports are usually glass plates of square or rectangular plates and the size of the plates generally 20 x 20 cm. Generally glass plates are used.
The glass plates should be cleaned i.e. prewash to remove dirt and dust particles. Glass plates should be cleaned with detergent and ethanol.
3. Preparation of slurry:
Slurry is normally made in water. Required amount of adsorbent powder is weighed out and dissolve in water e.g. silica gel powder are mixed with water in a slurry of silica gel with water in a ratio of 1:2. But 0.02 M Na acetate is used instead of water in case of silica to avoid cracking of adsorbent. Proper stirring is required during slurry preparation for homogenous preparation of the slurry (without grains). Magnetic stirrer can be used for mixing the slurry. The slurry should not be too thick or too thin. If it is too thin it will run too quickly and produce extensively thin layers and if it is too thick spreading produces lines along plate.
Two types: Thin layer: 0.25 mm
Thick layer: 1.20 mm
A layer of thickness of about 0.25 mm is used for analytical purpose. By mixing 25 gm of silica gel with 50 ml distilled water, a layer of 0.25 mm can be prepared. Thick layer of silica gel with water in ratio 25:40 is used instead of 25:50 ratio.
4. Preparation of Thin layer on plate:
Application of thin layer of adsorbent can be made by different ways/methods like by spreading method, Spraying method and Dipping method.
Dipping method: In this method, slurry of adsorbent is prepared then glass plates are dipped in the slurry (Adsorbent). After about 20 sec the plates are carefully withdrawn and allowed to dry. This method is not of much used.
Spraying method: In this method, slurry of adsorbent is prepared and then sprayed on to the glass plates with the help of an ordinary spray.
Spreading method: Most commonly used method to obtain thin and uniform layer. The slurry is placed in an applicator. This is either moved over the stationary plate or it is held stationary and plate is pushed or pulled through. In one method the adsorbent reservoir containing the slurry is held stationary and the plate to be coated is moved underneath. In another method the reservoir is moved over the plate.
5. Activation of Adsorbent:
After making thin layers on plate, the next job is to remove liquid associated with the layer as completely as possible. This is done by drying the thin layer plate for 30 min in air and then in an oven at 110°C for another 30 minutes and this drying makes the adsorbent layer active. Since water or other polar solvents greatly affect the development by adsorption chromatography. They should be removed from the chromatoplates and this is known as activation.
6. Sample Activation:
Methods of application of sample on chromatoplates are almost similar to these used in paper chromatography. Using micropipette, microsyringe or Pasteur pipette, the sample is applied in small spot above the edge (i.e. 2 cm) and 1.5 cm apart. Different amount of sample from 1-50 ml can be applied. The amount of sample that can be applied depends on thickness of plate and principle of chromatography employed. For example in adsorption TLC, thickness of 1.2 mm is used in which 10-50 ml of sample can be loaded. For 0.25 mm thickness TLC, ideal volume is 0.5 -5.0 ml.
7. Choice of Solvent
Mixtures of two or more solvents of different polarity are used. The choice of solvents depends on the two factors
I. Nature of substance to be separated
II. Material on which separation is to be carried out
For e.g. if the mixture to be separated consists of hydrocarbon, then active adsorbent is required together with non polar solvent.
Mixture of two or more solvents of different polarity often gives better separation than chemically homogenous solvent, i.e. one solvent is polar and another is non polar. Generally polar substances like alcohol, carboxylic acid, and amine solution should be made in polar solvent and cellulose or silica gel layers are selected. Polar solvent produce greater migration and thus give better separation. It has been observed that a combination of two solvents give better separation than obtained with single solvent.
Examples of Solvents system used in TLC
Solvent mixture Ratio
· Cyclohexane: ethyl acetate 95:5
· Benzene: ethyl acetate 95:5
· Benzene: methanol 95:5
· Cyclohexane: ethyl acetate 85:15
· Benzene: ethyl acetate 90:10
· Chloroform: ethanol 85:15
· Ethyl acetate: methanol 99:1
· Benzene: acetone 1:1
Solvent system for specific compounds
Compounds Solvent system (Ratio)
· Fruit juices Ethyl acetate: isopropanol: water: pyridine (26:14:7:2)
· Amino acids Butanol: acetic acid: water (4:1:5)
· Lipids Petroleum ether: diethyl ether: acetic acid (80:20:1)
· Free cholesterol Chloroform: methanol (2:1)
· Phosphotidyl choline Chloroform: methanol (4:1)
(Phospholipid) Chloroform: methanol: acetic acid: water (70:90:25:1)
· Neutral Lipids Hexane: diethyl ether: acetic acid (90:15:1)
Figure1: Separation of sugars in the Thin Layer Chromatography
8. Development of Chromatogram
It is usually carried out by ascending method in which solvent is allowed to rise to the height of about 10-16 cm above the origin. Then, the plate is removed from solvent tank and solvent front is carefully marked.
9. Visualization of spot
Location of compounds in TLC is easy because layers can be treated with corrosive substance without any adverse effect on thin layers. E.g. colorless compounds can be located with locating reagent. A solution of locating reagent of desired concentration is used where the plate is dipped or fine sprayed with such reagent.
I. Iodine is non destructive locating reagent and is very useful in identifying a large number of organic compounds. Either solution form or direct crystal forms of iodine can be used for locating.
a. Iodine is first dissolved in an organic solvent and then solution is sprayed on to the chromatoplate for detection of compound. Iodine is dissolved in 10 % methanol and this solution used for spray.
b. Cystals of iodine are placed in the tank during the development. Components on chromatogram may directly be exposed to iodine vapors contained in a tank for 10 min.
II. Sulfuric acid is also a good locating reagent but is corrosive in nature. It produces colored compound which are visible in daylight as well as in UV light.
III. Ninhydrin: Ninhydrin can be used as locating agents for amino acids.
Typical Spray Reagent:
Types of compounds Spray reagents
· Amino acids Ninhydrin
· Lipids Iodine vapor
· Reducing sugars Aniline diphenylamine reagent
· Carboxylic acids Bromocresol green
· Aldehydes and ketones 2,4 dinitrophenol hydrazine
· Phenols Ferric chloride
· Steroids, glycosides, Vitamins A Antimony trichloride
10. Qualitative Analysis of Spot
After spot are visualized, Rf values are calculated immediately because of the possibility of fading. Evaluation of Rf values can be used for qualitative purpose.
11. Quantitative Analysis
Quantitative evaluation of chromatogram can be carried out by 2 methods
a. Direct method:
Qualitative determination is undertaken directly on the layer. It includes visual comparison of spot. Photodensitometry method and Flourimetry method are used in direct method.
b. Indirect method
Spots are scrapped off from chromatoplates and then eluted with solvent e.g. ethanol is used.
Advantages of TLC:
TLC not only combines the advantage of paper and column chromatography but it is considered to be superior to either of these techniques.
a. It utilizes simple equipments. The technique is simple and cost is very low.
b. It is a versatile technique and can be applied to a wide variety of compound both organic as well as inorganic compound.
c. In TLC, development time is one hour or less. Excellence separation can be obtained within 15-20 mins. Identification and separation by TLC can be carried out quickly.
d. Wide choice of stationary phase can be used. The method may be employed for adsorption, partition or ionic exchange chromatography.
e. Sensitivity: The separation achieved by TLC is superior to those obtained by paper chromatography (10-100 times more sensitive). Due to its high sensitivity, compounds which are encountered in trace amount can be detected by TLC.
f. TLC gives sharper distinct spot. Diffusion of spot in TLC is low.
g. Easily recovery of component: It is possible to remove powdery coating of plate by scrapping with the knife.
h. Easy visualization of separated components.
i. Sometime, fluorescent indicators such as sodium salt of fluorescence, eosin or zinc silicates are added to the silica gel. Thin layer thus produce fluorescence on exposure to UV light. The main advantage is that non fluorescence but UV absorbent, substance can be obtained on a thin layer containing fluorescent indicator.
j. Corrosive reagent and acid can be spread on thin layer chromatography without adverse effect.
k. Capacity of thin layers of an adsorbent is higher than that of paper chromatography.
Limitation: The only limitation of TLC is that it can only be used for small scale production.
Application of TLC:
- · TLC is applicable for qualitative and quantitative analysis.
- · TLC has been used as analytic tool especially in organic chemistry. The main reason for popularity as an analytical tool in organic chemistry:
- o It can be used for most of the chemical compound
- o It has high speed separation i.e. development time is rapid in comparison to paper chromatography.
- · TLC can be used as a purification process. Micrograms of sample are sufficient for checking their purity by TLC. Because TLC has 10-100 times more sensitivity in comparison to paper chromatography. Due to its high sensitivity it often enables impurities to be observed in pure sample.
- · TLC can be used to assess that a reaction is complete or not.
- · TLC can be used for identifying organic compound for e.g. acids: citric acid and tartaric acid have been separated on layers of silica gel G by using a butyl acetate, acetic acid and water mixture.
- · Amino acids, proteins and peptides: A mixture of 34 amino acid, proteins and peptides has been successfully isolated from urine by using silica gel plate.
- · Vitamins, antibiotics and food products have been separated by TLC on Thin layer of silica gel G by using 2 solvent systems acetone and methanol or Benzene and formamide.
- · Antibiotics: Erythromycin has been separated on silica gel G using Methyl chloride, methanol, Benzene, formamide. Tetracycline by using 10 % citric acid, Butanol, methanol as a solvent system.
- · Vitamins A, D and E can be separated on silica gel G using 80 % cyclohexane and 20 % diethyl ether.
- · TLC can be used to separate inorganic ions, cations, anions and organometallo compounds.
- · TLC has been used in the analysis of urine and blood in pathological lab.