Jadavpur University

College :- Bengal Institute Of Pharmaceutical Sciences

Name :-Kuntal Mitra

Roll No :- 11

Class :- 3rd Yr 6th sem

Subject :-Pharmacognosy (Paper-2)

Topic :-TLC

Course :-B.Pharm (Ayu)

Separation of black ink on a TLC plate

CHROMATOGRAPHY

Introduction:-

Chromatography is the separation of a mixture into individual components using a stationary phase & a mobile phase.

Types of Chromatography:-

1. Based upon the nature of Stationary phase & mobile phase:

Gas –Solid Chromatography Gas –liquid Chromatography Solid –liquid Chromatography Liquid –liquid Chromatography

2. Based on the principle of separation:

Absorption Chromatography Partition Chromatography

3. Based on the modes of Chromatography:

Normal Phase Chromatography Reverse Phase Chromatography

4. Other Types of Chromatography:

Ion-exchange Chromatography

Gel Permeation Chromatography Chiral Chromatography

THIN LAYER

CHROMATOGRAPHY

(TLC)

TLC of Kalmegh Leaf extract in time of my WBPPDCL training.

History:-

The history of TLC dates back to 1938 when Izmailov & Shraiber separated plant extracts using 2mm. thick & firm layer of alumina set on glass plate. In 1944, Consden, Gorden & Martin used filter papers for separating amino acids. In 1950, Kirchner identified terpenes on filter paper & later glass fiber paper coated with alumina. Only in 1958, Sthal developed standard equipment for analyzing by thin layer chromatography.

Introduction:-

Thin Layer Chromatography (TLC) is a chromatography technique used to separate chemical compounds. It involves a stationary phase consisting of a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose immobilized onto a flat, inert carrier sheet. A liquid phase consisting of the solution to be separated is then dissolved in an appropriate solvent and is drawn up the plate via capillary action, separating the experimental solution based on the polarity of the components of the compound in question.

Principle:-

The basic principle of TLC mainly depends upon the adsorption chromatography.

The most commonly used solvents utilized in this form of chromatography are silica & alumina. As the components move through the sorbates their relative rate of migration are affected by their individual affinities for the sorbents. Separation occurs when one compound is more strongly absorbed by the sorbents than the other components. When sorbent is silica or alumina, polar natural products moves slowly compare to non-polar natural products. Adsorption takes place because of the interaction between the compound & groups associated with the sorbents. In case of silica, binding occurs between the compound & the free hydroxyl groups of the sorbates.In this

particular case adsorption involves the hydrogen bonding between compound functional groups & adsorbent surface hydroxyl group.

Types of Stationary Phase (Silica):-

Name MeaningG GypsumH Containing no foreign matterR Specially purifiedP For preparity workW Water tolerantC Channeled

RP Reverse phaseF Fluorescence (60F254)

Retention Factor (Rf):-

The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the solvent front travels 2.8 cm, the Rf is 0.75.

The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant:

solvent system adsorbent thickness of the adsorbent amount of material spotted temperature

Practical Requirements:-

Stationary Phase Glass Plates Preparation & activation of TLC plates Application of sample Development Tank Mobile Phase Development technique Detecting or visualizing agent

Plate preparation:-

TLC plates are made by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. This mixture is spread as thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic, and the resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. The thickness of the adsorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative

TLC. Every type of chromatography contains a mobile phase and a stationary phase.

Technique:-

The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different stationary phases. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction

products.

A small spot of solution containing the sample is applied to a plate, about one centimeter from the base. The plate is then dipped in to a suitable solvent, such as ethanol or water, and placed in a sealed container. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to

differences in their attraction to the stationary phase. Results also vary depending

on the solvent used. For example, if the solvent were a 90:10 mixture of hexane to ethyl acetate, then the solvent would be mostly non-polar. This means that when analyzing the TLC, the non-polar parts will have moved further up the plate. The polar compounds, in contrast, will not have moved as much. The reverse is true when using a solvent that is more polar than non-polar (10:90 hexane to ethyl acetate). With these reagent. Solvents, the polar compounds will move

higher up the plate, while the non-polar compounds will not move as much.

The appropriate solvent in context of thin layer chromatography will be one which differs from the stationary phase material in polarity. If polar solvent is used to dissolve the sample and spot is applied over polar stationary phase TLC, the sample spot will grow radially due to capillary action, which is not advisable as one spot may mix with the other. Hence, to restrict the radial growth

Chromatogram of 10 essential oils colored with vanillin

of sample-spot, the solvent used for dissolving samples in order to apply them on plates should be as non-polar or semi-polar as possible when the stationary phase is polar, and vice-versa.

Visualizing the Spots :-

As the chemicals being separated may be colorless, several methods exist to visualize the spots:

Often a small amount of a fluorescent compound, usually Manganese-activated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a backlight (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.

Iodine vapors are a general unspecific color reagent Specific color reagents exist into which the TLC plate is dipped or which are

sprayed onto the plate.

Stages of Development:-

Chromatography of an extract of green leaves (for example spinach) in 7 stages of development. Carotene elutes quickly and is only visible until step 2. Chlorophyll A and B are halfway in the final step and lutein the first compound staining yellow.

Factors influencing TLC:-

1. Polarity or nature of Stationary Phase2. Polarity or nature of mobile Phase3. Relative Humidity4. Saturation Chamber5. Particle size of the adsorbent used in stationary Phase6. Flow of the mobile Phase7. Temperature8. pH of the mobile phase9. Sample volume

Step 1 Step 2 Step 3 Step 4

Step 5 Step 6 Step 7

10. Thickness of solvent of the stationary Phase11. Solvent used in sample12. Migration distance13. Impurities in the mobile phase14. Rf value15. Spot size on application

Advantages of TLC over other Chromatographic techniques:-

Simple equipment Short development time Wide choice of Stationary Phase Early recovery of separated components Easy visualization of separated vaporizes

Troubleshooting TLC :-

All of the above (including the procedure page) might sound like TLC is quite an easy procedure. But what about the first time you run a TLC, and see spots everywhere and blurred, streaked spots? As with any technique, with practice you get better. One thing you have to be careful Examples of common problems encountered in TLC:

The compound runs as a streak rather than a spot

The sample was overloaded. Run the TLC again after diluting your sample. Or, your sample might just contain many components, creating many spots which run

together and appear as a streak. Perhaps, the experiment did not go as well as expected.

The sample runs as a smear or a upward crescent.

Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates.

The sample runs as a downward crescent.

Likely, the adsorbent was disturbed during the spotting, causing the crescent shape.

The plate solvent front runs crookedly.

Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crookedly run plates make it harder to measure Rf values accurately.

Many, random spots are seen on the plate.

Make sure that you do not accidentally drop any organic compound on the plate. If get a TLC plate and leave it lying on your workbench as you do the experiment, you might drop or splash an organic compound on the plate.

No spots are seen on the plate.

You might not have spotted enough compounds, perhaps because the solution of the compound is too dilute. Try concentrating the solution, or, spot it several times in one place, allowing the solvent to dry between applications. Some compounds do not show up under UV light; try another method of visualizing the

plate. Or, perhaps you do not have any compound because your experiment did not go as well as planned.

If the solvent level in the developing jar is deeper than the origin (spotting line) of the TLC plate, the solvent will dissolve the compounds into the solvent reservoir instead of allowing them to move up the plate by capillary action. Thus, you will not see spots after the plate is developed.

You see a blur of blue spots on the plate as it develops.

Perhaps, you used an ink pen instead of a pencil to mark the origin.

Application of TLC:-

1. (a) For separating of essential oils containing drug with the help of mobile phase (Benzene:ethylacetate=95:5) or (Benzene:ethylacetate=9:1), can be separated like Eucalyptus Oil.

TLC of Halud in time of my WBPPDCL training

(b) With the help of solvent system CHCl3:C2H5OH:Glacial CH3COOH =94:5:1, we can separate the pigments from Curcuma rhizome.

2. In case of alkaloidal drugs we can use the solvent system like CHCl3:diethylamine =9:1 for separation.

3. For the separation of cardiac glycoside (digitalis,digitoxin) with the help of CHCl3:CH3OH:H2O =64:50:10

4. For the separation of flavonoids we can use the solvent system or mobile phase CHCl3:ethylacetate =6:4.

5. When we can not separate the mixture of components by the Column Chromatography then the preparative TLC is performed to obtain the each compounds ,from the Rf of the spot or sample.

6. Determination of the pigments a plant contains 7. Detection of pesticides or insecticides in food 8. Analyzing the dye composition of fibers in forensics,

9. Identifying compounds present in a given substance .

References:-

Vogel's Textbook of Practical Organic Chemistry

Text book of Pharmaceutical Analysis

Pictures are taken from my training in WBPPDCL Drug Testing Lab.

Internet

Preparative TLC of Kalmegh to isolate andrographolide in time of my WBPPDCL training