Thin Layer Chromatography And Drugs, Report, Preparation Procedure

Thin layer Chromatography

Thin-layer chromatography becoming increasingly popular in the clinical lab because it is more efficient than paper chromatography. Thin-layer chromatography routinely used in the separation of sugars, amino acids, lipids, and various drugs. In addition, it takes less space and the results are reported much faster. The Thin-layer chromatography plate however is more expensive than the paper but the cost is becoming down as the raw materials for preparing the Thin-layer chromatography plates are easily available.


In thin-layer chromatography, the mixture of the test compounds in solution is applied as a spot on a sorbent. Which is the solid phase that holds or absorbs the specimen. The sorbent for the thin layer chromatography on the other hand is a thin layer of silica gel coated on a solid plate that can be prepared in the laboratory.

What Is Chromatography

Chromatography is a technique by which a mixture of organic compounds carried in the mobile phase (liquid or gas) is separated into its constituents on the stationary phase.


Preparation of Thin Layer Plate

Thin-layer chromatography is prepared by two following methods:

First Method:-


The procedure to prepare thin-layer chromatography plate thin-layer chromatography means- Thin layer chromatography plate In this one side is coated with adsorbents. The most common adsorbent is silica-alumina we have taken Silica gel- G as an adsorbent For the preparation of thin-layer chromatography first prepare a slurry of silica gel -G by mixing one part of silica gel- G with 2.5 parts of distilled water.


We have taken 5 gram of silica gel G into the glass pestle motor and add 12.5 ml of distilled water now mix it to prepare the slurry. There should not be any lumps on the slurry transfer the slurry into the clean and dry beaker with the help of doper transfer a small amount into glass slide move the plate around remove the excess slurry from plate procedure is same for big glass plate dry this plate at room temperature.

Place the dry plate in a hot air oven for about 1 Hr at 100 degrees centigrade to activate them after 1 hour remove the plate from the oven now plate is ready to perform separation experiments.

Second Method:-

1. Take a thin glass sheet of 2.5mm thickness and out it according to the tank available, these are usually 10 cm x 10 cm, 20 cm x 20 cm, or 10cm x 20cm. Thoroughly clean the plate preferable by washing with a good detergent, followed by hot water, and finally running with distilled water before drying. The glass plate should not have any greasy spots.


2.  Buy the absorbent from a reliable deal of scientific goods. The required quantity of the absorbent is mixed with distilled water to form a slurry (30gm silica gel and 60ml water) shake thoroughly for 1-2 minutes in a stoppered Erlenmeyer flask and then follow step 3.


3. Place the plate on a flat surface preferably, on a big piece of glass. A sheet of filter paper on the glass is recommended which will soak the overflowing absorbent suspensions.


Put a narrow strip of scotch tape across the edge of the four outer sides. Wrap the tape over onto the supporting surface so that the plate does not move during spreading shake the slurry gently and pour with a glass rod, over the plate along the untapped edge of the

plate.


4. Take a thin uniform glass rod, draw the slurry over the plate in one continuous glade along with the tape, care should be taken not to roll the glass rod excess following over the edge of this plate is dried immediately and therefore will not creep back on to the plate.


5. Dry the plate in the air for 30 minutes remove the tape carefully and put the coated plate in an oven at 110 degrees for 30 minutes to activate.


6. Always activate the plates by reheating in an over at 110°C for 50 minutes especially when the air is loaded with moisture.


The Procedure Of Thin Layer Chromatography


1. Make a line with a pencil 2-5 cm from the lower edge of the plate and parallel to the edge. Be careful to draw the line very lightly using only a dull pencil point. So that the sorbent is not scratched, mask the line for the spots. Usually, 1-3 cm apart a plastic spotting plate with holes can be used for the correct spacing of the sample along the starting line.


To ensure std development the upper edge of the plate can be kept at the constant distance (12-15 cm) flow the spotting. Remove the absorbent by scrapping beyond the scoring at the top.

2. Label the spot with a lead pencil. Apply a measurement quantity of specimen on the designated spots use a hair drier small area do not oven heat. Reserve one spot for the mixture of std with known compounds and their concentration.


3. Put the spotting plate in the Chromatography jar. The jar 1s pre-filled with the solvent mixture but the level of the fluid should be below the spotting line. It is recommended that one side of the jar should be lined with the filter paper Drings rapid equilibrium with the solvent vapor.


We are sonata with vapor the solvent should be slightly the spot line and if the solvent should be slightly below the solvent level is too low, pure fresh solvent carefully down one side of the jar with the help of a beaker and a glass rod. Bring the level close to the spot but it must not touch the spot. This starts the development of the Chromatogram.

4. Remove the plate when the solvent front has reached almost to the top edge. Mark the solvent line by the frame is the cupboard.


5 Spray the spot locating reagent on the dried plate by the measure of an atomizer ghazis spray.


6. Identify the spot and Rf calculation may be of additional help.


The Steps For Paper Chromatography and Thin Layer Chromatography Can Be Summarized As Follows:

1. Mixture of organic compounds is taken into solution.


2. A known quality of the substance is applied on the sorbent as a spot. The sorbent is the solid or stationary phase that holds the mixture.


3. The spot is thoroughly dried under a hot airflow.


4. The mixture of organic solvents is allowed to flow over the spot along the length of the sorbent. This is the mobile phase. The moving solvent picks up the mixture of the organic compounds dissolve it and as it moves over the solid phase.


It set up a competition between the solvents. The solute and sorbent particles on which they are observed. As a result these compounds originally present in the form of a mixture fall apart on the stationary phase.

5. Allow the solvent to reach close to the opposite edge.


6. Completely dry the sorbent and then apply the spotting reagent. When sprayed on paper and plate read with the compound separated by Chromatography enable to locate the spot.


7. Locate the spot and compare it with the standard which runs simultaneously. The flow of solvent can be unidirectional or bidirectional. If it is unidirectional, several specimens can be whereas if it is bidirectional only one spot of the specimen is applied.


Two solvent mixtures are used in sequence for the bidirectional Chromatography. The commonly used solvent is the chloroform method. Ammonium hydroxide (4:4:1) and butanol ethanol + water (7:2:2) in the same sequence after switching the site completely drying is necessary before the side is switched.

Rf expresses the selective mobility or various compounds as the radio of the distance traveled by the spot from the point of application and the distance traveled by the solvent under controlled specified conditions. The Rf remains fairly constant and is helpful in the identification of the compound.
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