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TLC Analysis of Analgesic Drugs

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Abstract: In this experiment, a thin-layer chromatography (TLC) was used to determine the composition of various over-the-counter analgesics.(ACETAMINOPHEN, Acetylsalicylic Acid, Caffeine, and ibuprofen). Chromatography is a technique that we used to separate and identify individual components in a mixture. These chromatography techniques focused on the fact that components of a mixture tend to move at different speeds along the flat surface from the paper used to differentiate them from each other. The different rates of movement were the results of differing attractions of the components to the coating material compared to the tendency of the components to remain in the moving fluid. The results obtained were compared using a Co-Spot method to ensure their identity.

Introduction: Thin-layer chromatography is one of the easiest of the many chromatography techniques. A thin layer of a suitable solid substance is coated on a sheet of glass or plastic. A very small sample of the mixture to be analyzed is “spotted” onto the sheet. By immersing one edge of the sheet in an appropriate liquid developing solvent, the solvent is drawn up the sheet by capillary action, and the compounds of interest are carried along at different rates, effectively separating the components. This is commonly called “developing” the plate. After the plate has been developed, it is examined under ultraviolet (UV) light, which allows you to note the location of the spots.

Experimental conditions in TLC include the solvent system used to dissolve the compounds, the absorbent coated onto the TLC plate, the thickness of the absorbent layer, and the relative amount of the material spotted onto the plate. Under an established set of experimental conditions, a given compound always travels a fixed distance relative to the distance the solvent front travels. This ratio of the distance the compound travels to the distance the solvent front travels is called the Rf value. The symbol Rf stands for “retardation factor,” or “ratio-to-front,” and it is expressed as a decimal fraction. The equation is shown below:

The purpose of this experiment is to identify an unknown proprietary drug using thin-layer chromatography. The unknown’s behavior in thin-layer chromatography will be compared with that of its possible component analgesics. The possible unknowns and their analgesic ingredients will be ASPIRIN, caffeine, acetaminophen, ibuprofen.

Materials and Safety:

Preheat a water bath to each bench to about 60 °C.
Collect two TLC plates and two spotting capillaries to spot the plates. Lightly draw a line from the bottom of the plate from 1- cm. mark the first late with 5 points where the samples will be spotted, four knwns and the mixture of knowns. Mark the second late with four marks for the unknows and one for the mixture of knowns. Prepare the developing jar by introducing enough solvent to cover the bottom of the jar to about 0.5cm height. Introduce a filter paper folded to fit in the jar. Allow the solvent to soak the filter paper and let the atmosphere in the close jar equilibrate. The eluent used is ethyl acetate containing 0.5% acetic acid. Crush the tablet using the mortar and pestle and transfer the transferred the tablet to a labelled centrifuge tube.

Add about 5ml of a 50/50 mixture of ethanol and methylene chloride. Heat the mixture gently for few minutes in hot water bath. Allow the mixture to rest for 10 minutes so that the insoluble fraction can settle to the bottom of the test tube. Centrifuge if necessary. Spot the first plate with the solution of the standard provided by the preroom. Place the TLC plate in the jar and allow the solvent to run up the plate to about 1 cm from the top. Indicate the position of the solvent front with the pencil as soon as you remove the plate from the jar. Develop the second plate as you did the first plate.

Visualize the spot under UV light and mark them with pencil. Place the plates in iodine chamber and mark the spots. Sketch the plates in your notebook with necessary comments. Calculate the Rf value of each spot and identify the ingredients of different analgesic drugs. The elution sequence is always dependent on the elution solvent very often sequence is as follows. Ibuprofen, acetylsalicylic acid, acetaminophen and caffeine. Calculation/ Results:

1. (Known solvents)
Distance travelled y the solvent on TLC plate= 5.3 cm
Caffeine = 0.283
Aspirin = 0.896
Acetaminophenm = 0.679

1. (1 knwn solvent and 1 unknown solvent)
Rf value
Rf value = 0.8
Rf value = 0.019
Rf value = 0.019

Rf value = 0.45

Rf value =0.19

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