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Thin-Layer Chromatographic Analysis of Drug Component



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    TLC is used because it is “sensitive, fast, simple and inexpensive analytical technique”2 (Mohrig 151). The unknown solid (unknown #3 in this case) used in the experiment was obtained and added to a 2. 5ml of Ethanol/dichloromethane in a test tube. This was the solvent used in the experiment because Dichloromethane volatility and ability to dissolve a wide range of organic compounds makes it a useful solvent for many chemical processes.

    The solid was already in powder form therefore we did not have to crush it but it the unknown was in a tablet form, one would need to reduce it to a powdery-like form in order to perform this Thin-Layer Chromatography experiment. The powder solution was thoroughly mixed with a glass rod in the test tube containing the solvent, until completely dissolved. The solution was then pour into a small vial for further used in the experiment. A developing chamber was then prepared to continue the TLC.

    The developing chamber is a critical technique of TLC for it becomes the principal reference to recording the Rf values of the solutions. 5ml of Ethyl Acetate Acid was poured inside a 250ml beaker. As stated in the introduction, CH3COOCH2CH3 was the developing solvent in our chamber for this solution. The beaker was then covered with a piece a foil and places under a hood. This was done so that the chamber would be ready as soon as the TLC plate prepared, as well as for security reason because of the level of toxicity of dichloromethane and it’s “possibility that prolonged inhalation […] may cause cancer. 1 (Lehman 145). For this reason, gloves and safety goggles were worn at all times. A TLC plate was obtained and with a pencil, 4 spots were made gently on its surface to serve as reference point to place the solutions. The points were respectively 1 cm from each other with the first and last point being at least 1. 5 cm from their corresponding sides and all points were 1. 5 cm from the bottom, to give time to the developing solvent to expand through all the points at the same rate once the TLC plate put in the developing chamber.

    Using capillary micropipettes, the solution were spotted to the plate. Spotting is the process of adding samples unto a TLC plate by simply applying the end of the pippette on the plate and letting the solvent adhere to the plate. It was important to ensure that the spots made were extremely small because the samples could mix in the developing chamber by having the diameter of the dots increase and overlap each other, ruining the experiment to compare the unknown sample to one of the unique on given.

    Correlating to the issue of ruining the experiment, one should make sure that the TLC place is being held on the side and that no fingers or other foreign object come in contact with the lateral part of the plate for it very probable that moisture, fingerprints and other debris come in contact with the Ethyl Acetic Acid in the chamber and obstruct the spots from the solutions. The 4 samples were spotted as such, from left to right: Sample 1- Caffeine, Sample 2 – Aspirin, Sample 3 – Acetaminophen and Sample 4 was the unknown.

    Once the TLC was spotted properly, it was put into the developing chamber, under the hood. Once the TLC plate put in the beaker, one should make sure the paper is sitting properly and evenly in the solution. One of the problems encountered in this experiment was that the TLC plate obtained was not cut evenly in the bottom therefore the solvent was rising up through Sample 1 and 2 at higher rate than 3 and 4, making the experiment biased and invalid.

    The developing solvent is to rise through all points on the plate at the same rate in order to get proper results. The chamber was to be watched closely because if the solvent was to go beyond the plate, we wouldn’t get the “distance traveled by the solvent front”1 (Lehman 668) value needed to calculate Rf. Once the development solvent reached about 5 cm from the top of the TLC plate, it was removed from the chamber yet still kept under the hood to dry momentarily and let the solvent evaporate.

    Once the plate was dry, it was removed from under the hood and observed under a UV light with short wavelength. This is called “quenching” of the TLC plate fluorescence and was a beneficial step in experiment because the samples absorbing this wavelength will “become darker against a green or white fluorescence because of the UV activation of the fluorescent indicator in the plate”4 (Ficsher, Funk, Jork & Wimmer).

    With the spots being visible, the final solvent front position was marked horizontally with a pencil as well as the final displacements of the sample spots on the plate. Using a ruler, the displacements of both the solvent and the samples were recorded in the data. The developing solvent travelled a distance of 4. 5 cm before being removed from the chamber. The sample travelled respectively at a distance of 0. 8 cm for Caffeine, 3. 7 cm for Aspirin, for Sample 3 – Acetaminophen, the distance travelled corresponded to 2. cm and for the unknown Sample #3, the distance recorded was of 3. 7 cm, similarly to Sample 2 – Aspirin. The Rf values of each sample was calculated by dividing the distance travelled by the samples by the distance travelled by the solvent front. The Rf values were found to be 0. 1778 for Caffeine, 0. 822 for Aspirin, 0. 5778 for Acetaminophen and 0. 822 for the unknown sample #3. The developing solvent and the Ethanol/dichloromethane were disposed of properly in the liquid waste container.

    Thin-Layer Chromatographic Analysis of Drug Component. (2017, Mar 02). Retrieved from

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