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PHE 1023 / CHE1023 Organic Chemistry LabB.Eng (Hons) in Pharmaceutical EngineeringB.Eng (Hons) in Chemical Engineering Trimester 2, AY 2017 / 2018Student Name(s)Seng Chia HueyDarryl Chua Zong RuDaniel Choy Chi SiongMatric Card Number170117117001571700730Group Number7Experiment CodeOC03Experiment TitleChromatographic Separation Process: Thin-Layer ChromatographyExperiment InstructorMs Sherlyn TanAbstractThe purpose of this experiment was to separate a dye mixture by Thin-Layer Chromatography (TLC) and perform Thin-Layer Chromatography on various dyes. The mobile phase used was ethyl alcohol while the stationary phase was silica gel. The analytes were Eosin, Fuchsine powder and Methyl Red. The average retardation factors obtained from this experiment was 0.795cm for Eosin, 0.688cm for Methyl Red and 0.06cm for Fuschine powder.IntroductionTLC was invented in the early 20th century and grew in popularity given how rapid and inexpensive it was 1. The technique uses a polar adsorbent as the stationary phase and a solvent as the mobile phase which works to separate compounds based on polarity2. In this experiment, the TLC foil, a sheet of aluminium coated with an adsorbent layer of silica gel was used. Silica gel, a relatively polar stationary phase due to the hydroxyl group on its surface interacts strongly with polar compounds. The mobile phase can be a polar or nonpolar organic solvent or a mixture of solvents depending on the compound that is being separated. Capillary action draws the mobile phase across the stationary phase, moving each analyte at a different rate depending on its strength of adsorption to the stationary phase and its solubility in the mobile phase. Polar compounds will be carried along more quickly by polar mobile phases as compared to non polar compounds resulting in a distribution of spots on the TLC foil that allow us to sort the compounds by polarity. The ratio of the migration distance of the analyte to the migration distance of the solvent is known as the retardation factor which can be used to identify compounds due to its uniqueness to each compound. It can also be used to compare between 2 different compounds in a TLC experiment. If a nonpolar mobile phase is used, the compound with a larger retardation factor would be less polar as it has a weaker interaction with the polar stationary phase. ProceduresThe TLC foil was cut into dimensions of 10cm by 15cm. The coating at the sides of the TLC foil was then lightly scraped to prevent lateral division. Using a soft pencil, a starting line was drawn on the silica gel coating that was approximately 20mm away from the bottom of the TLC foil. The 4 dye solutions were prepared in a test-tube each. The first test tube contained a small amount of Eosin while the second test tube contained a small amount of Fuchsine powder. Both were dissolved in 4ml of deionized water. The third test tube contained Methyl Red that was dissolved in 4ml of ethyl alcohol. In the fourth test tube, 1ml of solution was drawn from each of the first 3 test tubes and contained together to give a mixture. The mobile phase was then prepared by filling the separation chamber with 32ml of ethyl alcohol and 8ml of deionised water. A micro-capillary was then used to transfer the dye solutions from the test tubes onto the silica gel and immediately placed into the separation chamber and was allowed to be developed. Once the solvent has reached the desired solvent front, the TLC foil was removed from the separation chamber and left to dry. Observations were recorded and the migration distance of each substance was measured to calculate the retardation factor. ResultsThis experiment was conducted twice as the results from the first separation process was deemed unsatisfactory. Nonetheless, results from both experiments can be found in the Appendix, Figure 9. Photos of the actual Thin-Layer Chromatography separation outcomes can be seen in Figure 1 and 2.For Observations of Thin-Layer Chromatography and the retardation factor for each of the 3 dyes, please refer to Figure 9 in the Appendix. Average Rf Value of Eosin = 0.795cmAverage Rf Value of Methyl Red = 0.688cmAverage Rf Value of Fuchsine powder = 0.06cm Supplementary QuestionsQ1. The stationary phase was silica gel and the mobile phase was a mixture of 4 parts by volume of ethyl alcohol and 1 part by volume of water. Q2. Eosin is the most polar compound, followed by Methyl Red, then Fuchsine powder. From Figure 3, 4 and 5 in the Appendix, Eosin is the most polar compound as it has the most number of oxygen atoms. Oxygen is highly electronegative and can form hydrogen bonds, a relatively strong form of intermolecular attraction. Methyl Red is the 2nd most polar compound as it has 3 nitrogen atoms and 2 oxygen atoms. Nitrogen is also a highly electronegative atom that can form hydrogen bonds. Eosin is more polar than Methyl Red as Eosin has more oxygen atoms and oxygen is more electronegative than nitrogen. The least polar compound is Fuschine powder as it has the least number of electronegative atoms. If there is a greater polar attraction between the mobile phase and the dye than the silica gel and dye, the dye will then spend more time in the mobile phase and move up the TLC foil the fastest, rather than being adsorbed onto the silica gel. Ethyl alcohol was used as the mobile phase in this experiment. It is a very polar compound due the the presence of the hydroxyl group (-OH). Ethyl alcohol has a high eluting power.  The more polar an eluent is, the greater its ability to move compounds through the adsorbent2. The oxygen in the hydroxyl group of ethyl alcohol is also highly electronegative and can form hydrogen bonds. As the mobile phase used in this experiment was highly polar, the most polar dye, Eosin, eluted the fastest.Q3. Throughout the whole of the experiment, 2 main problems were encountered.In procedure number 4, the amount of Fuchsine powder to be added was not clearly specified in terms of mass. The resulting solution could have been too concentrated for the solvent to move. Therefore, the observation in which the dye failed to exhibit any significant movement along with the solvent on the silica gel can be explained.In this experiment, the silica gel was carefully handled by holding it on its sides. However, as the separation process proceeded, it was noticed that there were Fuchsine powder contamination around the center of the silica gel. As such, it is believed that contamination could have occurred prior to the commencement of the experiment. Q4. When recording the distance travelled by the dyes, the end point was determined by the center point of the most concentrated spot as shown below in Figure 6. However, in some cases where there are more than one concentrated spot, a certain consensus has to be developed on how we measured the values. In this case, it was decided that the concentrated spot that was the furthest away from the starting point would be taken. With all these factors in play, it would cause the retardation factor to be vary ever so slightly. Q5.  TLC has numerous limitations as an analytical technique for identifying compounds. The first limitation is that volatile compounds cannot be used with this technique as they tend to evaporate quickly and a large portion of the compound might evaporate while the foil is being left to dry, thus affecting the resolution of the compound. Another limitation of TLC is that the reference values for a compound to be identified using TLC must already be known in order to identify it. This makes novel compounds with no reference values hard to identify. This issue is further exacerbated when two or more compounds have similar retardation factor values, thus making it impossible for TLC to distinguish between chemically identical molecules such as enantiomers and isomers that have similar polarities.Lastly, TLC takes places in an open system and the experimenter has to take additional factors like humidity and air pressure into account which may obscure the results by reducing the reproducibility and credibility of the reference values, which were obtained under different conditions. DiscussionThe average retardation values obtained from this experiment was 0.795cm, 0.688cm and 0.06cm for Eosin, Methyl Red and Fuchsine powder respectively. Eosin has the highest retardation factor. Eosin being the most polar compound out of the 3 dyes eluted the fastest as a polar mobile phase was used.To ensure accurate results, the TLC foil should be carefully handled and there should be as little contact as possible with the silica gel surface. This is to avoid contamination that would lead to unexpected spots upon developing the TLC foil. Markings on a TLC foil should also only be done with a soft pencil as a mechanical pencil may cause the silica coating to be scraped off while a pen might introduce a new dye solution to the silica gel surface. After the addition of the mobile phase to the separation chamber, the chamber should be sealed off to ensure that the mixing ratio of the mobile phase does not change and also to equilibrate the separation chamber. An unsaturated chamber could result in the evaporation of the mobile phase, thus leading to a higher solvent consumption and consequently, a higher retardation value2. Also, during the developing of the TLC foil in the separation chamber, the separation chamber should not be agitated. Any movement to the separation chamber may cause the developing mobile phase to rise unevenly on the TLC foil, causing a skewness of the results. One of the experimental error was the overspotting of Fuchsine powder onto the TLC foil, which could have contributed to the compound having poor separation and running as a streak on the TLC foil, as seen in Figure 7. Experimental ErrorsDuring the transfer of Fuchsine powder from the container into the test tube containing 4 ml of deionized water, some of the powder could have gotten onto the gloves which would have contaminated the TLC foil while it was being handled.Instructions not being specific on the amount of Fuchsine powder to be added, causing the solution to be too concentrated. As a result, it was hard for the solution to elute. Thus, the observation in which the Fuchsine failed to show any signs of elution in the first experiment can be explained.More time might have been required for the dyes to show the true extent of the separation. However, since time was of limiting factor, the dyes’ (Eosin and Methyl Red) retardation factor could be quite close together.ConclusionIn conclusion, while this Thin-Layer Chromatography experiment has certainly proven to be an extremely fast and simple technique to carry out, it has also shown that it does come with certain limitations. As encountered in this experiment, a shortage of information regarding the specific amount of dye to be dissolved could have contributed to a number of unsatisfactory results. Furthermore, the technique presents its shortfalls when it tries to separate compounds that exhibit similar retardation values where, instead of the compounds showing clear signs of separation, they would all be located closely together. In addition, as the experiment is conducted in an open system, the reproducibility would be determined by the experimenter’s ability to control the environmental factors such as temperature and humidity.As such, while the technique can be extremely useful in certain industries such as pharmaceutical and food testing1, for it to be able to produce consistent and accurate results, a certain degree of information must always be available and be provided to the ones that are carrying out the experiment.Applied LearningThin-layer chromatography is widely used in the industry to carry out various tasks ranging from blood tests and qualitative purity tests for medicines.  During the preparation of the TLC experiment, the starting line 20mm away from the bottom of the TLC foil so that the dyes do not dissolve in the mobile phase. TLC is used extensively in three main industries, the pharmaceutical, food and clinical testing industries. In the pharmaceutical industry, TLC is used to determine the purity of a drug compound while in the food industry, TLC is used to analyze dyes and separate lipids. Risk AssessmentRefer to Appendix, Figure 8, for the Risk AssessmentAppendix and CitationsGeneral Internet Site1L. Editors, “Thin Layer Chromatography”, Chromatographyonline.com, 2018. Online. Available: http://www.chromatographyonline.com/thin-layer-chromatography-2. Accessed: 20- Jan- 2018.Technical Report2″Thin-Layer Chromatography”, Courses.chem.psu.edu, 2018. Online. Available: http://courses.chem.psu.edu/chem36/Experiments/PDF%27s_for_techniques/TLC.pdf. Accessed: 20- Jan- 2018Internet Documents3″Material Data Sheet – Basic Fuchsine MSDS”, Sciencelab.com, 2018. Online. Available: http://www.sciencelab.com/msds.php?msdsId=9923017. Accessed: 20- Jan- 2018.4″Material Data Sheet – Basic Methyl Red MSDS”, Sciencelab.com, 2018. Online. Available: http://www.sciencelab.com/msds.php?msdsId=9926095. Accessed: 20- Jan- 2018.5″Material Data Sheet – Basic Eosin MSDS”, Sciencelab.com, 2018. Online. Available: in http://www.sciencelab.com/msds.php?msdsId=9923911. Accessed: 20- Jan- 2018. Figure 1: Experiment 1 Figure 2: Experiment 2 Figure 3: Eosin Figure 4: Fuchsin Powder Figure 5: Methyl RedFigure 6Figure 7Figure 8: Risk Assessment Figure 9: Results