One of the many processes that you will use in forensic science is thin layer chromatography (TLC). This is a method which can be used on a wide variety of materials and is principally based on silica gel as the “stationary phase” and a mixture of solvents “mobile phase”. You will hear a great deal more about this method throughout your course, but it is essential, at this stage, to understand the underlying concepts of how it works. In this experiment, you will be asked to separate two drugs of differing polarity, but found in the same class, and then explain the basis of separation (consult literature and lecture notes). The structures of the drugs and silica gel was explained to you in the lecture on Thin Layer Chromatography in week 8 (4).
Safety points and the hazard assessments are at the end of this practical and must be printed off with this practical.
At the end of this experiment, you will have learned:-
• How to correctly set up, develop and interpret a thin layer chromatogram.
• How to explain the simple chemical processes involved in separating materials of differing polarity by chromatography on silica gel.
• How to apply your knowledge of differing functional groups on compounds to predictions about chromatographic separations you will achieve.
1. Prepare the TLC tank with the mobile phase (MeOH / conc. NH3, 100 / 1.5 by volume). (THIS WILL BE PROVIDED FOR YOU)
2. Gently mark a UV active chromatographic plate as described in the lecture. (DO NOT USE INK)
3. Spot (4-5 times) each of (i) methanol, (ii) cocaine solution (1mg/mL in methanol), and (iii) benzoylecgonine solution (1mg/mL in methanol) onto the chromatographic plate.
4. When the spots are dry, develop the chromatogram (put your TLC plate in the already prepared TLC tank)
5. When the chromatogram has developed (at least 2/3 of the way up the plate), remove it from the TLC tank, and IMMEDIATELY mark the solvent front using pencil.
6. Allow the chromatogram to dry at room temperature.
7. Draw any materials that you can see under white and UV (254 nm) light, spray the chromatogram with acidified potassium iodoplatinate and then draw the chromatogram again, noting the position of the spots and their colour reaction.
8. Calculate the Rf values (see lecture notes on how to do this) of any compounds that you can see.
9. Write the experiment up as a full experiment (as detailed on page one of this practical guide) and answer the following questions.
1. How much drug does 25 ?l of solution contain, if the original concentration is 1mg/mL?
2. Why does one drug move further than the other? Explain your answer in terms of the strengths of the interactions between the drugs and the silica gel.
3. Looking at the structures for the cocaine derivatives provided in the lecture, how would the Rf value for ecgonine compare to the values for cocaine and benzoylecgonine you have calculated here (e.g. bigger than both)? Explain your answer in terms of the strengths of the interactions between the drugs and the silica gel.
Word equivalent: 1000 words (including your answers)
Marking scheme for TLC practical: (word equivalent ~1000 words)
Aims: Just one or two sentences detailing the aims of the experiment (5 marks)
Should not be copied from the introduction given in the practical instruction (no marks)
Few paragraphs on analytical technique, and your analyte. (5 marks for technique, 5 marks for analyte), should also be cited properly (i.e. acknowledge the source of information).
Should not be copied from the method given (no mark)
Should not be bullet points
Should be summarised in paragraphs (as a journal article) and written in the past tense and third person
Results and calculations:
A) Table
Number and title to table
Table (8 marks) should include the solvent front measurement; distance travelled by the spot and then the Rf values for all of your analytes as well as standards. Blank must be included in the result table. Include the sketch of the TLC plate.
B) Rf calculation:
Formula to calculate Rf with one exemplar calculation
C) Sketch of plate number and title for the figure
Should include the spots and solvent front and the distances measured.
Discussion & Conclusion
Discussion (6 marks): Discuss what your results mean, number of spots, Rf values, nature of spots etc. How was your solvent front? Whether equilibrium was disturbed or not? Quality of your results etc.
Also discuss the significance of negative control and positive control used.
Conclusion: should only be brief, conclude your findings. Have you achieved your aim? The conclusion should mention that the cocaine has a higher Rf value than benzoylecgonine.