Sandra K. Koster, Ph.D. Lecturer
Department of Chemistry
4003 Cowley Hall
Office Hours: Summer hours are by appointment
(608) 785-8282
(608) 783-7013
CHM 300 Lab
Sandra Koster's Home Page Department of Chemistry UWL Home Page ANALYSIS OF ANALGESIC DRUGS
We will consider four possible analgesic ingredients: aspirin,
acetaminophen,
ibuprofen and caffeine. Make sure you have the structures of these
compounds
in your notebook. Solutions of each of these will be made up and
spotted
on a TLC plate against a solution of an unknown analgesic powder. By
comparing
Rf values, we will identify the ingredients in the unknown
analgesic.
More polar compounds hold more tightly to the polar silica gel on the
TLC
plates and therefore do not move up as high on the plate when the TLC
plate
is developed in a solvent. This results in a smaller Rf
value
for a more polar ingredient and allows the separation and
identification
of the components of an analgesic tablet. Carefully read the Thin Layer
Chromatography technique section in your book for more detailed
explanations
of this kind of analysis.
PROCEDURE
Solutions:
Using your own sample or a stock sample, prepare solutions of aspirin and caffeine in a 50:50 mixture of methylene chloride : ethanol. Each solution can be prepared in a small vial and should have a concentration of about 5 to 10 mg/ml. In addition, prepare an ibuprofen solution. Crushed ibuprofen tablets will be available for this solution. It should be prepared at about 20mg/ml concentration in the 50:50 methylene chloride : ethanol solvent. Note that not all the ibuprofen powder will dissolve since the tablets contain insoluble starch. Several small vials of acetaminophen solution in methylene chloride : ethanol will be available in the lab to use as the fourth reference solution or you can prepare a solution from the solid as you did for your aspirin. Make up a solution of unknown analgesic powder (crushed tablets) just as you did for the ibuprofen powder using a concentration of about 20mg/ml in the same solvent.
Plate Preparation:
Obtain a commercially prepared TLC plate (Silica gel on polyester with fluorescent indicator.) Draw a light pencil line across the bottom of the plate about 1½ cm up. Using the microcaps provided in the lab or capillaries you prepare, spot each solution at equidistant intervals along the pencil line: fill each capillary by dipping it in the solution and gently touch it to the plate to empty it. Use several short touches to empty each capillary so that the spots will be small. There will be four reference spots and your unknown. Use a different microcap or capillary for each spot. Putting the spots on the plate in alphabetical order will make it easier to remember which spot is which. Sketch the plate in your notebook with the identities of each spot labeled. After spotting, look at your plate under the UV lamp in the hood to make sure all five spots are easily visible. DO NOT LOOK DIRECTLY AT THE UV LIGHT! If any spots are weak, add more solution on top of the weak spots with the proper capillary and check under UV light again.
Plate Development:
Obtain a chromatography jar, place a filter paper upright against one wall of the jar and add about 10 mL of developing solvent (0.5% acetic acid in ethyl acetate). About a centimeter of the filter paper should be standing in the solvent. Adjust the amount of solvent in the jar if necessary. Cap the jar and allow it to stand a few minutes until the filter paper becomes wet with solvent. Then place the TLC plate in the developing jar with the spotted end at the bottom of the jar, leaning the top of the plate against the filter paper. The bottom of the plate should be sitting in the solvent but the solvent should not be deep enough to submerge the spots. Cap the jar and allow the solvent to rise up the plate undisturbed until about 80% of the plate is wet. Remove the plate and quickly draw a pencil line across the plate to mark the farthest reach of the solvent. This is called the solvent front. Allow the wet plate to dry in the hood.
Analysis:
Look at the plate under UV light and circle any spots that show up. You may note that some of the spots fluoresce different colors under UV light. There should be a single spot for each known, assuming each sample is pure, and the spots for the different knowns should have risen to different heights. Your unknown may have one or more spots, which should line up horizontally with the corresponding spot(s) for the knowns. For example, Anacin tablets contain both aspirin and caffeine so if you had Anacin powder as your unknown, two spots should show up for it, one rising as high as your caffeine spot and one rising as high as your aspirin spot.
Your instructor may have you place your plate in a closed jar containing some iodine crystals for further analysis. This should be done in the hood since IODINE VAPORS ARE TOXIC. After a few minutes the iodine vapor will preferentially dissolve in the organic spots and darken them. You should remove the plate from the jar with tongs to avoid SKIN BURNS FROM THE IODINE. Circle any additional spots if they appear.
Retention fractions (Rf):
Different compounds should rise to different heights on your TLC
plates,
however the exact height a particular compound rises depends on how
high
the solvent is allowed to rise up the plate. If the solvent travels
higher,
then the spots all travel higher too. To correct for this difference
and
generate a number which can be compared to reported values or to other
people’s work, the retention fraction or Rf value is
calculated.
The retention fraction is defined to be the fractional rise of the spot
compared to the rise of the solvent:
Rf = distance from baseline to spot
The Rf value for a compound will change if a different developing solvent or a different type of plate is used. Calculate Rf values for each spot on your plate. Spots with the same Rf values within experimental error should be the same compound. Draw a labeled sketch of your developed plate in your notebook.
In your conclusion, identify the ingredients in your unknown and detail the logic you used to make your decisions.
Email me at koster.sand@uwlax.edu
last modified 2/9/00