“Why Does Aspirin Cross a Cell? To Get To The Other Side!”
- To understand the definition of a weak acid or base
- To demonstrate the movement of ionized and unionized forms of aspirin between an aqueous phase (water loving) and organic phase (lipid loving) at different pHs.
- To understand how aqueous and organic phases mimic the biological membrane and the intracellular extracellular space.
Aspirin is a weak acid and it tends to ionize (give up a H atom) in an aqueous medium at high pH. Drugs do not cross biological membranes when they are ionized. In a low pH environment like the stomach (pH =2), aspirin is predominantly unionized and crosses membranes into the blood vessels readily. At higher pH, in the intestine (pH = 6), a greater proportion of aspirin is ionized, so it moves across membranes more slowly (however, due to the very large surface area for absorption in the intestine, all the aspirin does enter the bloodstream). The aqueous and organic phases can mimic the environment of the stomach or intestine and the cell membranes.
4 separation flasks (125 ml) or 150 ml beakers with small stir bars, 100 ml graduated cylinders, water, ethyl acetate (or an oil such as peanut oil), 1 M NaOH, pH meter, 4 aspirin (not buffered or enteric-coated), micropipets, filter paper, hand-held UV lamp, graph paper.
Place 1 aspirin tablet (325 mg) in 4 different beakers containing 50 mls of water. The tablet will start to dissolve immediately, but the filler will not dissolve very well. Adjust the 4 beakers with several drops of 1 M NaOH to make the pH of the solutions approximately 3, 5, 7 and 9. The acidic aspirin will counteract the more basic solutions, so several drops must be added to get to a pH of 7 and 9. Record the pH of each solution. Shake or stir well. Include a 5th beaker with no aspirin, just water, as a control.
This experiment can be performed with either peanut oil or an organic solvent such as ethyl acetate to mimic a biological membrane (a hydrophobic medium). A procedure using the ethyl acetate follows. Add ~50 ml of ethyl acetate to each beaker. Stir or shake vigorously for a few minutes. Let the solutions stand for several minutes to allow the phases to separate. The organic solvent is on the top and the water is on the bottom. Carefully insert a micropipet through the organic layer into the water layer and remove a small amount of the water phase. Apply it in a small spot on a piece of filter paper so that it spreads out to about the size of a penny. Allow the spot to dry a bit before adding more sample. The number of times the sample is spotted onto the filter paper must be the same for each of the different pH solutions. Write the pH of the aqueous phase next to the spot. Once the spots dry, the aspirin can be detected as a fluorescent spot by shining a UV lamp (short wavelength setting, 354 nm) over the paper.
Another way of detecting the presence of aspirin in the water phase is to use a UV spectrophotometer or a colorimeter. This is especially useful for quantitation (this would be a good exercise for an advanced chemistry class). Or, students might arrange to visit a scientist’s lab equipped with a UV spectrophotometer to carry out the analysis. Instead of applying test or standard aliquots to filter paper, aliquots can be added to cuvettes and placed in the instrument to obtain a reading of the absorbance of UV light (the absorbance wavelength should be around 275 nm). If quantitation is desired, a standard curve can be constructed and used to estimate the amount of aspirin in the different aqueous aliquots. To make the standards, first make a stock solution of 1 aspirin tablet in water adjusted to a pH of at least 7.0. Calculate the concentration of aspirin in the stock solution (325 mg in 25 ml or 13 mg/ml). After stirring with a stir bar for several minutes, filter the solution into a clean beaker to get rid of the undissolved filler at the bottom. Organic extraction is not necessary since all the aspirin will remain in the water phase at high pH. Make 3 standards by diluting the stock aspirin solution 1:1, 1:2 and 1:10 with water into clean beakerscalculate the new aspirin concentration for each dilution. Include a control beaker with no aspirin. Put an aliquot of each standard into a cuvette and scan the absorbance with the UV spectrophotometer from 200-400 nm.
If the filter paper method is used, the more fluorescent the spot, the more aspirin present in the sample. So, at the lower pH, the aspirin will become unionized and move into the organic phase. The pH 3 spot should not be fluorescent (the control spot should not fluoresce either). As the pH increases, more aspirin will be ionized and stay in the water phase. The pH 7 spot should be intensely fluorescent (Figure 6).
If a spectrophotometer is used to scan across a wavelength ranging from 200-400 nm, an absorbance peak will be present at approximately 275 nm. The absorbance level (peak height) is increased as the pH of the aqueous samples increases. (Note: at higher pH, the peak tends to move to higher wavelengths and it picks up a shoulder. This is probably due to contamination by the ethyl acetate which can become hydrolyzed at the more basic pH). For quantitation of the results, construct a graph of the absorbance area (or peak height) vs aspirin concentration in each of the standards. The amount of aspirin in each of the samples dissolved at different pHs can be estimated from this standard curve (i.e. for a given absorbance reading for each sample use the graph to find the aspirin concentration in the sample).
Discussion of Results:
Students can discuss how the effect of decreasing the pH caused the aspirin to move into the organic phase (it was unionized at low pH) while increasing the pH caused the aspirin to stay in the water phase (it became ionized). This is exactly how it happens in the body, where the cell membrane is the “organic” phase and the extracellular or intracellular space is the water phase. They can predict where the aspirin is more likely to be absorbed into the bloodstream (stomach better than small intestine). Students should be able to describe the properties of a biological membrane (i.e. lipophilic core) that allow drugs in their non-polar (unionized) form to move across, along the concentration gradient.
Have students provide a definition of a weak acid in the context of the experiment. Determine if students can apply the concepts learned from the example with aspirin, a weak acid, to a drug that is a weak base, such as cocaine. The students should be able to hypothesize how the results would have differed if they had performed the experiment with cocaine. Without actually carrying out the experiment, they should be able to predict whether cocaine that is orally administered by chewing coca, is absorbed more readily through the stomach or the small intestine. If they quantitated their aspirin data, they should be able to plot hypothetical data for cocaine, with the expected results (with decreasing pH the cocaine should stay in the aqueous phase).
Figure 6 Aspirin fluoresces on the filter paper under a UV lamp. At low pH, aspirin resides in the organic phase. At increasing pH, aspirin becomes charged and resides in the aqueous phase, causing more fluorescence.