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How to Determine the Concentration of Paracetamol in a Sample

Paracetamol, or acetaminophen, is a common over-the-counter painkiller found in drugs like Tylenol. You can determine the concentration of acetaminophen in a sample with a variety of methods. The one most convenient for you will depend on the kind of sample and the level of sensitivity and precision you require. One of many possible approaches involves spectrophotometry -- shining light of a particular wavelength through your sample and using the absorbance to figure out how much acetaminophen was present. The method described here was reported in the "Journal of Food and Drug Analysis" in 2008.

Things You'll Need

Gloves, goggles and lab coat (put these on before you start)
Calibrated micropipette with disposable plastic tips
4 percent copper (II) sulfate in water (reagent C)
Test tubes &test tube rack
4 percent bicinchoninic acid (BCA) in water (reagent B)
Parafilm
Ethyl acetate
Fume hood
Sodium chloride
Spatula
Pasteur pipettes with rubber bulb
Standard solution of acetaminophen with known concentration
Cuvette
Spectrophotometer

Instructions

- 1
  Measure out 200 microliters of reagent C with your micropipette and add it to a clean test tube. Measure out 5 milliliters of reagent B, add them to the test tube and swirl gently to mix.
- 2
  Cover the test tube with Parafilm and keep it for later use.
- 3
  Pipette 0.5 mL of your unknown into another test tube.
- 4
  Transfer your test tubes to the fume hood.
- 5
  Add one milliliter of ethyl acetate to the unknown, followed by a few crystals of sodium chloride. Cap the test tube and shake it vigorously for 30 seconds to mix the contents, being sure to hold your thumb on the cap at all times as you do so. Immediately afterwards, remove the cap to release any pressure, then recap the tube.
- 6
  Allow the contents of the tube to separate into two layers. The ethyl acetate is less dense and thus will form the upper layer or "organic phase." Add 2 mL of the solution you prepared in Step 1 to another clean test tube.
- 7
  Using one of your Pasteur pipettes, carefully transfer the bottom layer to a clean test tube, then transfer the organic layer to the tube containing the 2 mL of mixed reagent. Take this tube and swirl it gently or cap it and vortex it to mix. Allow it to stand at room temperature for five minutes.
- 8
  Take a clean cuvette and add 1 mL of deionized water. Place it in the spectrophotometer and use it as a blank to correct the spectrophotometer reading. The precise instructions to follow for using your spectrophotometer will depend on the manufacturer. See their guidelines for details.
- 9
  Clean out the cuvette and dry it thoroughly (or use another clean cuvette) and add some of your mixed reagent to a clean cuvette until you reach the full 1 mL mark. Place the cuvette in the spectrophotometer and measure its absorbance.
- 10
  Add 1 mL of the standard solution to a clean cuvette (either clean and dry the old one or use a new one), then test it in the cuvette and record its absorbance.
- 11
  Add 1 mL of your unknown to a clean cuvette, place it in the spectrophotometer and measure its absorbance.
- 12
  Subtract the absorbance of the reagent mixture blank from the absorbance of the unknown. Do the same for the standard.
- 13
  Divide the result for the unknown by the result for the standard, then multiply by the concentration of acetaminophen in the standard. This will give you your paracetamol concentration.

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