The scenario is that a woman has been stopped by the police due to the condition of her driving. She tells the police that her driving was erratic due to trying to take the lid of a bottle of aspirin which she required for a severe headache. The police could not find the aspirin bottle within the car and the woman then told the police that it got thrown out of the vehicle. Serums samples were taken from the woman to do alcohol and drug tests.
Aspirin is an analgesic drug that provides pain relief without causing unconsciousness and anesthesia. Aspirin is used to help with pain, fever, osteoarthritis, inflammatory conditions, migraine headaches and many other things.
Salicylates are the main substance in aspirin and this is what is being tested for. To test for this substance visible spectroscopy will be used. Spectroscopy has a variety of methods that are the interaction between light and matter. For this practical the method used is visible spectroscopy via the use of a spectrophotometer. This equipment measures the amount of light absorbed by passing a beam of light through the sample and the amount of light is measured by a detector. This produces an absorbance value which can be used to calculate concentrations.
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The aim of this test is to calculate the concentration of the serum samples to find out whether the levels of salicylic acid in these samples are toxic or not.
Methods and Materials
The materials that were used are 0.2mg/ml Salicylic acid, Ferric Nitrate solution, 0.039M HNO3, water and a sample of the woman's serum. The equipment used was a spectrophotometer, a graduated pipette and cuvettes.
To be able to find out the salicylic acid concentrations of the serum samples, six standard solutions had to be created first. Each standard solution was composed of a combination of H2O, salicylic acid, Ferric nitrate and HNO3 all totalling up to 2ml in each solution.
Salicylic acid (ml)
Ferric nitrate (ml)
Contents and volumes in the six standard solutions
Table 1: a table showing the volumes in the six standard solutions.
The components of the solutions seen in table 1 were placed into test tubes using a graduated pipette. The solutions were then mixed thoroughly and left for 5 minutes. After five minutes a sample of each solution was placed into cuvettes using the pipette. The spectrophotometer was then set a 540nm.Using solution 1 as the blank; the spectrophotometer was then set to zero. Every other standard solution was then inserted into the spectrophotometer to obtain absorption measurements using the blank as a zero reference.
Three 2ml samples of the woman's serum were then created using a combination of the materials seen above.
Contents and volumes in the serum samples
Serum sample 1
Serum sample 2
Ferric nitrate solution (ml)
Table 2: a table showing the volumes in the serum samples.
The components of the serum solutions were then placed into test tubes at the volumes shown in table 2. These samples were then mixed thoroughly and left for five minutes. After five minutes the samples were then placed into cuvettes, and then the spectrophotometer. The spectrophotometer was again set at 540nm and the serum blank used as the blank. The other two serum samples were then placed into the machine and the absorption results recorded.
The measurements from the spectrophotometer where recorded and concentrations of Salicylic acid calculated for the standard solution. These concentrations then created a calibration curve to allow the serum sample concentrations to be discovered.
Absorbance at 540nm
Concentration and absorbance measurements for the six standard solutions
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Table 3: a table showing the absorbance at 540nm and the concentration of the solutions.
The concentrations in table 3 where calculated using C1V1=C2V2. Where C1 is 0.02mg/ml, the original concentration of salicylic acid, V1 is the volume of salicylic acid, C2 is the unknown concentration and V2 is 1ml, the total volume of H2O and Salicylic acid. For example standard 3 has the concentration 0.06mg/ml. From looking at table 1 it is seen that C1 is 0.02mg/ml andV1 is 0.3ml. it is then known that C2 is unknown and V2 is 1ml. The equation is then rearranged to C2 = C1V1/ V2 to produce C2=0.02mg/ml x 0.3ml / 1ml = 0.06mg/ml.
The concentration and absorption results are then put into a graph to create a calibration curve.
Figure 1: A calibration curve formed from the 6 standard solutions measurements for absorbance and concentration
The calibration curve shown in figure 1 was plotted from the results in table 3. This curve was then used to calculate the concentration of the serum solutions.
Concentration and absorbance results for the three serum samples
Serum Sample 1
Serum Sample 2
Table 4: a table showing the results obtained from the serum samples absorbances, concentrations and an average.
The results in table 4 where done as a duplicate to make the results more accurate and reliable but due to an error within the practical. An average was later calculated to correct the inaccuracy created by the error, but ideally the test should have been done with 3 samples.
Using the results from table 4 and the calibration curve from figure one it can be determined if the results agree with the drivers story. The average serum sample had a salicylic acid concentration of 0.116mg/ml. In relation to the standard solutions this result is quite high therefore the results confirm she did in fact take aspirin. From this it cannot be fully determined if the level is toxic or not.
Salicylic acid toxicity causes many bio-chemical that target no specific organ. An acute overdose would cause symptoms like nausea, vomiting and tinnitus. If the overdose was chronic there would be symptoms like confusion, fever, hypoxia, dehydration and metabolic acidosis. Other symptoms of toxicity are respiratory alkalosis, alkaline urine and headaches. Comparing the symptoms with the scenario some of the symptoms could explain the reasons for her erratic driving but there still could be other reasons.
Serum levels of salicylic acid provide if the ingested amount was therapeutic, toxic or chronic toxicity. A therapeutic level is 15-30mg/ml and a toxic level is 40-50mg/ml. Any serum level above 60mg/ml is a chronic level. To compare the concentration of the serum to toxicity levels the serum concentration must be converted to mg/dl. For this it is need to be known that there are 100 millilitres in 1 decilitre, therefore 0.116mg/ml needs to be multiplied by one hundred. The serum level is then 11.6mg/dl; this level is not a toxic level but does confirm that aspirin was taken at a possible therapeutic level. This confirms that the woman was telling the truth about taking aspirin but does not confirm why her driving was erratic.
Other test could also be done to test for salicylic acid, these as urine pH, a bedside ferric chloride test and arterial blood gases. Also high-performance liquid chromatography could be used but this requires time that would not be available in a serious toxicity. Salicylic acid has many related compounds, for example, benzoic acid, phenol, magnesium salicylate and aminosalicylic acid. Test for these could also be done to help determine the source.
In conclusion the results from the visible spectroscopy show that the woman did not have salicylic acid toxicity even though she did take aspirin. As her levels where not toxic there has to be another reason for her driving. To confirm this more test could be done like the urine pH and the arterial blood gasses. Also test on the related compounds could be carried out to confirm is she did take aspirin or if the levels in of salicylate in her blood are from elsewhere. The next thing to do would be to test for alcohol and other drugs both legal and illegal. Other drugs to test for could be things like cocaine, opiates, amphetamines, marijuana, methadone, tramadol and oxycodone. These could be testing for either through the blood, urine or hair.
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