Experimental Procedure
Spectrophotometric Determination of Salicylate
in Acne Medication
A stock solution of approximately 100 mM salicylate is
prepared from its dried sodium salt and is then appropriately
diluted to yield five other standards for the calibration curve. To
100 μL of each standard, triplicate samples of face wash, and the
synthetic unknown, 10.00 mL of acidic iron(III) nitrate solution
is added. The absorbance of these solutions is then measured
in plastic cuvettes. This step may be modified depending on
available instrumentation. In one class, each pair of students
had access to an Ocean Optics absorbance instrument that
provided the entire spectrum for each sample. This allows for
simple determination of λmax and the absorbance values at λmax.
In another laboratory setting, one scanning spectrophotometer,
and several single wavelength “spec 20” units were available. In
this situation, each pair of students collected a spectrum of one
standard to determine λmax. Then the spec 20 units were set to
this wavelength and the absorbance of each calibrant and test
solution was determined. If only single wavelength instruments
are available, instructors may provide students with the λmax
value to expedite the measurement process (~535 nm). From the
absorbance data collected for the standards, a linear calibration
curve is constructed (Figure 1), and the concentration of each
unknown is determined using a least-squares fit line derived from
the calibration data. The absorbance data are plotted against the
undiluted standard concentrations to simplify quantitation of
salicylate in each unknown. Since all standards and unknowns