THE CASE OF SULFAMETHOXAZOLE

Let’s consider the theory again to establish the calibration range:
Specific absorbance of sulfamethoxazole is 673 in an alkaline solution at a λmax of 256 nM.
This implies that a solution of 1 g sulfamethoxazole in 100 mL (1%) of an alkaline solution should have an absorbance (or OD, Optical Density) of 673. This value cannot be measured with the usual equipment. It is therefore necessary to consider dilutions in order to fit within the analysis range of spectrophotometers which is around an OD of 1. Starting first with a 1/50 dilution:

  When weighing 20 mg on a precision balance the error is negligible, and the conditions are optimal for making the reference sulfamethoxazole stock solution. In a 100 mL volumetric flask, the 20 mg sulfamethoxazole standard is added, dissolved in a few mL of ethanol and made up to 100 mL with basic water (NaOH 0.05M). This solution should theoretically have an OD of 13.46 according to the above equation. This value being too high for a direct reading on the spectrophotometer, additional dilutions need to be made.
Working solutions 1, 2, 3; corresponding to 1/10, 1/20 and 1/40 diluted solutions are prepared.