Question: If we have six standard solutions of phosphorus with concentrations of 0, 10, 20, 30, 40, and 50 ppm, their corresponding absorbance values on a spectrophotometer are recorded as 0, 8, 18, 28, 38, and 48, respectively. After developing a regression equation based on this data, we analyze a sample of unknown phosphorus concentration and obtain an absorbance of 65. Since the sample's absorbance exceeds the range of the standard curve, should we prepare higher concentration standards for calibration, or should we dilute the sample before measurement?

Answer: Since the absorbance of the unknown sample (65) is higher than the highest absorbance of the standard curve (48), the concentration is beyond the calibrated range. In this case, the appropriate approach is to dilute the sample before measuring it again. This ensures that the absorbance falls within the established standard range, where the regression equation remains valid. If the sample concentration is too high, extrapolating beyond the standard curve may lead to inaccurate results due to potential deviations from linearity in the spectrophotometric response. To proceed, the sample should be diluted (e.g., by a factor of 2 or more), and the absorbance should be measured again. The obtained concentration from the regression equation must then be multiplied by the dilution factor to determine the actual concentration. While preparing higher concentration standards is an alternative, dilution is the preferred method in routine laboratory analysis, as it is quicker and avoids potential non-linearity at higher absorbance values.