Spectrophotometry is a technique used to measure the concentration of a substance in a sample by analyzing the amount of light absorbed by the sample at a specific wavelength. Here is a step-by-step protocol for determining the concentration of an unknown substance using spectrophotometry:1. Prepare a series of standard solutions: Prepare a series of standard solutions with known concentrations of the substance of interest. These solutions will be used to create a calibration curve, which will help determine the concentration of the unknown sample.2. Select the appropriate wavelength: Determine the wavelength at which the substance of interest absorbs light most strongly. This can be done by measuring the absorbance of a standard solution at various wavelengths and selecting the wavelength with the highest absorbance value.3. Measure the absorbance of the standard solutions: Using a spectrophotometer, measure the absorbance of each standard solution at the selected wavelength. Make sure to zero the spectrophotometer using a blank solution a solution without the substance of interest before measuring the absorbance of the standard solutions.4. Create a calibration curve: Plot the absorbance values of the standard solutions against their respective concentrations. The resulting graph should be a straight line, with absorbance on the y-axis and concentration on the x-axis. Perform a linear regression analysis to obtain the equation of the line y = mx + b , where m is the slope and b is the y-intercept.5. Measure the absorbance of the unknown sample: Using the same wavelength as before, measure the absorbance of the unknown sample. Make sure to zero the spectrophotometer with a blank solution before measuring the absorbance of the unknown sample.6. Calculate the concentration of the unknown sample: Using the equation of the calibration curve, solve for the concentration of the unknown sample x by plugging in the measured absorbance value y into the equation. Rearrange the equation to solve for x: x = y - b / m.7. Report the concentration: Report the concentration of the unknown sample, including the appropriate units e.g., moles per liter or grams per liter .Note: It is important to ensure that the spectrophotometer is properly calibrated and maintained, and that all glassware used is clean and free of contaminants. Additionally, it is crucial to work in the linear range of the spectrophotometer to obtain accurate results. If the absorbance of the unknown sample is outside the range of the calibration curve, it may be necessary to dilute the sample and repeat the measurement.