# Spectrophotometry

## Determination of the Concentration of a Dye

One of the most common applications of spectrophotometry is to determine the concentration of an analyte in a solution. The term analyte refers to the compound for which one is analyzing. In this virtual experiment, you will analyze a solution containing the dye indigo carmine to determine the concentration of the dye. Therefore, the dye is analyte.

The experimental approach exploits Beer's Law, which predicts a linear relationship between the absorbance ( A ) of the solution and the concentration ( C ) of the analyte (assuming all other experimental parameters do not vary).

In practice, a series of standard solutions are prepared. A standard solution is a solution in which the analyte concentration is accurately known. The absorbances of the standard solutions are measured and used to prepare a calibration curve, which is a graph showing how the experimental observable (the absorbance in this case) varies with the concentration. For this experiment, the points on the calibration curve should yield a straight line (which is the prediction of Beer's Law). The slope and intercept of that line provide a relationship between absorbance and concentration:

A = slope C + intercept

The unknown solution is then analyzed. The absorbance of the unknown solution, Au, is then used with the slope and intercept from the calibration curve to calculate the concentration of the unknown solution, Cu.

Cu =
Au - intercept slope

## Experiment

The objective of this experiment is to determine the concentration of indigo carmine in an unknown solution. The spectrometer is set to 611 nm, which is λmax for indigo carmine. All measurements will be made with a 1.000 cm cell.

Select the solution to be analyzed. In addition to the blank (which contains no indigo carmine or anything else that would absorb 611 nm light) and the indigo carmine unknown solution, a series of standard solutions are available. The concentration of indigo carmine in each standard is provided. Measure the absorbances of the blank ( C = 0 ) and each standard solution. Use the known concentration and measured absorbance to add a point to the calibration curve. Once the data has been plotted, graph the line-of-best fit and calculate the slope and intercept. Finally, measure the absorbance of the unknown solution and calculate the concentration of indigo carmine in the unknown solution.

Operation of the Spectrophotometer: Select the desired cell path length and tartrazine concentration. Then start the simulation. Once photons begin reaching the detector, start the Data Acquisition. The intensity of light (photons per second) reaching the detector will be displayed. Note that the simulation employs more photons than are shown on the screen.

Reminder: μM = micromolar = 1 μmole / liter = 1 x 10-6 mole/L

Questions

1. What is the concentration of indigo carmine in the unknown solution in units of μM ?
2. If there were no experimental error, what value does Beer's Law predict for the intercept?
3. On the basis of the calibration curve, is Beer's Law obeyed? Explain your answer.

Light Source
Cell
Detector
Solution

Spectrometer

Data Acquisition

photons/sec

UnknownSolution.html version 3.0
© 2000, 2014, 2023 David N. Blauch