Spectrophotometry

Determination of Analyte Concentration

One of the most common applications of spectrophotometry is to determine the concentration of an analyte in a solution. The experimental approach exploits Beer's Law, which predicts a linear relationship between the absorbance of the solution and the concentration 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 in this case is a plot of absorbance vs concentration. The points on the calibration curve should yield a straight line.

The unknown solution is then analyzed. The absorbance of the unknown solution is used in conjunction with the calibration curve to determine the concentration of the analyte. The data obtained from the standard are used to plot a straight line. The slope and intercept of that line provide a relationship between absorbance and concentration:

A = slope c + intercept

The absorbance of the unknown solution, A_u, is then used with the slope and intercept to calculate the concentration of the unknown solution, c_u.

c_u =

A_u - intercept

slope

Experiment

Objective:

Determine the concentration of an unknown solution.

Approach:

Measure the intensity of transmitted light for various standard solutions.
For each standard solution, calculate the absorbance of the solution.
Construct a calibration curve.
Plot the line-of-best-fit through the experimental points. The computer will give you one here, but you will also plot the same graph using Excel. A nice write up on how to do such can be found here: Putting in data and getting equation Using Excel
Measure the intensity of transmitted light for the unknown solution.
Calculate the absorbance of the unknown solution.
Use the calibration curve to determine the concentration of the unknown solution.

Run each simulation sufficiently long to detect at least 1000 photons. (Not all photons are shown on the screen.) Because the intensity for the blank is used to calculate all absorbances, it is especially important that the intensity for the blank be known accurately. If possible, wait until at least 4000 photons are detected for the blank.

			Concentration: blank 20.0 mmole L^-1 standard solution 40.0 mmole L^-1 standard solution 60.0 mmole L^-1 standard solution 80.0 mmole L^-1 standard solution unknown solution Number of Photons Detected: Elapsed Time: sec Intensity of Detected Light: photons/sec
Light Source	Cell	Detector
			c = mmole L^-1 A = Plotted Points: slope = intercept =

Beer's Law Carbon-Hydrogen Elemental Analysis

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