# Using a Spectrophotometer

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```					                                  Using a Spectrophotometer

Each wavelength is perceived by us as a specific color (see below). For example a wavelength of
450 nm is __________________ in color, the color of orange has a wavelength of
__________________, and a wavelength of 650 nm is ____________ in color.

Three things can happen to light that strikes a solution. Wavelengths that are reflected by the
solution are responsible for its color. Other wavelengths may be transmitted through the
solution and some may be absorbed by it.

A spectrophotometer measures the amount of light absorbed by a solution. The
spectrophotometer separates white light into a spectrum of colors (wavelengths). It then directs a
specific wavelength of light at a tube that contains a solution. The spectrophotometer calculates
the amount of light the solution absorbs. The amount of light absorbed by a solution depends on
the wavelengths of light and on the concentration of the solution.

Exercise 1: Develop an Absorption Spectrum for a Red Solution

In this exercise, you will learn to use a spectrophotometer. The spectrophotometer will project
different wavelengths of light at a red solution. The spectrophotometer will display a number that
indicates the amount of light absorbed by the solution at each wavelength. You will record and
graph these date. The graph, called an absorption spectrum, shows how each wavelength was
absorbed by the solution.

Go to the spectrophotometer and locate the following feature of the machine: the sample
compartment, the wavelength selector, zero control, power switch, display. Your lab instructor
will demonstrate how to use a spectrophotometer correctly.

1. Calibrate the machine at 400 nm. Insert the blank into the sample compartment. Close the
cover.
2. Measure the absorbance at 400 nm. Remove the blank, insert the red sample and close the
lid. Record the absorbance of 400 nm light by the red sample.
3. Change the wavelength by 20 nm. Recalibrate the machine with the blank. Remove the
blank and insert the red sample. Record the absorbance.
4. Repeat step 3 until 660 nm.
5. Return the blank and sample to the container beside the spectrophotometer.

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Absorbance values for a red dye solution
Wavelength (nm)              Absorbance
400
420
440
460
480
500
520
540
560
580
600
620
640
660

6. On the graph paper below, plot the absorbance values for each wavelength of light
directed at the red sample. The curve depicts the absorption spectrum for a red dye
solution. The x axis is wavelength (nm); the y axis is absorbance.
a. Which wavelength of light was absorbed most by the red sample?
b. Explain why the color of the solution is red.

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Exercise 2: Develop a Standard Curve
A standard curve is a line on a graph that shows the relationship between the absorbance value
for a solution and the concentration of solute it contains. To construct a standard curve, a series
of solutions is prepared with different, known concentrations of solute. The absorbance of each
solution is measured with a spectrophotometer. The absorbance values for all the solutions are
plotted on the graph to form the standard curve. The line is then used to determine the unknown
concentration of solute in a solution when its absorbance value is known.

The purpose of this experiment is to learn how to construct and use a standard curve. You will
prepare six solutions with different concentrations of a red dye, measure their absorbance, and
plot these values on a graph. You will then use this standard curve to determine the unknown
concentration of this red dye in various solutions.

1. Prepare 6 test tubes as indicated below. Label the test tubes with a Sharpie. Accurate
measurements are essential.

Test tube                      Contents
W                              5 ml DI water
1                              1 ml red dye and 4 ml DI water
2                              2 ml red dye and 3 ml DI water
3                              3 ml red dye and 2 ml DI water
4                              4 ml red dye and 1 ml DI water
5                              5 ml of red dye

2. Refer to your absorption spectra to select the best wavelength of light to use for
measuring absorbance by red dye.
3. Measure the absorbance of the solution in each tube with a spectrophotometer and
record the absorbance values below. You will need to transfer the solutions into
spectrophotometer tubes. Rinse the tubes with DI water before adding a new solution.

Test tube                      Absorbance             mg/ml of red dye
W
1
2
3
4
5

4. In the laboratory, we frequently express the concentration of a solute in
milligrams/milliliter (mg/ml). Each ml of red dye solution contains 10 mg of red dye.
Convert the concentration of red dye in each test tube to mg of dye/ml of solution. Use
the equation C1V1=C2V2. Your instructor will demonstrate how to make this
calculation. Fill in the last column of the table (above).

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4. The absorbance values in the table represent the data points used to draw a standard
curve for red dye. Plot these values. The x axis is labeled: Concentration of red dye
(mg/ml). The y axis is labeled: Absorbance.

6. Determine the absorbance of red dye in the spectrophotometer tube labelled
“unknown.” Using your standard curve, determine the concentration of the unknown.

7. Clean all test tubes and spectrophotometer tubes.

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