The spectrophotometer is an instrument used to measure the effect of a sample on a beam
of light.
We can learn a lot about a substance by studying the way it interacts with light.
A lamp provides the source of light.
The beam of light strikes the diffraction grating, which works like a prism and separates
the light into its component wavelengths.
The grating is rotated so that only a specific wavelength of light reaches the exit slit.
Then the light interacts with the sample.
From this point, the detector measures the transmittance and absorbance of the sample.
Transmittance refers to the amount of light that passes completely through the sample
and strikes the detector.
Absorbance is a measurement of light that is absorbed by the sample.
The detector senses the light being transmitted through the sample and converts this information
into a digital display.
Let's practice by performing a simple experiment.
In this exercise, we'll determine the absorbance spectrum of red food coloring.
To begin, set the wavelength to 380 nanometers.
With the sample chamber empty and the lid closed, set the display to 0% transmittance
by using the zero control knob.
There are two cuvettes.
One contains only milli-Q water.
This is our blank.
Wipe the blank cuvette with a kimwipe to remove any fingerprints and place the cuvette into
the sample chamber by aligning the guide marks.
Remove the curvet with the blank solution.
Now set the display mode to absorbance by pressing the mode control key.
The second cuvette contains milli-Q water and red food coloring.
This is our sample.
The absorbance of the sample is .845.
Press the mode control key to select transmittance.
The transmittance for the sample is 14.2%.
Record this in your lab notebook.
Increase the wavelength by 25 nanometers to 405nm.
Wipe the blank cuvette again and place it in the sample chamber.
Adjust the transmittance control knob until it displays 100%.
Now remove the blank.
Wipe the sample cuvette and place it in the sample chamber.
The transmittance for the sample at 405 nanometers is 7.4%.
The absorbance for the sample is 1.130.
Increase the wavelength by 25 nanometers to 430.
Repeat these steps with the blank and sample, writing each value in your lab notebook.
the wavelength by another 25 nanometers to 455 and test the sample again.
By repeating these steps up to 680 nanometers, we can plot the absorbance spectrum for red
food coloring.
Now that we’ve finished our first practice exercise using the spectrophotometer, you’re
ready to enter the lab and try it for yourself.