A spectrophotometer is a device used to measure light at a specific wavelength. It consists of two parts: a spectrometer and a photometer. The spectrometer provides light at a specific wavelength. The photometer measures how intense the light is. By calculating the amount of light that a solution is able to absorb and applying Beer's Law, the spectrophotometer can determine the concentration of a colored solution.
Plug in and power on the spectrophotometer. Run the machine for five to 10 minutes to allow it to warm up.
Find the wavelength knob beside the sample compartment and rotate it to set the wavelength.
Turn the filter wheel to select the corresponding filter. Use violet for wavelengths between 300 and 375 nm, blue for wavelengths between 375 and 520 nm, yellow for wavelengths between 520 and 740 nm, and red for wavelengths of 740 to more than 900 nm.
Press the mode button located at the front of the spectrophotometer to select the mode that displays Percent Transmittance and Absorbance simultaneously.
Open the sample chamber to ensure it is empty, then close it. Turn the left front dial to set Percent Transmittance to 0 percent.
Don gloves and clean a cuvette with a lab wipe to ensure cleanliness and reduce the risk of erroneous results. Insert the cuvette filled three-quarters of the way with solvent into the sample chamber and close the door.
Rotate the right front dial until it reads 100 percent T.
Remove the solvent cuvette and replace it with a sample cuvette. Close the sample chamber.
View the meter to determine the reading and record it in your records.
Plug in and turn on the spectrophotometer. Allow it to warm up for 15 minutes. This is necessary for the machine to perform properly.
Adjust the wavelength knob located beside the sample compartment to the desired wavelength.
Check the sample compartment to ensure it is empty and closed. Adjust the zero control knob located at the left front of the spectrophotometer by rotating it until it reads 0.
Don gloves and wipe a blank cuvette with a lab wipe. Insert the cuvette into the sample compartment and close the door.
Adjust the light control knob located at the right front of the spectrophotometer by rotating it until it reads 100. Remove the blank cuvette and insert a sample cuvette. Record the value shown on the meter.
Plug in and turn on the spectrophotometer. Allow it to warm up for 15 minutes.
Press the Percent T/A selector to select Percent Transmittance or Percent Absorbance mode. Locate the wavelength dial beside the sample chamber and set it to the desired wavelength.
Don gloves and wipe a cuvette with a lab wipe to clean it and remove any fingerprints. Insert a clean, solvent-filled cuvette into the sample chamber and close the door.
Adjust the right front dial to read 100 percent if in Percent Transmittance mode or 0 if in Absorbance mode.
Replace the solvent-filled cuvette with a sample cuvette. Record the value shown by the spectrophotometer.
Special light filters may be required on some spectrophotometers if working at certain wavelengths.
The machine must be zeroed out after each sample or if changing the wavelength.
Failure to allow the spectrophotometer enough time to warm can result in erroneous results.
Make sure cuvettes are free of any particles, smudges or fingerprints, as these can throw the machine’s calculations off.
Spectrophotometers are expensive machines. Take care to not inadvertently damage the machine while using it.
Check the sample chamber to ensure it is empty and that both the chamber and test tube access doors are not open. Press the power button located on the back of the spectrophotometer to turn on the machine. Wait 10 minutes to allow the machine to warm up.
Type the desired wavelength on the keypad and press the “go to” key.
Don gloves and wipe the blank solution cuvette carefully with a lab wipe to remove any residue or fingerprints. Insert the blank solution cuvette into the sample chamber, aligning the clear face of the cuvette to face the front of the machine.
Locate the “auto zero” button on the spectrophotometer keypad and press it.
Replace the blank solution cuvette with a sample cuvette that has been cleaned with a lab wipe. Record the absorbance displayed on the screen.
- Purdue University Department of Chemistry: Visible Spectroscopy
- University of Cincinnati Clermont College; Microbiology: Spectrophotometer Use; David B. Fankhauser; July 2007
- Rice University; Principles of Spectrophotometry; David R. Caprette; May 2005
- Bates College; Use of the Spec 20; Greg Anderson; September 2008