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2.1: Basic Spectrophotometry

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    OBJECTIVES

    Upon completion of this exercise, appropriate discussion, and related readings, the student will be able to:

    1. Locate the power switch, zero adjustment (where applicable), fine adjustment, coarse adjustment, sample chamber, wavelength adjustment, and readout device on a spectrophotometer.
    2. Check a spectrophotometer for proper wavelength calibration and adjust if necessary.
    3. Properly adjust (set) wavelength, zero (dark current) and 100% transmittance (zero absorbance) of a spectrophotometer.
    4. Measure the absorbance of several solutions.
    5. Calculate the value of an unknown solution by using it’s absorbance and that of a known standard.

    glossary

    • Blank: the solution placed in the sample compartment of a spectrophotometer when adjusting the transmittance to 100%
    • T: The absorbances of all other test solutions are then measured against the blank. Often the word is used as verb, “to blank a spectrophotometer”.
    • Dark current adjustment: adjusts current through detector to “zero” when no light is present.
    • Fine and coarse adjustments: similar to “zero adjustments’ on Certain instruments, allows finer control of 100% transmittance settings.
    • Wavelength adjustment: device which adjusts angle of grating, prism, or mirror to achieve a desired nominal wavelength setting.
    • Zero adjustment: sets instrument’s current reading to 100% transmittance (zero absorbance) when a blank sample is present in the sample compartment.

    MATERIALS:

    • Spectrophotometer(s) Didymium Filter
    • Test solutions Cuvettes
    • Wavelength verification solutions

    PROCEDURE:

    Spectrophotometer Components

    1. Locate the power switch on the spectrophotometer and turn the instrument on.
    2. While the instrument warms up (10 minute minimum), locate the following: fine adjustment, coarse adjustment, zero (dark current) adjustment, sample chamber, wavelength adjustment, and readout device.
    3. Move the fine, coarse, zero, and wavelength controls to familiarize yourself with their operation.

    Wavelength Calibration

    There are several methods that can be used to verify proper wavelength adjustment. Your instructor will demonstrate the use of a Didymium Glass Filter and a Color Calibrator. Specific instructions will vary depending on the materials that are used. Following the instructions given by your instructor, check the wavelength calibration of your spectrophotometer and adjust if necessary.

    Adjustment

    Before using a spectrophotometer, there are several simple adjustments that must be made. These are:

    1. Adjust wavelength adjuster to the desired wavelength
    2. Adjust the zero (dark current) if instrument is so equipped.
    3. Adjust for 100% transmittance (zero absorbance) using a blank solution (distilled water or other material as instructed)
    4. Recheck the zero and 100% T, adjust if necessary.

    These adjustments should be made before each use of a spectrophotometer. Step #3 must always be performed following any adjustment or change in the wavelength.

    Unknown Readings

    Your instructor will demonstrate the proper use of a cuvette in a spectrophotometer. Using this information and steps 1 through 4 from above, measure the absorbance of the standard and unknown solutions provided at a wavelength of 540 nm. Use distilled water as your blank solution. Record your results on the data sheet for this exercise.

    Determining Concentration

    There are three methods commonly used to determine concentration. They are instrument calculation, manual calculation, and use of a standard curve. Instrument calculation and standard curves will not be dealt with in this exercise.

    Manual Calculation

    To calculate an unknown concentration manually, the absorbance of the unknown is compared to the absorbance of a standard using the following formula.

    A = Absorbance, [ ] = concentration

    \[\frac{A_{unknown}}{A_{standard}} \times [standard] = [unknown]\]

    Calculate the values for solutions 1 and 2 using the reading obtained earlier. The value for the standard is 200 g/L. Record your results on the data sheet for this exercise.

    OPTIONAL EXERCISE:

    Effect of incorrect instrument settings

    1. Incorrect dark current setting
      1. Adjust the zero (dark current) so that the instrument setting is not on infinity, but on 2.0 absorbancy reading at 540 nm.
      2. Now measure the absorbance of the standard and unknown solutions provided. Record results on the data sheet.
      3. Calculate the concentration of the unknown.
    2. Incorrect blank setting
      1. Adjust the dark current back to its proper setting.
      2. Place blank solution in instrument and set the 100% T (zero absorbance) to read 90%T.
      3. Repeat steps A2 and A3 above.
    3. Comparison of different spectrophotometers.
      1. Follow the above Procedure.
      2. Calculate concentration of unknowns.
      3. Record results on data sheet.
    Data Sheet, Exercise #1

    NAME:____________

    DATE: ____________
    Absorbance and Concentration of Solutions
    Material Absorbance Concentration
    Standard _________ 200 g/L
    Unknown # _________ _________
    Unknown # _________ _________
    OPTIONAL EXERCISES Absorbance Concentration
    Standard _________ 200 g/L
    Unknown # _________ _________
    Unknown # _________ _________
    B Absorbance Concentration
    Standard _________ 200 g/L
    Unknown # _________ _________
    Unknown # _________ _________
    C Absorbance Concentration
    Standard _________ 200 g/L
    Unknown # _________ _________
    Unknown # _________ _________

    DISCUSSION QUESTIONS:

    1. What is the advantage of using a calculation method to determine an unknown value?
    2. Why must the 100% T be adjusted after the wavelength has been changed?
    3. Why will the zero setting not change significantly as the wavelength is changed?

    OPTIONAL QUESTIONS:

    1. How does an incorrect zero (dark current) setting affect the calculated unknown concentration?
    2. How does an incorrect zero absorbance setting affect the calculated unknown concentration?
    3. Do two different spectrophotometers give the same values for unknowns after proper calibration?

    This page titled 2.1: Basic Spectrophotometry is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by Lawrence Kaplan & Amadeo Pesce.

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