Appendix C/F12

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Wellesley College

BISC110/112- Introduction to Cell Biology Lab- Fall 2012


APPENDIX C: Use of the Beckman DU®530 and Beckman DU 730 Spectrophotometers for Wavelength Scans

APPENDIX C Part I Use of the Beckman DU®530 Spectrophotometer for Wavelength Scans (e.g., Absorption Spectra)
The Beckman Spectrophotometers are used to measure the absorbance of a sample over a range of wavelengths.

    1. From the Main Menu, press the soft key under “USER PROGRAM.” Select one of the following:
                Program #5 (350-700nm, absorption spectra of a green colored solution) for Lab 8 or for the practical; or
                Program #6 (400–700nm, % transmittance spectra of color filters) for Lab 10.
    1. NOTE: Make certain that you are using the proper type of cuvette and insert it properly into the light path. We will use plastic disposable square sided cuvettes in these spectrophotometers.
    2. Place a cuvette filled with the appropriate blanking medium into the cuvette holder oriented so that the clear sides are facing the light path , then press the soft key under Baseline to initiate the baseline scan of the blank material. It will take several minutes to complete the baseline scan.
    3. When the baseline scan is completed the screen will read Ready to Scan.
    4. Remove the cuvette containing the blank and insert the cuvette containing the sample. Make sure the clear side of the cuvette faces the light path
    5. Press the soft key under Start Scan to initiate the wavelength scan, which will be displayed on the screen as it is being generated.
    6. To find out the peak absorbance wavelength, press the cursor soft key. You will get a menu with arrows to move left or right to find your peak. The absorbance of the sample at this peak wavelength is indicated next to the peak in nm. Record it in your lab notebook.
    7. To print your absorbance spectrum, press the PRINT key on the keypad, followed by the GRAPH ONLY soft key to print out the absorption spectrum. Do not print out the table, as it takes a long time and several pages.
    8. Don't forget to exit the program to the main menu!

    APPENDIX C Part II: Use of the Beckman DU 730 Spectrophotometer for Wavelength Scans

    Use of the Beckman DU®730 Spectrophotometer for Wavelength Scans (e.g., Absorption Spectra)

    1. In the main menu, choose wavelength scan
    2. Wavelength scan (350nm - 700nm). (Make sure that the y axis is showing Abs (absorbance) rather than % transmittance.)
    3. Put in cuvette with the solution used as your instrument blank. Make sure that the clear side of your cuvette is directed so that the clear side is facing the light path (top/bottom) for this spec and not right/left as you would if using the DU530 specs.
    4. Press Blank
    5. When the blanking is complete put in cuvette with sample. Make sure that the clear side of your cuvette is directed facing the light path.
    6. Press Read
    7. When scan is complete use the two arrows found to the right of the display to find the peak absorbance wavelength and/or the absorbance at that wavelength.

    To print:

    1. Press Options
    2. Press More
    3. Press Send Data
    4. Unclick Table
    5. Press OK
    6. Don't forget to exit the program to the main menu.