Appendix C/S12

From OpenWetWare
Jump to navigationJump to search
Wellesley College

BISC110/112- Introduction to Cell Biology- Spring 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. If the machine is set to your desired program, start at step 5. If not, start at step 2.
2. From the Main Menu, press the soft key under “USER PROGRAM.” Select one of the following:
            Program #5 (400–700nm, absorption spectra of various photopigments) for Lab 8 or for the practical; or
            Program #6 (400–700nm, % transmittance spectra of color filters) for Lab 9.

If you have found your parameters pre-programed skip steps 3 & 4 and go directly to step 5.

3. From the Main Menu Screen use the More soft key and then the Scan λ soft key to select the wavelength scan mode.

4. Select Options under Scan λ and set the following parameters. The first 2 numbers given below (λ min and λ max) determine the scan range. In this example, the range includes the visible spectrum and a portion of the IR spectrum for the copper peak. You may of course set a different range.

λ min. @ 400nm; press ENTER key
λ max. @ 700nm; press ENTER key
λ step @ 2.0nm; press ENTER key
view @ ABS; press ENTER key

scale @ AUTO; press ENTER key and then the EXIT key

5. NOTE: Make certain that you are using the proper type of cuvette for your application. We have 3 types: disposable plastic, regular glass and quartz glass cuvettes. Plastic cuvettes may be used with visible light and a water blank. Regular glass cuvettes are used with visible light and an acetone (dissolves plastic) blank. Quartz glass cuvettes are used with UV light (can pass through quartz but not regular glass or plastic) and an acetone blank.
6. Place a cuvette filled with the appropriate blanking medium into the cuvette holder with the clear sides to the left and right, 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.
7. When the baseline scan is completed the screen will read Ready to Scan.
8. Remove the cuvette containing the blank and insert the cuvette containing the sample. Make sure the clear side of the cuvette faces East/West
9. Press the soft key under Start Scan to initiate the wavelength scan, which will be displayed on the screen as it is being generated.
10. When the scan is completed, it is stored in a file numbered 0–9. If you want to make an overlay scan, record the file # in your lab notebook next to the pigment that was scanned.
If you have additional scans, repeat steps 6–11.
11. If you NOT wish to overlay the spectra you recorded, but you want 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. Press the Print Key on the Keypad, followed by the Graph ONLY key to obtain a print out of your absorption spectrum. (DO NOT print out the Table.)
12. Press the RECALL key.
13. Select the first file # and press ENTER. The first scan should appear on the screen. If you do wish to print a graph with all of your spectra overlayed on one graph, follow steps 14-16 BEFORE you hit the Print key.
14. Press the MORE and OVERLAY SCAN soft keys.
15. Select the second file # and press ENTER. A second scan should appear on the screen, atop the first scan. Repeat for other scans, if necessary.
16. When finished, 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. Don't forget to exit the program to the main menu unless your instructor asks you to do otherwise.

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 (400nm - 700nm). Make sure that the y axis is showing Abs (absorbance) rather than % transmittance.
  3. Put in cuvette with blank (water, acetone, or whatever is appropriate). Make sure that the clear side of your cuvette is directed North/South for this spec and not East/West 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 North/South
  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 when you are done.