UV-VIS Spectroscopy

Ultraviolet (UV) and Visible (VIS) light can cause electronic transitions. When a molecule absorbs UV-VIS radiation, the absorbed energy excites an electron into an empty, higher energy orbital. The absorbance of energy can be plotted against the wavelength to yield a UV-VIS spectrum. UV-VIS spectroscopy has many uses including detection of eluting components in high performance liquid chromatography (HPLC), determination of the oxidation state of a metal center of a cofactor (such as a heme), or determination of the maximum absorbance of a compound prior to a photochemical reaction. Most organic compounds that absorb UV-VIS radiation contain conjugated pi-bonds. Both the shape of the peak(s) and the wavelength of maximum absorbance (l_max) give information about the structure of the compound.

Ultraviolet radiation has wavelengths of 200-400 nm. Visible light has wavelengths of 400-800 nm. Plastic cuvettes can be used to hold a sample if you wish to scan only the visible region. Since plastic absorbs UV radiation, more expensive quartz cuvettes are used when ultraviolet scans are desired.

Our department has two Hewlett Packard UV-VIS instruments, the HP 8342A and the HP 8453. Both are located in Salem 104. The HP software ChemStations is used to operate the instruments.

8432 UV-VIS spectrometer	8453 UV-VIS spectrometer

To use the UV-VIS spectrometers:
1. Open ChemStations by double-clicking the “HP UV/Vis Online” icon. 2. Press the “Cancel” button when prompted to enter a password. No login or password is needed.
3. Use the “Instrument” dropdown menu to turn on the lamp(s). There is only one lamp on the 8432. It serves as both the visible and UV light source. There are two lamps in the 8453. Turn on the deuterium lamp for scanning the UV region and the tungsten lamp for visible scans. 4.  At the top right of the ChemStations window, make sure the Mode is set to standard. 5. In the Task window, select “Spectrum/Peaks” from the dropdown menu if it is not already selected. Choose the Setup icon in the Task window. The data type should be set to Absorbance. For a visible spectrum, make the lower wavelength limit 400 nm and the upper 800 nm. For scanning the ultraviolet region, make the limits 200 and 400 nm. 6. In the Sampling window, select Manual from the dropdown menu if it is not already selected.
ChemStations Main Window
7. Fill a cuvette with solvent and place the cuvette into the sample holder. Lock the cuvette into place using the lever on the side of the holder. 8. Obtain a background spectrum by clicking the word Blank in the Sampling window. If a “Last Blank Spectrum” window appears, simply close it. 9. Remove the cuvette from the sample holder and fill the cuvette with your sample. Return the cuvette to the sample holder. 10. Obtain a spectrum by clicking Sample in the Sampling window. 11. The spectrum should appear in the display. If it is satisfactory (absorbance <1 for most intense peak), then select the “Overlaid Sample Spectra” window and print your spectrum.
Error Messages: If you see a red error message bar at the bottom of the ChemStations window, read the text and correct the problem. To export the data to Excel: 12. Select the spectrum itself by clicking on the curve. (Data point boxes should appear on the curve once it is selected.) Use the File dropdown menu and choose “Export Selected Spectrum as” then “CSV format.” Save your spectrum to a floppy disk. What's the advantage? You can easily determine lmax, even for a shoulder, by viewing the data points in Excel. You can also format the graph in Excel and easily insert it into a Word document. When you are done: 13. If others are waiting for the instrument, click the “Clear” icon on the toolbar to clear your spectrum from the window. If no one is waiting to use the instrument immediately, turn off the lamp(s) and close the HP software. 14. Dispose of any waste and clean your cuvette.

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