Operation of the PE Lambda 35 UV-Visible Spectrometer

WARNING: Please do not place any liquid containers on or near the PE Lambda Spectrometer or the associated computer. Liquid spills will destroy the electronic components of these instruments.

1. Open the sample compartment and make sure that both cell holders are empty. Then Power on the spectrometer (rocker switch at back right on top).
2. The computer is in "always on" mode--it is not necessary to turn it on.
3. Move the mouse to wake up the computer, and log in.
4. From the Windows desktop, double click (DC) on "UV-Vis" icon. This will open a window called "UVWinLab". Superimposed on this will be a grey window called "UVWinLab/Methods".
5. In the lower left corner of the UVWinLab window you will see "Instrument initializing". Wait until this changes to "Instrument ready".
6. The Methods window lists the methods that have been set up for collecting spectra. A method is a set of parameters, such as wavelength range, spectrum resolution, automatic saving of spectrum, and so on. DC on the method of choice. If you are not sure, DC on "scan1.msc", which is an all-purpose method that can be modified to suit your needs. Double clicking on a method will open a new gray window titled with the name of the selected method (e.g., "UVWinLab/Methods/scan1.msc").
7. The Method name window has 3 tabs at the bottom called "Scan", "Inst", and "Sample". The scan tab should be colored white, indicating that the scan parameter window is open; the other tabs are grey.
   • In the Scan window, enter
     • a "Start" wavelength (usually 1100 nm, unless you know where the sample absorbs)
     • an "End" wavelength (usually 200 nm, unless you know where the sample absorbs)
     • a "Data" interval (usually 1 or 2 nm)
     • an "Ordinate max" value for the output
     • an "Ordinate min" value for the output
     • a "Display mode" for the output (usually "serial", meaning that only one spectrum at a time can be viewed).
   • At the bottom of the window, click the "Inst" tab. In the Inst window select
     • a scan speed (usually 960 nm/min)
8. If you plan to run a lot of spectra using the same parameters, you may want to save the method under a new name, so that you can use it again. If so, C "File", C "Save As", enter a new file name. In this window you can also choose whether or not to run a baseline every time you want to run a spectrum. If so, make sure the box beside "Autozero on Start" is selected. If not, deselect this box. Then C OK. If you have selected "Autozero on Start", the instrument will automatically prompt you to load a blank sample prior to every spectrum. If you have deselected this option, you will have to run the baseline before collecting your first spectrum, and periodically after that.
9. Assuming that you have deselected "Autozero on Start", C the "Autozero" button at the top of the window. This will bring up a small window that instructs you to load a blank sample into the instrument. A blank can be any of the following:
   • Nothing in either beam.
   • A set of empty matched cells, one in each beam
   • A set of matched cells containing the solvent used to dissolve the sample, one in each beam
   Open the sample compartment cover and insert the desired blank. Then C OK.
10. Allow the baseline scan to complete. This requires 1-2 minutes. When "Instrument ready" appears in the bottom left corner of the UVWinLab window,
11. C the "Start" button at the top of the window. This opens a small window prompting you to insert a sample.
12. Open the sample compartment cover and place your sample in the holder nearest you. Close the sample compartment cover.
13. C OK. The spectrum will be scanned and will be shown in a white "Graph" window that opens on the screen. A label for the scan (such as scan002) will be shown in the bottom left corner of this white screen. Wait until "Instrument Ready" appears in the bottom left corner of the UVWinLab window.
14. Save the file if desired using the "File, Save As" menu selections.
15. Print the spectrum if desired using the "File, Print" menu selections.
16. To run another spectrum with the same parameter settings, C "Start". To run another spectrum with different parameter settings, minimize the "Graph" window, open the Method name window (C:\UVWI...) and return to step 7. When parameters have been reset, click "Start".

    NOTE: When "Start" is clicked, the previous spectrum will be lost and replaced by the new one. To preserve the old spectrum, you must save it before running another one.

Normally a UV-Visible spectrum is presented so that

• The wavelength range is defined by convenient numbers (e.g., 900, 300 rather than 876, 294) such that the range of absorption is fairly closely bracketed.
• Absorbtion bands have intensities of at least 0.25 and at most 2 absorbance units. In order to achieve this requirement, it may be necessary to do several scans using solutions of different concentrations, then overlay the resulting scans so that all absorption bands are properly presented.

Normally, one begins with a fairly concentrated solution and uses the entire wavelength window of the instrument (1100-200 nm). Once the spectrum has been run, the wavelength window may be narrowed appropriately by modifying the output parameters set earlier

1. Open the Graph window
2. C "View" in the top menu
3. In the View menu, C "Format Graph"
4. Enter desired values of parameters. Here you can also choose colors and specify a spectrum grid if you want.
5. Click OK. The spectrum should now be modified according to the new parameter selections.
6. You can now save or print.

• Close all windows.
• LOG OFF of the computer.
• Turn off the UV-Visible spectrometer.
• Remove sample and reference cells from sample compartment.