The UV/Vis Spectrometer

Theory - Startup & Shutdown - Basics of Operation - UV/Vis Operation for the Kinetics Experiment

The UV/Vis spectrometer consists of a light source, a sample compartment, a diode-array detector, and a data acquisition computer. The sample compartment is between the light source and the detector. The spectrometer measures the amount of ultraviolet and visible light transmitted by a sample placed in the sample compartment. Typically liquid samples are used, contained in a transparent "cuvette" or "cell". A flow-through cell for the kinetics experiment is currently in the sample compartment, but another standard cuvette can easily be substituted for it. The sample compartment in our spectrometer is made for 1 cm cuvettes.

The wavelength at which a chemical absorbs light is a function of its electronic structure, so a UV/Vis spectrum can be used to identify some chemical species. For a particular chemical, the amount of the light absorbed is related to the amount of the chemical between the light source and the detector, so a UV/Vis spectrum can be used to quantify some chemical species. To use UV/Vis data for quantitation, "Transmittance" (T, the percentage of light transmitted by the sample) must be be converted to "Absorbance" (A = -log(T)). When 1 % of the light is transmitted (T=0.01), then Absorbance=2. When 100 % of the light is transmitted (T=1), then Absorbance=0. Our spectrometer will automatically record absorbance.

"Beer's Law" states that absorbance and concentration in solution are linearly related for a given chemical, so long as the length of the path through the solution is fixed. Actually, this linear dependence only holds below Absorbance=2. When less than 1 % of the light is transmitted (A > 2), the detector behaves non-linearly. A UV/Vis spectrum is only useful for quantifying chemical species after "calibration". A series of solutions of known concentration must be analyzed and their absorbances recorded. From this data, a mathematical relationship between absorbance and concentration can be determined. For CVD in water when absorbance is measured at 592 nm,
[CVD] = (0.00001169 +/- 0.00000015) * A.
This calibration was determined by V.L. Young in December 2001.

Startup and Shutdown (Return to top)

· Startup.
Remove the cell/cuvette from the sample holder. (Move the lever in the sample compartment and it will lift out easily.) This will allow maximum light through the sample compartment while the spectrometer initializes.
Turn on the computer. Press Ctrl-Alt-Del to log on.
Do not change the User name. Request the Password from the Lab Coordinator if you don't know it.
Wait for Windows to load. Ignore the Acrobat Reader window that opens automatically.
Turn on the UV/Vis by pressing the button on the bottom left front of the rear "box".
Double-click on the "8453 UV-Vis Online(2)" icon to start the Chemstation software and communicate with the instrument.
Type in your name when it asks for the Operator Name. Don't type in a Password, unless you plan to remember it to access your data later. Click the "OK" button.
Once Chemstation has started, return the cell/cuvette to the sample holder.

· Warm up.
Allow the UV/Vis to warm up for 45 minutes before collecting spectra.

· Shutdown.
Close the Chemstation software and the "CAG Bootp Server" software that started automatically.
Turn off the UV/Vis by pressing the button on the bottom left front of the rear "box".
Shut down Windows. Turn off all peripherals.

Basics of Operation (Return to top)

· All control of the spectrometer is done through the Chemstation software. It is Windows-based. There are several modes of operation. A different mode is selected by clicking on Mode in the top menu. Manuals are available from the Lab Coordinator (Jim Caesar).

· Before a sample spectrum can be collected, a "blank" must be collected. A "blank" is a spectrum with no sample in place. It is the reference spectrum for the spectrometer. When you collect your blank, you should try to have the cell exactly like it will be for a real sample except that the compound to be measured is missing. Each time the spectrometer collects a sample spectrum, it automatically subtracts the blank from it. The blank is lost when you close the Chemstation software, so a new one will be needed when you start it again. However, so long as you don't close Chemstation, the software will continue to use the same blank, even if you change modes. There is no need to collect a new blank before each sample!

Procedure for Collecting Spectra with the UV/Vis for the Kinetics Experiment (Return to top)

1. The spectrometer should already be on and warmed up when you arrive. If not, follow the directions for "Startup" and "Warmup" above.

2. Look near the top of the screen, where the current Method and Mode should be displayed. You should see "Method: CVDEXPT.M" and "Mode: Kinetics". If you don't . . .

o Click on Mode > Kinetics.

o Click on File > Load Method. Select CVDEXPT.M

3. Check to make sure the instrument setup is correct. Click in Instrument > Setup Spectrometer. The following values are correct

o Wavelength range from 190nm 1100nm

o Integration time 0.5. This means that 0.5 s of spectra are averaged to make one spectrum, and is a good compromise between reducing noise and measuring "instantaneous" concentration.

o Interval 1nm

o Use only these values!

4. Check to make sure the method is correct. The method tells the spectrometer how to run. It has two parts which should be checked.

o Click on Method > Time & Calculation.

§ Use wavelength(s) 592

§ Background correction none

§ Y-scaling from 0 to 2 AU

§ Run time 1800s. This is the length of time the spectrometer will collect data.

§ Cycle time 30s. This is the time between spectra.

§ You may change the Y-scaling, the run time, and/or the cycle time. During a run, you may abort data collection before the total run time has elapsed, and the software will save the data already collected. You may also tell the software to automatically calculate a rate of reaction for a specified reaction order.

o Click on Method > Options & Info.

§ x Straylight correction on

§ x Autosave spectra to file UOLabA.kd

§ x Prompt for sample information before run

§ You may change the default filename.

5. Now collect a blank spectrum. (Also see Basics of Operation, above.)

o Place the inlet side of the tubing to the flow-through cell in a beaker full of distilled water and turn on the sipper pump. Leave the outlet valve on the reactor open so the water from the cell can drain out. Wait for clear water to begin dripping from the outlet.

o Click on the "Blank" button at the lower left of the screen. The blank spectrum will shortly be displayed. Ideally, it will be a horizontal, flat, slightly noisy line between about 400 and 700 nm, and the y-axis range should be about -0.005 - .005. If it is not, wait a couple of minutes and try again.

o When you think you have a good blank, check it. Wait one minute, then click on the "sample" button on at the lower left of the screen. This will collect one spectrum and automatically subtract the blank from it. The spectrum will be displayed. It should be flat between 400 and 700 nm. If it is not flat, wait another minute and see if it gets worse. Collect a new blank when it stops getting worse and check it. The most important part of the blank is near 592 nm, because this is the wavelength used to quantify the CVD.

o You will only collect one blank for the whole lab period. All of your data depends on it. It is worth getting a good one.

6. Turn off the sipper pump. Return the inlet tubing for the flow-through cell to the reactor.

7. Click on the "Time-based Measurement..." button at the lower left of the screen. Fill in whatever sample information you want, then click on the "OK" button.

8. Prepare your reaction mixture. Start the sipper pump.

9. Click on the "Start" button when you want to start taking data. It will be replaced by an "Abort" button, which you can click on to stop taking data.

o Each spectrum will be displayed as it is collected. You should see a big peak at 592 nm, but it should go back to zero by about 700 nm. If the absorbance is above zero at 700 nm, there is probably an air bubble in the cell that is interfering with the light. Don't stop the spectrometer; you can't stop the reaction, and you may be able to salvage enough data to avoid starting over. Remove the cell from the holder and try to release the bubble by inverting the cell and/or tapping it GENTLY with your finger. If you can remove the bubble before the reaction proceeds too far, you can just omit the data collected while you were working on the cell.

o A plot of Absorbance vs. Time will be displayed as the spectra are collected. A sudden jump in this plot is another indication of an air bubble in the flow-through cell.

10. When data collection for the current run is finished, the spectrometer will automatically calculate a reaction rate for the order you selected. This number may be useful to you, but it is NOT all that you need, and it MAY NOT be accurate. The automatic calculation provides no estimate of uncertainty, and does not know how to handle data that should be omitted (e.g., data collected before you added the second reactant, data collected when the absorbance was outside the linear range, data collected when the flow-through-cell was not in place, data collected when there was an air bubble in the flow through cell). Do not include the spectrometer's automatic calculation in your report!

11. Save your file so that you can access the original spectra again if you want. Click on File > Save > All Data As... Specify a filename different from the default; the default will be overwritten on the next run. The file will automatically save to c:\HPCHEM\1\DATA. You can same it to another folder or disk if you want.

12. Export your data to a comma-separated-variable (.CSV) format to make a text file that can be read by Excel or Matlab. Click on File > Export Result Table As > CSV format. Specify a filename and a folder or disk. Each line of the file will have one value of Time (s) and the corresponding value of Absorbance (AU) separated by a comma.

13. When you are ready to collect data for a new run, return to step 7. Do not collect a new blank!. After data collection for the new run is complete, save the data with a new name.

14. When you are done for the day, clean out the reactor and flush the flow-through cell with distilled water. The Lab Coordinator will shut down the computer and spectrometer.

· Send mail to Dr. Young: valy@bobcat.ent.ohiou.edu.

· Return to top of UV spectrometer page.

(Last modified on 12/14/01)