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  -Before using any instruments,    make sure the lab instructor (TA    or professor) has instructed you    on its use.   DO NOT use any of    the instrument unless you are    supervised by a lab instructor,    graduate instrument assistant,    or by a professor.   All of these    instruments are very expensive,    and printing out and reading    the directions alone is not    enough information to be able    to properly use any of the    instruments.

Ultraviolet Visible Spectroscopy

HP 8453 Diode Array

Operating Instructions
Diode Array Spectrophotometer

Turning on the Instrument:

     1. Turn on the Gateway computer. (Monitor button should be on.)
         a. The NT screen appears, giving you a choice of setups. Hit the Enter              key.

         b. When prompted, hit the Ctrl, Alt, and Delete keys to log on.

         c. Don't enter any password, and then click OK.

     2. Turn on the HP 8453 Diode Array. (Power button directly below model          plate.) Wait for the yellow light on the front of the instrument to turn          green. (This takes a few minutes.)

     3. Double click on the HP Diode Array icon. When the window, UV-Visible          ChemStation, appears, click on Cancel, and the software program will          continue to load.

Taking Spectrophotometric Data:

     1. For data to be taken, a task must be selected. Your selections are          found by clicking on the down arrow u of the Task box: (The following          selections appear.)

              Fixed Wavelengths (Default)
              Spectrum/Peaks
              Ratio/Equation
              Quantification

Determination of Lambda max (Spectrum/Peaks Task)

     1. To determine a solution's wavelength of maximum absorption (Lambda          max), click on Spectrum/Peaks. The Spectrum/Peaks Parameters          window will appear.

     2. For normal use, the following Spectrum/Peaks parameters should be          utilized:
              Peak/Valley find
              Find and annotate up* to 1 peaks (default is on X and 3)
              Find and annotate up* to 3 valleys (default is on X and 3)
              Prompt for sample information* (default is off)
              *(to turn parameter off _ or on X click on parameter)
              Data type*          Display spectrum**
              Absorbance From: 190 nm To: 1100 nm
              *(transmittance data, 1st , 2nd, 3rd, and 4th derivative data may also               be obtained, just click on the down arrow u and then click on the               desired data type)
              **(you may select any data range from 190 nm to 1100 nm)

     3. Click on OK when finished, if you need to change any parameters click          on Setup, and enter the new parameters.

     4. You are now ready to take data.
          a. Taking a spectrum of your blank: Place your blank in a clean cuvette,                inspect for bubbles, and then carefully wipe the exterior surface.                Place the cuvette into the holder in the diode array 8453 (make sure                cuvette is positioned correctly), and secure the locking mechanism                by gently pressing down on the lever. Locate the Sampling box (left                side of screen), and confirm the Manual setting. Click on Blank .                This spectrum displays where the cuvette or blank is absorbing.

          b. Taking a spectrum of your sample: Rinse the cuvette well, and fill it                with sample. Then, inspect for bubbles, and carefully wipe the                exterior surface (with a Kimwipe). Place the cuvette into holder, and                secure it. Click on Sample .

          c. Printing your sample spectrum: Locate the print icon (upper left               corner of screen), and click on it.

Determining an analyte concentration: Beer's Law plot (Quantification Task)

     1. To construct a Beer's Law plot, click on Quantification. The          Quantification Parameters window will appear.

     2. For normal use, the following Quantification parameters should be          used:
              Wavelengths:
                   Use wavelength nm (generally enter lmax of your sample)
              Background correction:
                   None (unless instructed leave on none)
              Calibration:
              Analyte name:
                   (enter the name of your analyte)
              Calibration curve type:
                   LinearOffSet (default is Linear, if you are using absorbance                    LinearOffset is best)
              Enter Concentration:
                   Concentration: Unit (enter concentration unit: ppm, mol/L, mmol/L)
                   Weight & Volume Weight / Volume Unit
                    (to change from concentration to weight & volume just click on it)
                    x Prompt for standard information
                    x Prompt for sample information
                    Data type* Display spectrum**
                    Absorbance From: 190 nm To: 1100 nm
                    *(transmittance data, 1st , 2nd, 3rd, and 4th derivative data may                    also be obtained, just click on the down arrow u and then click on                    the desired data type)
                   **(you may select any data range from 190 nm to 1100 nm)

     3. You are now ready to take data.
         a. Taking a spectrum of your blank: Place your blank in a clean cuvette,              inspect for bubbles, and then carefully wipe exterior surface. Place              the cuvette into the holder in the diode array 8453 (make sure cuvette              is positioned correctly) and secure the locking mechanism by gently              pressing down on the lever. Locate the Sampling box (left side of              screen) and confirm Manual setting. Click on Blank . This spectrum              displays where the cuvette or blank is absorbing.

         b. Taking a spectrum of your standards: Rinse the cuvette well and fill              with sample, inspect for bubbles, and then carefully wipe exterior              surface. Place the cuvette into the holder, and secure it. Click on              Standard . Enter the standard information and click on OK .

         c. Printing your standard curve and statistical data: Click on File, Print              then Current View. To print a single window, click on the window (the              title box will turn blue), and then click on File, Print then Current                        window.

         d. Taking a spectrum of your sample: Rinse the cuvette well, fill it with              sample, inspect for bubbles, and then carefully wipe the exterior              surface. Place the cuvette into the holder and secure it. Click on              Sample .

         e. Printing your sample spectrum: Locate the print icon (upper left              corner of screen), and click on it.

Shutting Down the Instrument:

     1. Click on File, and then click on Exit ChemStation.

     2. When the window, Close HP 845x UV-Visible [1], appears, make sure          you DO NOT Save Configuration, and then click on OK.

     3. Click on CAG Bootp Server (lower left corner of screen), click on File          then Exit.

     4. Click on Start (lower left corner of screen), then Shut Down. When the          window Shut Down Windows appears click on Yes .

     5. When the window Shutdown Computer appears turn off Vectra VL          computer but NOT the MONITOR.

     6. Turn off the HP 8453 Diode Array.