Instructions for Cary 500 – Concentration Measurements

Adapted from Cary Help Files

Susan J. Kohler

August 31, 2000

 

Start the Concentration application and turn on the spectrophotometer:

Step 1

Make sure that there are no other applications open which run the Cary spectrometer. Then double click on the Cary Concentration icon on the desktop.

Step 2

If the spectrometer is not already turned on, turn it on with the on/off switch at the lower left corner of the instrument. The spectrometer will go through a series of self checks. Messages appear in the lower left corner telling you what is happening. When the spectrometer is ready, the traffic light icon will turn green.

Set up data collection parameters:

Step 3

Click the Setup… button on the left side of the window to display the Setup dialog and specify the method parameters for a new method. To do this:

Set up instrument parameters such as wavelength, spectral bandwidth etc.:

Step 4 [Setup dialog | Cary tab]

  1. In the Wavelength field enter the wavelength that you want monitored.
  2. In the Ave Time field enter the required value. A good starting point is 0.1 sec.
  3. In the SBW field enter the required spectral bandwidth. Use the maximum setting (1.5) unless your method specifies another value.
  4. Select Replicates and set the value to the number of replicates that you are using. Alternatively you may select sample/standard averaging to make multiple measurements on the same sample.
  5. Select the Y mode you require. Click Abs to specify the Absorbance mode. Use Emission mode if you are measuring fluorescence.
  6. It is not necessary to enter a value in the Y min and Y max entry fields to specify the displayed ordinate range as no calibration will be performed or displayed.

 

Set up the calibration:

Step 5 [Setup dialog | Standards tab]

  1. Select the Standards tab.
  2. Select ‘Calibrate During Run’. Calibration will be performed and all measurements will be performed in Absorbance.

 

Set up lamp options:

Step 6 [Setup dialog | Options tab]

  1. Select the Options tab.
  2. Check the Auto lamps off checkbox if you want to automatically turn off the lamps at the end of the collect. This option enables you to preserve the lifetime of the lamps. It is especially useful for when performing runs overnight or unattended for long periods of time.
  3. Click on the UV/Vis radio button if you want both the lamps on during the run.
  4. Enter the wavelength at which you would like the source lamp to change from the UV to the Vis/NIR lamp. The recommended changeover is 350 nm for lamps with a UV cutoff. If you are using a Cary 500, enter the detector changeover wavelength as well.
  5. In the Beam Mode group, select the beam mode that you require. In most cases this should be Double Beam and Normal.

 

Set up reporting and printing requirements:

Step 7 [Setup dialog | Reports tab]

  1. Select the Reports tab and specify your reporting requirements for this method.
  2. Enter your name in the Name entry field.
  3. Enter a comment relating to your experiment in the Comment entry field.
  4. Set up your report style by selecting the appropriate checkboxes in the Options group.

    For example, select the AutoPrint checkbox to obtain a printout of your report automatically.
    Select the Parameters checkbox to include your experimental parameters in the report.
    Select the Graph checkbox to include a calibration graph in the generated report.

Set up your standards:

Step 8 [Setup dialog | Standards tab]

  1. Select the Standards tab. This dialog box allows you to enter a list of concentrations that will be used during your analysis.
  2. Enter the number of standards that you are going to use in the Number of Standards field. The table below this field will expand or contract to match your choice.
  3. In the table, enter the concentration of each standard.

Set up your samples and increment names:

Step 9 [Setup dialog | Samples tab]

  1. Select the Samples tab. This dialog box allows you to enter a list of sample names that will be used during your analysis.
  2. Enter the number of samples that you are going to use in the Number of Samples field. The table below this field will expand or contract to match your choice.
  3. In the Samples table, enter the name of each sample. You can enter up to 20 characters for each name.
  4. If the samples have the same name with a different numeric extension, enter the name in the first sample position and then press the Increment button.

 

Set up storage of collected data:

Step 10 [Setup dialog | Auto Store tab]

  1. Select the Auto Store tab.
  2. Select Storage off. The method (with calibration), collected data and report will not be automatically saved. However, you can manually save it all at the end of the collection.

 

Finish Setup:

Step 11

Once you are satisfied with your method setup click OK to confirm any changes you have made and close the Setup dialog.

Zero the instrument:

Step 12

  1. Click the Zero button to zero the system. Alternatively, select Zero in the Commands menu to perform a zero. The Zero dialog box will appear.
  2. Place a blank in the sample compartment and press OK.

 

Measure absorbance of standards and samples:

Step 13

  1. Press the Start button or select Start in the Commands menu. The Standard/Sample Selection dialog box will appear.
  2. Select the samples to be used in the analysis. By default, all samples are selected and appear in the right half of the window.
  3. Click OK to exit the Standard/Sample Selection dialog box. You will be prompted to insert your standards to generate your standard curve, and then will be prompted to insert your samples.
  4. Click OK to measure the sample and display the absorbance. (If replicates have been nominated, then the mean absorbance is calculated after the final sample replicate is read.)
  5. Repeat for the remaining samples.

 

Save your data and/or print your results:

Step 14

  1. Click on the File menu item and select Save Data As.
  2. Enter the file name for the run in the File name field.
  3. Click Save.
  4. Click on the File menu item and select Print.