See the Cary WinUV Software Manual reference on page 49. Fluorescence Spectroscopy Background Information

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1 1 Fluorescence Spectroscopy Background Information Instructions for the Operation of the Cary Eclipse Fluorescence Spectrophotometer See the Cary WinUV Software Manual reference on page 49. Fluorescence Spectroscopy Background Information Fluorescence is an excitation and emission process. The absorption of electromagnetic radiation excites atoms or molecules to a higher energy state. Emission of this radiation, in the form of electromagnetic waves, then follows as the atoms or molecules return to their ground state (Skoog 137). Several factors affect whether a compound will fluoresce, namely the substance s structure, including its rigidity, the temperature of the environment, the ph of the solution, and the concentration. It has been found, for example, that substances with aromatic functional groups and that have rigid structures fluoresce better than other compounds. As a result, fluorescence spectroscopy may be best utilized for compounds with an aromatic structure. Substitution on rings can also have an effect on fluorescence. An example of this would be with the halogens. As one moves down the periodic table for the halogens, a decrease in fluorescence is observed. Fluorescence can be useful in helping identify compounds by providing information about the aromaticity of the compound in question, which subsequently aids in the determination of the structure. (Skoog ).

2 2 Cary Eclipse Fluorescence Spectrophotometer Before starting any runs, you first must turn on the fluorescence spectrophotometer. The power switch is below the Cary emblem. Scan Click on the Cary Eclipse icon Scan. On the top toolbar, click Setup and then from the drop-down menu, click Setup. There will be several tabs underneath the main Setup tab. Make sure the parameters are set as follows: o Under the Cary tab: Under the Instrument setup section: Data mode highlight Fluorescence from the drop-down menu. The Scan setup should have the Emission or Excitation button clicked depending on which spectrum is desired. X mode should be set for Wavelength (nm). Set the Excitation (nm) to a wavelength that your molecule will absorb light at. These values can typically be found in literature, or your instructor may provide them. Enter values for the Excitation slit (nm) and Emission slit (nm). These will most likely be provided for you. If not, start out trying 5 nm as a value for each slit.

3 3 Enter the region of the spectrum that you want to scan by typing in values for Start (nm) and Stop (nm). Again, these may be provided for you. If not, take your Excitation value previously entered and add this to the sum of the slits. This will become your Start (nm) value. Designate your Stop (nm) value ~ nm higher than the Start (nm) value. Make sure the 3-D Mode box is not selected. Under Scan Control, click Medium. o Under the Options tab: In the Display options box, set the Y Minimum and Y Maximum. Check the Overlay traces box to display the results on one graph. CAT or S/N Mode should not be checked. Cycle mode should be unchecked. Smoothing should be unchecked. Excitation filter should be set for Auto. Emission filter should read Open. PMT Detector voltage should be at Medium. Corrected spectra should not be checked. o Under the Accessories tab: None of the boxes in this tab should be checked. o Reports tab: Within this dialogbox, the format of the reports can be set. Ask your instructor for specific instructions regarding this section. o Auto-store tab: To set up data storage before you begin your run, under the Storage box, click On; prompt at end. In the Auto-convert box, click the ASCII (CSV) toggle. This will store your data in the Cary format and the ASCII format, which can be read on different computers. After you have completed your setup parameters, click OK. The change in parameters should now be displayed in the Status Display box in the bottom right-hand corner of the screen. Wipe off the cuvette (make sure you are using the correct type) and carefully place it in the instrument, making sure not to touch the sides of the cuvette. Zero the instrument by placing your blank in the cell holder. Press the Zero button on the left-hand side of the screen. When the instrument has zeroed, the absorbance reading will read and zeroed in the top left corner. To start scanning your sample, press the Start button. A box title Sample Name will appear. Put your sample in the compartment and close the lid. Enter the name of your sample and press OK. After the scan has completed, a Save As box will appear. Type in a name and press Save.

4 4 From the emission scan you can find the maximum wavelength of excitation if you weren t sure about the wavelength value that you entered before. The same can be done to double check your emission wavelength. Analysis Once a plot has been drawn, you can use several buttons on the toolbar to manipulate the graph. Cursor mode Free mode : the cursor (which appears as a +) can be moved in any direction without any restrictions. Track mode : along with the cursor, a set of intersecting lines will appear. As you drag the cursor to the left or right, the horizontal line rides along the line produced by the data points. You can monitor the X and Y values that result from specific data points by looking in the right-hand corner beneath the graph. Track Preferences This displays the names of the lines generated from your data points and their corresponding colors and filenames. Graph Preferences This allows you to change the color and width of the axes, as well as the font and the way the data is plotted (i.e. dots or solid lines). Scale Graph This allows you to change the scale of the graph by typing in the area you would like to focus on. Add Label This feature allows you to add labels to your graph. Sometimes no peaks will appear. This may be due to the peak threshold. To adjust the peak threshold, go to Graph Peak Labels Threshold and adjust the threshold accordingly.

5 5 Sometimes only a vertical line will appear even though you don t see any other trace on the graph. This indicates that you need to adjust the Intensity (y scale). To do this, go to the Axes scales button and change the scale to a more appropriate number. Simple Reads This feature allows you to run samples at one wavelength. To enter the program, go to the Cary Eclipse icon Simple Reads Setup. A Setup box will appear with several tabs. In order to adjust the experimental parameters, fill in the information as needed. Under the Setup box: o Cary tab: Instrument setup: Data mode Fluorescence should be in the drop-down box. Wavelength setup enter the Ex. Wavelength (nm) and the Em. Wavelength (nm). (Ex. stands for excitation and Em. stands for emission). The Ex. Slit (nm) and Em. Slit (nm) must also be adjusted according to the method. o Options tab: The Excitation filter should be set for Auto. The Emission filter should be set for Open. The PMT Detector voltage should have the Medium button clicked or it may be changed to suit the experiment. After all of the experimental parameters have been set, press OK. Zero the instrument. A window titled Zero will open instructing you to load the blank. Do so and then press OK. The instrument is now zeroed. Put your sample into the cell holder and press the Read button. The instrument will take measurements and the results will appear in the bottom of the box. Concentration To enter the program, go to the Cary Eclipse icon Concentration Click on the Setup button on the left side of the window. A Setup box will appear with several tabs. In order to adjust the experimental parameters, fill in the information as needed. Setup Under the Setup box:

6 6 o Fill out the Cary tab with the necessary parameters. Under the Instrument Setup: Data Mode box, make sure the drop-down button reads Fluorescence. Under the Wavelength Setup box: Fill out the Ex. Wavelength (nm), Em. Wavelength (nm), Ex. Slit (nm), Em. Slit (nm), and Ave Time for your method. Average time is automatically set for seconds but can be adjusted if needed. o Under the Options tab: In the Display Options section of the window, adjust the Y minimum and Y maximum values as needed. The Excitation filter should indicate Auto is selected. The Emission filter should read Open. The PMT Detector voltage should have the Medium button clicked. o Accessories tab: See your instructor. The single cell holder will have to be switched to a well-plate set-up if multiple samples are to be run at the same time. o Standards tab: In the Standards box: Units selection change accordingly. Standards enter the number of standards you have. Replicates toggle should be 1 unless otherwise instructed. Std. averaging toggle should not be clicked. In the box indicating Std. and Conc., enter the various concentrations in the appropriate boxes. Fit Type Highlight the box for the type of fit you want for your data. Min R 2 This is automatically set at R 2 is called the correlation coefficient and is essentially a measure of error. It can be adjusted according to how closely you want your fit to be to the desired curve previously selected. o Samples tab: Sample Names box: Number of Samples This should be adjusted accordingly. Replicates This is automatically set at 1 unless otherwise instructed. Sample Ave should be off in the majority of cases. There will also be a box in this tab indicating sample names. These can be changed from Sample to whatever you want by deleting the text in the box and typing. Weight/volume Corrections

7 7 This aspect probably won t be utilized unless otherwise instructed. o Reports tab: This programming has the option of formulating reports, but be sure to ask your instructor about the proper procedure for these. o Auto Store tab: Storage On; prompt at end Autoconvert ASCII (CSV) After all of the parameters have been setup, click OK. Running Standards/Samples Zero the instrument as previously indicated. Put the first standard in the cell holder and press Start. A Standard/Sample Selection will appear. Select the standards and samples for your calibration so that they are in the Selected for Analysis portion of the box. Move any other solutions over to the Solutions Available box by using the arrows. and will move the selected standards or samples to the left side (Solutions Available) and right side (Selected for Analysis), respectively. and will move all standards and solutions to the left and right side, respectively. When the standards and samples are in the correct positions, press OK. o A Present Standard box will appear. Press OK if it indicates that the correct standard is selected. Once a reading has completed, the Present Standard box will appear again indicating the next standard. Repeat until all standards have been run. After the standards have been run, a calibration curve (or line) will be drawn and a correlation coefficient will be displayed. In some cases, however, a box labeled Concentration will appear with Warning; Min R 2 test failed. This means that your data did not fit with the indicated correlation coefficient (i.e.

8 8 you had bad data). You proceed by pressing OK or Cancel. If your press OK, a Standards box will appear saying, There is no valid calibration. Proceed in Intensity (a.u.)? By pressing OK, the intensity of subsequent samples will be measured, but no concentration will result since the calibration failed. A Present Sample box will then appear. The procedure for running the samples is the same as the standards just listed. Saving Once the samples have finished running, a Save As box will appear. Type in a filename, select a folder, and press Save. Retrieving Saved Data Click on the Cary Eclipse icon click on the operation heading you want to open data in (i.e. from a Scan, or Simple Read, etc.) File Open select your file Open. Closing Down Close all windows and logoff. Turn off the instrument and the computer monitor.

9 9 Toolbar for analysis Excitation wavelength Intensity reading Changes to a Start button when online Emission wavelength Listing of experimental parameters