UV-VIS and Fluorescence
Introduction:
Ultraviolet-visible spectroscopy is based on measuring transmittance and absorbance of a
molecule’s binding electrons. This is carried out in the wavelength region between 190 and 800 nm.
Instrumentation includes a source, wavelength detector, sample container, radiation transducer, and
signal processors and readout devices. UV-VIS molecular absorption is widely used for quantitative
analysis in chemical, environmental, forensic, and clinical lab settings. Fluorescence is used to detect low
concentrations of analyte. However, not all molecules have fluorescent capabilities. The purpose of this
lab is to determine levels of salicylic acid in aspirin samples by UV-VIS and to determine an unknown
based on fluorescence of quinine and sulfuric acid standards.
Procedure:
On day one, dilutions of a salicylic acid stock solution (10 ml) will be diluted in hexane (1oooppm). An
appropriate wavelength will be determined and a calibration curve will be created. On the second day,
an aspirin sample will be filtered and then run so that the sample falls in the range of the calibration
curve. In the fluorescence lab, sulfuric acid will be used as a solvent and the 1000ppm solution will be
diluted to 100ppm. Four different concentrations will be tested. Then, an unknown will be tested.
UV-VIS
1. Power on the instrument by pressing the switch on the left side near the power cord
2. Turn on the monitor and double-click VisionPro
 Instrument will run through startup check for about 10-15 minutes
 In toolbar frame, select the Scan icon
 In dialog box, enter wavelength from 190-400nm, choose Absorbance, and Tungsten Lamp
 Click on Baseline Correction and make sure 100%T is selected
 Click on the Baseline/Zero icon
3. Wash a quartz cuvette with hexane 3 times, fill ¾ way and dry with kim wipes, place cuvette in first
slot with frosted sides facing you and the window
4. Click the Run icon
5. To obtain peak and abs, click on the graph window, graph menu at the top and choose Peak Labels
and select Wavelength and Value. Select Track at the top of the list and click on Graph.
Fluorescence
1. Turn on the instrument and computer
2. Open the PX-150x program
 Check lower right corner to see if instrument says the instrument is on
3. Set the instrument to Quantitative Analysis by going to Acquire Mode and selecting Spectrum
4. Fill cuvette with first standard and run the instrument under these settings
 Spectrum Type: Excitation
 EM Wavelength: 400nm
 Ex Wavelength Range: 220 (start)-900(end)
 Sensitivity: Low
 Scan Speed: Fast
 Recording Range: -10.00 (low), 500.00 (high)
 EX/EM Slit Width: 10/10
 Response Time: 0.02
 Repeat Scan/Auto File: No setting needed
5. After first run, click Search ʎ and click Search. Ensure the default range for the EX and EM are 230450nm and 240-650nm respectively. Once the optimal wavelengths are found for EX and EM, note
them. Change these parameters:
 Spectrum Type: Emission
 EX Wavelength: The ideal wavelength
 EM Wavelength Range: The ideal wavelength
6. Run scan of each concentration and tonic water
UV-VIS Standards
Wavelength (nm)
311
311
310
310
310
Aspirin Sample
311
Concentration (ppm)
40
25
10
7
2
15.8
Absorbance
0.558
0.406
0.210
0.144
0.040
0.259
Absorption
UV-VIS Calibration
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.0133x + 0.0484
R² = 0.9817
0
10
20
30
Concentration (ppm)
40
50
Flourescence Standards
Wavelength (nm)
449
493
448
447
Tonic Water
450
Concentration (ppm)
2
3
4
5
21
Intensity
834.125
1014.657
695.762
857.634
416.727
Fluorescence Calibration
1200
Intensity
1000
800
600
y = -24.837x + 937.47
R² = 0.0602
400
200
0
0
1
2
3
4
5
6
Concentration (ppm)
Discussion:
Our UV-VIS calibration curve came out very well. Our aspirin sample fell in the range of our
standards and according to the curve the concentration of salicylic acid in the aspirin sample was about
15.8 ppm. The fluorescence calibration did not end up as reliable as the UV-VIS. The curve has a negative
slope and a very bad R2 value. We did have some problems with the fluorescence part of the lab,
though. The instrument was not finding the optimum wavelength and we realized that we made the
dilutions of the standards incorrectly and they were more concentrated than they should have been.
Once we made the dilutions correctly, we still had problems with the instrument finding an optimum
wavelength. This made it hard to find the range needed on the spectra and see the peaks. Since we had
to dilute the standards twice, it is possible there was some error, resulting in intensities that do not
follow the increasing concentrations. Since there were problems with the instrument and calibration
curve, the calculated concentration of tonic water is not likely to be accurate. If we had more time, it is
possible that preparing the correct dilutions and running the new standards could yield more accurate
results.