UV-VIS and Fluorescence
Purpose:
This lab is meant to familiarize us with using the UV/VIS and the Fluorescence. This
was done by making different standards and dilutions and running them.
UV/VIS background:
The Ultraviolet/Visible Spectroscopy can be used on an electromagnetic spectrum
ranging from 200nm to 800nm. This is typically a quantitative technique measuring
absorbance and %T. From this you are able to find concentration of a solution. It is also
possible to use the UV/VIS for qualitative data but it much more useful for quantitative data.
The bonding of electrons in a molecule helps the energy absorb. This instrument is typically
used with inorganic, organic, and biological species. The instrument has a source providing
energy, a monochromator, a reference cell, a photomultiplier tube, an amplifier, and some
type of read out device. The UV/VIS can either be used on its own or as a detector for other
instruments.
Fluorescence background:
This is a quantitative technique typically used to determine large elements that form
vapors and hydrides. Fluorescence is used to measure excitation and emission. Unlike the
UV/VIS the Fluorescence has two monochromators and two photomultiplier tubes. This
instrument tends to be more expensive and is not any less sensitive than any other options
so it is less commonly used.
Procedure:
-Using the UV/VIS
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Turn on the instrument
Open the “VisionPro” program on the computer
Instrument will make a start up check
Select the scan icon on the tool bar
4 viewing windows should appear
Settings should be
 Wavelength: 190nm-400nm
 Scanning for: Absorbance
 Lamp: Tungsten
 Set the baseline correction for 100%T
Place 1 blank in each sample holder
 Use a quartz cuvette filling it ¾ full with hexane
Approve the blank request which will start the baseline scan
 If denied then press the baseline/zero icon
Set the results dropdown menu to peak pick
Prepare sample and standards and place it in the front holder
Press the run icon
Run each sample 3 times with out opening the instrument
Click on sample name on left and right click on plot samples
 This puts all samples on one graph
When finished close out of the program and turn off computer monitor (leave the
computer on)
-Using the Fluorescence
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Turn on the instrument and computer
Open up the PX-150X program
Check the lower right corner to make sure the indication says that the instrument is
on
Then go to configure menu instrument and then make sure that on is selected
Set to Quantitative analysis by going to acquire mode selecting spectrum
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Fill a cuvette with 2ppm solution and run with the settings
 Spectrum type: excitation
 EM wavelength: 400nm
 EX wavelength: 350nm – wavelength containing peak of interest
 Sensitivity: low
 Scan Speed: fast
 Recording Range: -10 to amt. showing entire peak
 EX/EM Slit width: 10/10
 Response time: 0.02
 Repeat Scan/Auto file: no setting needed/disable
Then click Search wavelength and click search
 Set as 350-600nm and 240-650nm
 Record optical wavelengths
Run each standard 3 times recording wavelengths found for EX and EM
Data: Refer to page 52 for the data from the Fluorescence.
Results:
We ran multiple samples using the UV/VIS but after two days and running different
samples at different concentrations we were unable to come out with any usable results. We
never actually received any absorbances therefore I was unable to create a calibration
curve.
Fluorescence Calibration Curve:
Calibration Curve for Quinone
1050
Intensity (c/s)
950
850
y = 241.9x - 39.907
R² = 1
750
650
550
450
350
1
Concentration
(ppm)
1.5
2
2.5
3
3.5
Concentration (ppm)
4
Intensity (c/s)
2
443.887
3
685.778
4
927.679
Tonic Water had an intensity of 463.827, which = y
463.827 = 241.9𝑥 − 39.907
𝑥 = 2.08𝑝𝑝𝑚
Therefore Tonic Water has a concentration of 2.08ppm of quinone
4.5
Conclusion:
I found running the UV/VIS to be very complicated because no matter what we tried
we were unable to get results and it took us a while to actually start using the instrument
due to issues with finding a usable cuvette. We made all of our samples both lab periods and
tried running them but never got results. The Fluorescence gave us odd results at first but
after trying it multiple times we finally got the instrument to work. We ran less samples
than we wanted but we were still able to make a calibration curve with great linearity. I
found the Fluorescence to be an easy instrument to use for the most part the most difficult
part was the age of the computer and the fact that it kept freezing. I would like to try using
the UV/VIS again so that I can get usable results.