UV-VIS and Fluorescence
Purpose: The purpose of this lab was to teach us how to use the UV-VIS and the
Fluorescence. We ran various standards and dilutions on both instruments.
Background:
UV-VIS:
The UV-Vis spectroscopy can be used on an electromagnetic spectrum ranging from
200nm to 800nm. This is a quantitative method that measures absorbance and %T. Using
this information you can calculate the solution concentration. The UV-Vis can also be used
for qualitative data, but is more used for quantitative data. The electron-electron bond helps
with the absorption of energy in a molecule. The instrument generally uses inorganic,
organic, and biological species as samples. The instrument has an energy source, a
monochromator, a reference cell, a photomultiplier tube, an amplifier, and a read out
device. The UV-Vis can either be used on its own or as a detector for other instruments.
Fluorescence:
Fluorescence is the emission of light by a substance that has absorbed light or
other radiation. Fluorescence is a quantitative method used to measure both excitation and
emission. The fluorescence has two monochromators and two photomultiplier tubes, unlike
the UV-Vis. The instrument is less commonly used because it is more expensive and is not
any less sensitive.
Procedure:
UV-VIS
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Turn on the instrument
Open the “Vision Pro” program on the computer
Select the scan icon on the tool bar
Four viewing windows should appear
Select parameters:
a. Wavelength: 190nm-400nm
b. Scanning for: Absorbance
c. Lamp: Tungsten
d. Set the baseline correction for 100%T
Place a blank in the back holder
a. Use a quartz cuvette and fill it ¾ full with hexane
Approve the blank request which starts the baseline scan
a. If denied, press the baseline/zero icon
Set the results by clicking the “drop-down” menu to peak pick
Prepare samples and place in the front holder
Press the run icon
Run each sample three times
Click on sample name on left and right click on plot samples
Close the program and turn off computer monitor (leave the computer on)
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 instrument is on
Go to configure menu instrument and then make sure that on is selected
Set to Quantitative analysis: acquire mode select spectrum
Fill a cuvette with 2ppm solution and run with the settings
a. Spectrum type: excitation
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b. EM wavelength: 400nm
c. EX wavelength: 350nm – wavelength containing peak of interest
d. Sensitivity: low
e. Scan Speed: fast
f. Recording Range: -10 to amt. showing entire peak
g. EX/EM Slit width: 10/10
h. Response time: 0.02
i. Repeat Scan/Auto file: no setting needed/disable
Then click Search wavelength and click search
a. Set as 350-600nm and 240-650nm
b. Record optical wavelengths
Run each standard three times recording wavelengths found for EX and EM
Data: Refer to page 33 in my lab notebook for the data from the Fluorescence
Results:
Over the two days, we ran many samples with various concentrations on the UV-VIS.
We were unable to get any reliable results and spectra. We never received any absorbance
data therefore it was impossible to produce 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
1.5
2
2.5
3
3.5
Concentration (ppm)
Concentration
(ppm)
Intensity (c/s)
2
443.887
3
685.778
4
927.679
Tonic Water: intensity = 463.827 = y
463.827 = 241.9𝑥 − 39.907
𝑥 = 2.08ppm
Tonic Water: concentration = 2.08ppm (quinine)
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4.5
Conclusion:
The UV-VIS was very complicated to work with because we were unable to get any
results no matter what we tried. Also, the quartz cuvettes were hard to find which took us
extra time before we could actually start using the instrument. We made samples during
both days that week and still tried running them, but never got any results.
At first, the Fluorescence kept giving us weird results but after multiple times we
finally were able to get it to work. We weren’t able to run all of our samples but we were
still able to create a calibration curve. I really didn’t work on the Fluorescence because I was
trying to fix the issues with the UV-Vis. Our biggest issue was how old the computer was and
due to the age of it, the computer kept freezing.