David Millard
UV-Vis/ Fluorescence
Purpose:
The goal of this lab was to acclimate ourselves with the UV-Vis as well as fluorescence
instruments. Using fluorescence, quinine standards will be created and used to make a
calibration curve. Then tonic water will be tested for the concentration of quinine. The UV-Vis
will have standards of salicylic acid made and create a calibration curve. Old aspirin and new
aspirin will be tested to see how much salicylic acid has formed over the age of the pill.
Introduction:
The UV-Vis uses the amount of light that is absorbed by a solution to determine the
concentration of the sample. The fluorescence is similar to the UV-Vis, but uses fluorescence to
excite the molecules, giving the user two sets of data, an excitation and an emission. UV-Vis is a
very inexpensive way to determine the quantitative data of a sample, whereas the fluorescence is
a little more pricy, but finds a little more quantitative data.
Procedure:
UV-Vis
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Turn on the instrument.
Click on “Vision Pro Software.”
Allow the instrument to initialize for 10-15 minutes.
On the toolbar, click the scan icon.
o Enter the wavelength (190-400nm).
o Absorbance.
o Select tungsten lamp.
Set baseline correction to 100% T.
Find the baseline.
Wash the quartz cuvette with hexane multiple times and then fill ¾ full, and wipe outside
of cuvette with a Kim wipe and place one cuvette in each holder.
Run the sample.
To view the peaks, go to the graph window, go to graphs in menu, select peak labels,
wavelength and value.
Use the keypad to find the peak and record wavelength and absorbance.
After the sample is finished (we ran 3 trial for each sample), empty the front cuvette,
wash and rinse with the next concentration standard and then wipe with a Kim wipe and
place in the cuvette holder in the front of the instrument.
o Repeat with scans and rinsing for all other samples.
Fluorescence
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Turn on the instrument.
Open “PX-150x” program on the desktop.
Menu – Configure – PC Configure – Port 1.
Menu – Configure – Select instrument – on.
Set to quantitative analysis – Acquire mode in menu and select spectrum.
Obtain a stock solution and dilute to the designated standards.
o Wash quartz cuvette with the standard you will be using multiple times before
running the sample.
Go to parameters – configure – parameters.
o Spectrum type: excitation
o Scan speed: fast
o FM wavelength: 400nm
o Record range: -10.00 to 500
o FX wave range: 200 – 900
o Ex/Em slit width: 10/10
o Sensitivity: Low
o Response time: 0.2
Run the sample.
After first run, click search lambda.
o The range of the absorbance
 Ex: 230-450nm
 Em: 240-650nm
Search for optimal wavelengths for Ex and Em.
Change parameters – configure – parameters
o Spectrum type to emission
o Excitation wavelength to the optimal found
o Be sure emission wavelength range encompasses the optimal emission
wavelength found.
Run other samples, record the wavelength and intensity of the largest peak.
o To view peak, go to the menu, manipulate – peak pick. Expand the window.
Data:
Fluorescence
Standards were diluted from a 1000ppm stock solution of quinine.
(1000ppm)(V1) = (50mL)(1ppm)
V1 = 0.050mL
(1000ppm)(V1) = (50mL)(2ppm)
V1 = 0.100mL
(1000ppm)(V1) = (50mL)(3ppm)
V1 = 0.150mL
(1000ppm)(V1) = (50mL)(4ppm)
V1 = 0.200mL
(1000ppm)(V1) = (50mL)(5ppm)
V1 = 0.250mL
Standard Conc.
5ppm
4ppm
3ppm
2ppm
1ppm
Blank
Unknown
Excitation 363nm
Wavelength nm (avg.) Intensity (avg.)
360
15.639
359.67
7.9.5
359.3
74.6
359
8.19
359
9.512
359
7.40
359.67
11.339
Emission 448nm
Wavelength nm (avg.) Intensity (avg.)
449.3
978.911
447.3
822.9313
447.67
499.0217
447
403.4177
448
209.584
411
1.421
449
447.8413
Calibration Curve
y = 195.94x + 34.835
R² = 0.9792
1200
Intensity
1000
800
600
400
200
0
0
1
2
3
Concentration (ppm)
4
5
6
The unknown was tonic water which was diluted to a 5% solution with the 0.1M sulfuric acid.
y=195.94x+34.835
average intensity 447.8413
Calculated concentration of quinine is 2.108 ppm for a 5% solution.
For the bottle, 42.16ppm was quinine.
UV-Vis
Standards were diluted from a 1000ppm stock solution of salicylic acid.
(1000ppm)(V1) = (10ppm)(10mL)
V1 = 0.1mL
(1000ppm)(V1) = (20ppm)(10mL)
V1 = 0.2mL
(1000ppm)(V1) = (30ppm)(10mL)
V1 = 0.3mL
(1000ppm)(V1) = (40ppm)(10mL)
V1 = 0.4mL
(1000ppm)(V1) = (50ppm)(10mL)
V1 = 0.5mL
Solution Concentration
10ppm
20ppm
30ppm
40ppm
50ppm
Blank
Aspirin exp. 2006
Aspirin exp. 2010
Lambda average (nm)
310
310
310.67
311.67
312
310
310.33
310
Absorbance average
0.124
0.254
0.334
0.456
0.594
0.000
0.260
0.041
UV-Vis Standards
y = 0.0114x + 0.0118
R² = 0.9956
0.7
0.6
Intensity
0.5
0.4
0.3
0.2
0.1
0
0
10
20
30
40
50
60
Concentration (ppm)
y = 0.0114x + 0.0118
Aspirin 2006 avg absorbance 0.260
Calculated concentration 21.74ppm
Aspirin 2010 avg absorbance 0.041
Calculated concentration 2.53ppm
Conclusion:
The first day of this lab was a little frustrating. We did not run all of the samples on the
UV-Vis because of the non-appearance of data on the computer. Also, on the fluorescence we
were getting very strange results with our standards, our blank were a higher intensity than our
1ppm and 2ppm standard. We later found out that we had been using plastic cuvettes instead of
the quartz cuvettes. The next day when we ran our samples with the quarts cuvettes, our data
was amazing. The lab on the second day seemed to run almost flawlessly. The data we received
was very consistent and the running of the instrument was fairly simple.
The result of the lab showed that the calculated quinine concentration in the bottle that
was tested was 42.16ppm. Also, the aspirin that was tested showed results that were just as nice.
The aspirin that had expired in 2006 had a much higher level of salicylic acid than the aspirin
that had expired in 2010. The 2006 aspirin had a calculated concentration of 21.74ppm whereas
the aspirin that had expired in 2010 had a salicylic acid level much lower, calculated at 2.53ppm.