SOP for UV-Vis and Fluorescence
By Dan Palumbo and EJ Habina
Introduction
A UV-Vis spectrophotometer is an instrument commonly used in lab that analyzes compounds in
ultraviolet and visible regions of electromagnetic spectrum. Unlike infrared which looks at vibrations,
UV-Vis looks at electronic transitions. The UV-Vis instrument allows for the determination of wavelength
and maximum absorbance of compounds.
The Beer-Lambert Law, A = εbc, where A is Absorbance, ε is molar extinction, b is path length,
and c is concentration, directly applies to the UV-Vis instrument.
The instrument used is the Thermo Evolution 600 UV-Vis spectrophotometer. “The optical
design of the Evo 600 includes dual deuterium and tungsten sources and a high performance
photomuliplier detector. Its features provide enhanced high-level performance for the Material Science
market and general research applications”
(http://www.thermo.com.cn/Resources/200802/productPDF_1838.pdf).
While UV-Vis has limits in detection, fluorescence can find trace concentrations as low at 1 ppt.
Fluorescence uses two wavelengths along the spectrum to determine the quantitative measurements of
an analyte. The fluorescence instruments send light into a sample and excite the electrons in a molecule.
The instruments then read the radiation released when the electrons return to their ground states.
The instruments used are the Shimadzu Spectrofluorophotometer and the PE Luminescence
Spectrophotometer.
Purpose of the instrument
As previously stated, the UV-Vis instrument allows for the determination of wavelength and
maximum absorbance of compounds. The instrument has a wide variety of uses including life science
biochemical, pharmaceutical and quality control, and material science. It can be used in nucleic acid
tests, to test for protein concentration, thermal denaturation/renaturation, and kinetics.
The fluorescence instrument is a type of electromagnetic spectroscopy which analyzes
fluorescence from a sample. Fluorescence spectroscopy is used in biochemical, medical, and chemical
research fields for analyzing organic compounds.
Experimental Purpose
UV-Vis:
The purpose of this lab is to determine the absorbance levels of Salicylic Acid in aged aspirin
samples by running samples of SA in hexanes through the UV-Vis instrument.
Fluorescence:
The purpose of the next part in the lab is to determine the amount of quinine in tonic water
through the fluorescence instruments.
Experimental Procedure
UV-Vis:
1. Prepare standard solutions of 40, 25, 10, 7 and 2ppm from a 1000ppm stock solution of Salicylic
Acid (SA) in hexanes. Use 50mL volumetric flasks to dilute the stock solution with hexane
2. Run 3 samples of each standard through the UV-Vis spectrometer
3. Create calibration curves from the data returned from the UV/vis
4. Dissolve and filter an aspirin tablet through hexane. Run this new solution for SA absorbance on
the UV-Vis instrument
Fluorescence:
1. Prepare standard solutions of 2ppm,3ppm, 4ppm, 5ppm solutions of quinine in .1M H2SO4 using
the 1000ppm stock solution
2. Run 3 samples of each concentration through the fluorescence instruments and record the
wavelength and intensity of the largest peak
3. Make a solution of tonic water by diluting 5mL of tonic water with 95mL of H2SO4 and run 3
scans of the solution through the fluorescence instruments. Record the wavelength and
intensity of the largest peak
Instrument Procedures
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’
3. Instrument will run through a startup check for about 10-15 minutes
4. In the toolbar frame, select the scan icon (looks like a scatter plot with curved lines)
5. In the dialog box enter the wavelength from 190-400nm, choose absorbance, and Tungsten
Lamp
6. Click on Baseline Correction and make sure 100%T is selected
7. Next Click on baseline/zero icon (looks like a graph with arrows pointing down)
8. Wash a quartz cuvette with hexane 3 times, then fill 3/4th of the way and dry with kim wipes.
Place the cuvette in the first slot with the frosted sides facing you and the window
9. Next click on the run icon (looks like a man running)
10. To obtain peak and abs, click on the graph window, then go to the graph menu at the top and
choose peak labels and select wavelength and value. In the menu graph again select track at the
top of the list and click on graph and using the arrow keys find the peak around 310nm and
record the abs value
11. Repeat steps 8-10 with each standard and aspirin solution
Fluorescence:
1. Shimadzu:
a. Turn on the instrument and computer
b. Open the PX-150x program
c. Before starting anything, check the lower right corner and see if the indication says the
instrument is ON. If not:
i. Go to the Configure menu and go down to the PC Configuration and make sure
the FIRST PORTAL is selected for the instrument
ii. Then also under the Configure menu, go to the Instrument and make sure that
ON is selected
d. Set the instrument to quantitative analysis by going to Acquire Mode and selecting
Spectrum
e. Obtain stock solution of 1000ppm quinine in .1M H2SO4. With the stock solution make
2ppm,3ppm, 4ppm, 5ppm solutions of quinine in .1M H2SO4
f. Fill a cuvette with some 2ppm solution, run the instrument under these settings
i. Spectrum Type: Excitation
ii. EM Wavelength: 400nm
iii. Ex Wavelength Range: 220 (start); 900 (end)
iv. Sensitivity: Low
v. Scan Speed: Fast
vi. Recording Range: -10.00 (low); 500.00 (high)
vii. EX/EM Slit Width: 10/10
viii. Response Time: 0.02
ix. Repeat Scan/ Auto File: No setting needed
g. Then click Search λ and click Search. Ensure that the default range for the EX(Excitation)
and EM(Emission) are 230-450nm and 240-650nm respectively. Once the optimal
wavelengths are found for EX and EM, note them. Then reset the parameters to be:
i. Spectrum Type: Emission
ii. EX Wavelength: The ideal wavelength found in the optimal wavelength search
iii. EM Wavelength Range: Make sure that the optimal EM wavelength is included
in the range
iv. Sensitivity: Low
v. Scan Speed: Fast
vi. Recording Range: -10.00 (low); 500.00 (high)
vii. EX/EM Slit Width: 10/10
viii. Response Time: 0.02
ix. Repeat Scan/ Auto File: No setting needed
h. Run a scan of each concentration (2ppm, 3ppm, 4ppm, and 5ppm) and record the
wavelength and intensity of the largest peak. Repeat 3 times for each
i. Make a solution of tonic water by diluting 5mL of tonic water with 95mL of H2SO4
j. Run a scan of this solution and record wavelength and intensity of largest peak. Repeat
3 times
2. Perkin Elmer:
a. Turn on instrument and computer
b. Open the WINLab program
c. Fill a cuvette with your blank (H2SO4) and run a scan with the same parameters as
before except change the slit from 8 to 3 for both
d. Run your samples from the other fluorescence instrument each three times. Record the
wavelength and intensity of the largest peak. To find these numbers, click the button
with a peak with a vertical line through it and drag the line to the middle of the largest
peak. The wavelength and intensity are found towards the bottom of the screen
e. Repeat for your tonic sample 3 times
Data
UV-VIS:
1. Day 1+2+3:
 After talking with a classmate who was in the lab period before us and was also on UVVIS, it was understood that the 1000-ppm stock solution that was available for this
procedure was not working properly, and therefore it was in our best interest to make
our own stock. We attempted unsuccessfully to create our own 1000-ppm stock
solution of salicylic acid in hexane using 0.5 g of salicylic acid and dissolving in 500 mL of
hexane. We attempted over several class periods to try to dissolve the salicylic acid but
with no success. In an attempt to at least follow the procedure, we decided to use the
stock solution anyway to see if any results could be obtained.

Calculations for stock solutions:
o 40 ppm:
 40 mL of stock w/ 10 mL hexane in 50 mL vol. flask
o 25 ppm:
 25 mL of stock w/ 25 mL hexane in 50 mL vol. flask
o 10 ppm:
 10 mL of stock w/ 40 mL of hexane in 50 mL vol. flask
o 7 ppm:
 7 mL of stock w/ 43 mL of hexane in 50 mL vol. flask
o 2 ppm:
 2 mL of stock w/ 48 mL of hexane in 50 mL vol. flask
Concentration of Sample (ppm)
Wavelength
Absorbance
40 ppm
25 ppm
10 ppm
7 ppm
2 ppm
310
311
311
311
311
>3
>3
>3
>3
0.613
Fluorescence
2. Day 4:
 After the disappointing results and problems which occurred with the UV-VIS section of the
procedure, the fluorescence segment must now be completed in order to finish the lab.



We prepared 2, 3, 4, and 5 ppm samples from the original 1000 ppm stock solution by taking
10 mL of this stock solution and diluting it in 100 mL vol. flask with sulfuric acid.
After finding the optimal Excitation/Emission wavelengths, each sample was run three times
recording the wavelength and intensity for each.
A tonic water/sulfuric acid sample is also prepared using 5 mL of tonic water w/ 95 mL of
sulfuric acid, which was also scanned in the fluorescence instrument three times.

Calculations for stock solutions:
o 5 ppm:
 5 mL of new stock w/ 95 mL sulfuric acid in 100 mL vol. flask
o 4 ppm:
 4 mL of new stock w/ 96 mL of sulfuric acid in 100 mL vol. flask
o 3 ppm:
 3 mL of new stock w/ 96 mL of sulfuric acid in 100 mL vol. flask
o 2 ppm:
 2 mL of new stock w/ 98 mL of sulfuric acid in 100 mL vol. flask


Optimal Excitation Wavelength: 345 nm
Optimal Emission Wavelength: 451 nm
Concentration of Samples (ppm)
2 ppm
3 ppm
4ppm
5 ppm
Wavelength (nm)
#1
#2
#3
#1
#2
#3
#1
#2
#3
446
448
448
448
446
448
447
446
449
#1
#2
#3
451
448
450
447
447
447
450
Intensity
336.596
334.9
336
336.494
752.36
745.179
748
747.173
932.592
936.238
934
934.253
977.786
980.562
979.948
979
Fluorescence
1200
Intensity
1000
800
600
y = 213.93x
R² = 0.8687
400
200
0
0
1
2
3
4
5
6
Concentration of Sample (ppm)
Tonic Water w/ Sulfuric Acid
Trial #1
Trial #2
Trial #3
Wavelength
(nm)
448
448
448
Intensity
324.239
277.996
280.189
Conclusion:
UV-VIS
Unfortunately for this section of the experiment, the problems with creating the standards due
to the problematic nature of the current stock solution really prohibited us from being able to truly
conduct the experiment successfully. We attempted at first to simply dissolve the 0.5 g of salicylic acid in
the hexane in a 500 mL volumetric flask using a magnetic stir bar. Since we could not heat the solution
too much due to its volatility as well as it being in a volumetric flask, we simply attempted to turn up to
the stir bar rpm to make up for this. However, after watching the solution mix for over two and a half
hours, it was obvious that it was not a success. The next class period we again tried to make this 1000
ppm stock solution, except this time we added the salicylic acid and hexane in a beaker so that along
with the stir bar a slight amount of heat could be added to hopefully aid in dissolving the salicylic acid.
However, once again, the salicylic acid did not dissolve and it was then we decided to use the stock
solution we knew to be problematic. This solution gave us inconsistent results as expected, and
thoughts into how to improve this procedure were considered. Firstly, the idea of creating a 1000 ppm
stock solution is unnecessary if the greatest concentration needed is only 40 ppm. One possible solution
is to make a 100 ppm stock solution, which would be considerably more easily to create, and make the
various solutions and run them from this solution in one day. This is so that no degradation of the stock
solution can occur since the stock is made and tested in the same day. We believe that if we had more
time we could have successfully completed this part of the procedure.
Fluorescence
This part of the procedure was much less problematic than that of the UV-Vis section of the
experiment. We were able to not only produce results that were clear and easily read on the
instrument, but the consistency of the results also showed that the results were desirable. However, as
stated previously, the time constraint in which we were forced to complete this procedure did not allow
us to test an “unknown sample” against our various standards. However, both the consistency as well as
the time in which we were able to gain these results leads us to believe that if given more time this
procedure could have been completed more thoroughly.