Nikki DeLuca
Lab 4: UV/VIS and Fluorescence
Purpose
To learn how to use the UV/VIS and Specrofluorometer instruments. Known samples will be
tested along with unknown samples. The UV/VIS will be used to sample aspirin and the
Spectrofluorometer used to sample tonic water.
Introduction
UV/VIS spectroscopy measures the transmittance and absorbance of solutions. It has an energy
source, monochromater, reference cell with blank solution, detector, amplifier, and output. The
instrument is most often used for quantitative information about the analyte in a solution.
Spectrofluorometers measure emission and can be used for quantitative information. It is most
effective for large elements. They consist of an energy source, a two wavelength selectors, a
detector, amplifer, and output. They are not frequently used due to more simpler and less
expensive instruments being available.
Procedure (Thermo Evolution 600 UV/VIS)
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Turn on the instrument by flipping the power switch on the left side
Open the “VisionPro” program on the computer
Select the “Scan” icon on the tool bar
Wavelength: 190nm-400nm
Scanning for: Absorbance
Lamp: Tungsten
Set baseline correction for 100%T
Prepare Blanks:
o Wash a quartz cuvette with hexane multiple times, fill 3/4th full, and dry with Kim
wipes
o Place one blank in each sample holder
o After setting the Baseline Correction the program will have had a request for
blanks, and a simple approval of the request will start the baseline scan, if denied,
simply press baseline/zero icon
Set the results dropdown menu to peak pick
Prepare sample/standard and place in front holder, leave blank in the back one
Press the “Run” icon
Run each sample/standard three times without opening the instrument
Click on sample name on left and right click. Click “Plot Sample” to put all samples on
one graph.
Close program on computer, turn off instrument, turn off computer monitor. Leave actual
computer on!
Procedure (Schimadzu Spectrofluorometer)
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Turn on the instrument (switch on bottom right) and computer
Open the PX-150x program
Check lower right corner on the program screen to see if it says the instrument is on
Clock “Configure” and go down to “PC Configuration.” Click the “First Portal”
Set the instrument to “Quantitative Analysis” by going to “Acquire Mode” and select
“Spectrum”
Make a 100 ppm stock solution
Make 0.2 ppm, 0.1 ppm, 1 ppm, 5 ppm, and 10 ppm standards of quinine in 0.1M H2SO4
Fill a cuvette with 0.2 ppm solution and put into instrument holder
Scan under these settings:
o Spectrum Type: Excitation
o EM Wavelength: 400nm
o Ex Wavelength Range: 350 nm to wavelength that contains peak
o Sensitivity: Low
o Scan Speed: Fast
o Recording Range: -10.00 to an amount that shows entire peak
o EX/EM Slit Width: 10/10
o Response Time: 0.02
o Repeat Scan/ Auto File: No setting needed/ disable
Click “Search λ” and “Search.” Set the default ranges for the EX (Excitation) and EM
(Emission) to 350 - 600 nm and 240 – 650 nm. Record the optimal wavelengths found for
both.
Perform a new run under these settings:
o Spectrum Type: Emission
o EX Wavelength: The ideal wavelength found in the optimal wavelength search
o EM Wavelength Range: Make sure that the optimal EM wavelength is included in
the range
o Sensitivity: Low
o Scan Speed: Fast
o Recording Range: -10.00 (low); 500.00 (high)
o EX/EM Slit Width: 10/10
o Response Time: 0.02
o Repeat Scan/ Auto File: No setting needed/disable
Run each standard and record wavelengths and intensities of the largest peak. Run three
trials.
Make tonic water solution by diluting 5mL of tonic water with 95mL of H2SO4
Record wavelength and intensity of largest peak. Run three trials.
Data
UV/VIS and Fluorescence graphs are in lab notebook.
UV/VIS Absorbances
0.02 ppm
0.1 ppm
0.032
0.062
0.059
0.068
0.083
0.073
0.5 ppm
0.139
0.119
0.116
1.0 ppm
0.056
0.059
0.055
Unknown
0.212
0.209
0.215
Fluorescence Intensity
0.2 ppm
1.0 ppm
30.706
177.849
30.789
178.256
30.802
178.482
5.0 ppm
838.670
840.650
840.495
10 ppm
1015.008
1015.008
1015.008
Unknown
387.763
386.985
386.410
Calculations
Standard Preparation for UV/VIS:
M1V1 = M2V2
0.02 ppm * 50 mL = 100 ppm * x mL
0.1 ppm * 50 mL = 100 ppm * x mL
1.0 ppm * 50 mL = 100 ppm * x mL
5.0 ppm * 50 mL = 100 ppm * x mL
10 ppm * 50 mL = 100 ppm * x mL
x = 0.1 mL
x = 0.05 mL
x = 0.5 mL
x = 2.5 mL
x = 5.0 mL
Standard Preparation for Fluorescence:
M1V1 = M2V2
0.2 ppm * 50 mL = 100 ppm * x mL
1.0 ppm * 50 mL = 100 ppm * x mL
5.0 ppm * 50 mL = 100 ppm * x mL
10 ppm * 50 mL = 100 ppm * x mL
x = 0.01 mL
x = 0.05 mL
x = 2.5 mL
x = 5.0 mL
UV/VIS Calibration Curve
UV/VIS Calibration Curve using Salycylic Acid
0.2
0.15
y = 0.127x + 0.0558
R² = 0.9999
0.1
0.05
0
0
0.2
0.4
0.6
Concentration (ppm)
0.8
1
1.2
UV/VIS Calculations for Unknown
y = 0.127x + 0.0558
0.212 = 0.127x + 0.0558
x = 1.23 ppm
Fluorescence Calibration Curve
Fluorescence Calibration Curve using Quinone
1200
1000
Intensity
800
y = 102.2x + 102.28
R² = 0.8966
600
400
200
0
0
2
4
6
8
10
12
Concentration (ppm)
Fluorescence Calculations for Unknown
y = 102.2x + 102.28
386.376 = 102.2x + 102.28
x = 2.78 ppm
Conclusion
Compared to other instruments used in lab, the UV/VIS and Spectrofluorometer were
fairly simple to use and analyze data. There were no problems during out experiment that
required troubleshooting, and the data all seemed reasonable. For the objective of learning how
to use the instruments for the future and how to analyze the output, the experiment was very
successful. If the objective were to accurately quantify the aspirin and tonic water unknowns, the
procedures and sample preparations might have to be revised.