UV-Vis and Fluorescence
Introduction:
The UV-Vis spectrometry’s main purpose is to detect quantitative results by
having energy being absorbed by the bonding electrons of a molecule. The range of
the electromagnetic spectrum the UV-Vis can detect is 200 nm to 800 nm. The UVVis can detect various wavelengths with corresponding absorbencies.
The way the Fluorescence function is by detecting analyte molecules at very
low concentrations. The compounds that are tested with the Fluorescence naturally
have the ability to give off light without being apart of a chemical reaction. The
molecules within a compound that has the ability to fluoresce first absorbs light and
then changes energy states to give off that “glow.”
Purpose:
In this experiment, we used the UV-Vis to investigate the absorbance
properties of salicyclic acid (SA) by preparing stock solutions (100 mL) of SA in
hexane (1000 ppm). For the Fluorescence, the purpose was to investigate the
fluorescing properties of quinine by preparing a stock solution (1000 mL) of quinine
in sulfuric acid (1000 ppm).
SOP for UV-Vis:
1. Turn on the instrument (switch located on the left side).
2. Turn on monitor and double-click VisionPro.
a. If not logged in, username is student and there is no password.
3. Allow the instrument to turn on for about 10-15 minutes.
4. In the toolbar frame, select the Scan icon.
5. In dialog box, enter wavelength from 190-400 nm.
a. Choose Absorbance.
b. Choose Tungsten Lamp.
6. Click on Baseline Correction.
a. Make sure 100% T is selected.
7. Click on Baseline/Zero icon.
8. Wash a quartz cuvette with hexane three times, then fill ¾ of the way with sample
and dry with chem wipes.
9. Place cuvette in the first slot with the frosted sides facing you and the window.
10. Click the Run icon.
11. To see the peaks and absorbances, click on the graph window. From there, go to
the graph menu at the top and choose Peak Labels. Select the Wavelength and
Value.
a. In the menu graph again, select Track at the top of the list and click on
Graph.
b. Using arrow keys, find the highest peaks for each of the graphs for each
standards and find the common wavelength. The wavelength should be
around 240-310 nm.
SOP for Fluorescence: Shimadzu:
1. Turn on instrument (which should be located in the front right) and computer.
2. Open PX-150x program on computer.
3. Check lower right corner to make sure the instrument is on:
a. If not, go to Configure Menu and go down to PC Configuration to make
sure First Portal is selected.
b. After the First Portal is selected, go under Configure Menu, go to the
Instrument and make sure ‘On’ is selected.
4. Go to Acquire Mode and select Spectrum.
5. Make sure standards are prepared before continuing.
a. Make sure you wash the cuvettes three times with the smallest
concentrated solution (0.1 ppm) before testing each standard. Also, make
sure to clean the cuvettes with a chem wipe before hand to reduce the
fingerprints.
6. Fill cuvette with solution and run under these settings:
a. Spectrum type: excitation
b. EM wavelength: 400 nm
c. Ex wavelength range: 220 (start), 900 (end)
d. Sensitivity: Low
e. Scan speed: Fast
f. Recording range: -10.00 (low), 500.00 (high)
g. EX/EM slit width: 10/10
h. Response time: 0.02
i. Repeat Scan/Auto File: No setting needed
7. Click Search λ and then click Search.
a. EX (excitation): 230-450 nm.
b. EM (emission): 240-650 nm.
8. Reset parameters:
a. Spectrum type: emission
b. EX wavelength: (ideal wavelength found)
c. EM wavelength range: (include optimal EM wavelength)
d. Rest of the parameters are the same as before
9. Open left hatch and pull out platform to insert cuvette. Press Start.
10. Run scan of each concentration. Make sure to record wavelength and intensity of
largest peak.
a. In order to see the peaks, click Manipulate and then press Peak Pick.
11. Run scan of tonic water solution and record wavelength and intensity of largest
peak.
UV- Vis Procedure:
1. Prepare standard solutions of 10, 20, 30 40 and 50 ppm from 1000 ppm stock
solution of salicyclic acid (SA) in hexane.
2. Run three samples of each standard on instrument.
3. Create calibration curves from data.
4. Dissolve and filter an aspirin tablet through hexane. Run new solution on
instrument.
Fluorescence Procedure:
1. Prepare standards of 0.1, 1, 5 and 10 ppm solutions of quinine in 0.1M H2SO4
using 1000 ppm stock solution.
2. Run three 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 5 mL tonic water with 95 mL H2SO4.
4. Run three scans of solution through both instruments and record the wavelength
and intensity of largest peak.
Calculations:
The following calculations were for determining the amount of SA needed to
dissolve in hexane in a 100 mL volumetric flask.




10 ppm:
o (1 ppm)(100 mL)= (1000 ppm)(V2)
 = 1.0 mL SA
20 ppm:
o (2 ppm)(100 mL)= (1000ppm) (V2)
 = 2.0 mL SA
30 ppm:
o (3 ppm)(100 mL)= (1000ppm)(V2)
 = 3.0 mL SA
40 ppm:
o (4 ppm)(100 mL)= (1000ppm)(V2)
 = 4.0 mL SA

5 ppm:
o (50 ppm)(100 mL)= (1000ppm)(V2)
 = 5.0 mL SA
Blank: 0 mL stock diluted to 100 mL with distilled water

Dilute stock amount to 100 mL in 100 mL volumetric flask.
Fluorescence calculations:
The following calculations were to determine the amount of quinine in sulfuric acid.
The first calculation was to find the volume to make 0.1 M sulfuric acid.
( 18 M)(x)= (0.1 M)(1000 mL)
x= 5.5 mL
Making the standards using 25 mL volumetric flasks:
10 ppm:

(1000 ppm)(x)= (10 ppm)(25 mL)
o
= 0.25 mL quinine
5 ppm:

(10 ppm)(0.25 mL)= (2.5)(5 ppm)= 12.5 mL
1 ppm:

(10 ppm)(0.25 mL)= (2.5)(1.0 ppm)= 2.5 mL
0.1 ppm:

(10 ppm)(0.25 mL)= (2.5)(0.1 ppm)= 0.25 mL
For the 5, 1.0 and 0.1 ppm solutions, the way it was achieved was simple. Instead of
recreating new standards and measure out precise amounts of quinine, we used the
10 ppm standard as our foundation. For example, we used the 10 ppm
concentration and what volume needed from the quinine was 0.25 mL. From there,
we took the concentration needed and multiplied the product of the 10 ppm and
0.25 mL and determined the amount needed from the 10 ppm to make the rest of
the standards.
Data:
UV-Vis:
Calibration Curve for UV-Vis Conentration vs. Abs: Part 1
Samples
1
2
Standard Concentrations (ppm)
10
20
Absorbance
0.154
0.386
Intensity
240
240
3
4
5
30
40
50
0.377
0.816
1.038
240
240
240
UV-Vis Part 1
1.2
y = 0.2198x - 0.1052
R² = 0.9247
1
0.8
UV-Vis Part 1
0.6
Linear (UV-Vis Part 1)
0.4
0.2
0
10
20
30
40
50
Calibration Curve for UV-Vis Concentration vs. Abs Part 2
Samples
1
2
3
4
5
Unknown
Standard Concentrations (ppm)
10
20
30
40
50
Unknown
Absorbance
0.15
0.371
0.573
0.448
0.936
0.212
Intensity
241
241
241
241
241
241
The unknown concentration was about 12.8 ppm, based on the equation of the line.
1
0.9
0.8
y = 0.1649x + 0.0009
R² = 0.8065
0.7
0.6
0.5
Series1
0.4
Linear (Series1)
0.3
0.2
0.1
0
10
20
30
40
50
Fluorescence:
Calibration Curve for Fluorescence Shimadzu Concentration vs. Intensity
Samples
Standard Concentrations (ppm)
1
2
3
4
Tonic Water
0.1
1
5
10
Unknown
Wavelength (nm)
448
450
447
493
499
Intenisty
29.354
183.714
1014.471
1014.49
1014.49
Base on the graph, the concentration for the tonic water is about 10 ppm.
1200
1000
y = 378.62x - 386.03
R² = 0.8571
800
600
Series1
Linear (Series1)
400
200
0
0.1
1
5
10
-200
Conclusion:
UV-Vis:
Overall, this experiment was both successful and not so successful. On the
positive note, we achieved a concentration for the aspirin “unknown,” which was
around 12.8 ppm (used the equation of the line). However, this experiment’s R
squared value for the first experiment for the UV-Vis was 0.92472, while the second
experiment had an R squared value of 0.80647. The goal for the R square is to be
close to 1 as possible.
Another problem that was encountered was the 30 ppm standard. The
absorbance for the 30 ppm was much higher then the 40 ppm (0.573 vs. 0.488).
However, maybe the 30 ppm wasn’t the problem. I believe the 40 ppm was the
problem because in comparison to the previous experiments, the absorbance for the
40 ppm decreased (0.816 to 0.448).
Fluorescence:
Overall, when working with the fluorescence, it was difficult. The
calculations for determining the amount of 10 ppm solution for each 5, 1 and 0.1
pmm was at first difficult, but was overcome.
There were some instrumental difficulties as well. Some difficulties included
running samples. For example, the 0.1 ppm solution had a greater intensity then the
1.0 ppm.