Tim Abell
UV-Vis and Fluorescence
Introduction
UV-Vis is a spectrophotometer that operates in the ultraviolet and visible range of the
electromagnetic spectrum. It is used to analyze compounds through absorbance of radiation. It can be
used to determine the maximum absorbance of a compound.
Fluorescence detects the emission of radiation after if excites the electrons of the compound. It
can detect very low concentrations of analytes. It can be used to determine the concentrations of
analytes.
Purpose
The purpose of this experiment is to explore the absorbance properties of salicylic acid (SA) in
hexanes with the UV-Vis. Then determine the concentration of SA in old aspirin. The concentration of
quinine in tonic water will be determined with fluorescence.
Method (UV-Vis)
1.
2.
3.
4.
Turn on instrument (switch located at the rear left)
Turn on computer and double click on Vision Pro Software on the Desktop
Allow the instrument to initialize for 10-15 minutes
In the toolbar frame, select the scan icon
a. In the menu, enter: wavelength 190-400nm, absorbance, and Tungsten lamp
5. Click Baseline Correction
and select 100% T
6. Click on the Baseline Zero icon (looks like a graph with two arrows pointing down)
7. Insert quartz cuvette with hexane
a. Wash the cuvette 3 times, fill ¾ full, then wipe with Kimwipe
8. Click run icon
9. To see peaks, click on the graph window, then Graph in the menu. Select peak labels.
Select wavelength and value.
10. Click graph in the menu and select track, then click the graph window
11. Use the keypad to find the peak and record wavelengths and absorbance
12. Repeat from step 7 using standard solutions
Data (UV-Vis)
Concentration
(ppm)
Absorbance
10
20
30
40
50
0.154
0.386
0.372
0.816
1.038
UV/Vis Calibration
1.2
y = 0.022x - 0.1062
R² = 0.9216
Absorbance
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
60
Concetration (ppm)
Sample
Mass (g)
Aspirin
0.0045
Volume hexane
(mL)
100
Absorbance
0.212
SA Concentration
(ppm)
14.46
Method (Fluorescence)
1. Turn on instrument (switch located at the front right)
2. Turn on computer and open the PX-150x Program
3. Make sure the instrument is on:
a. In the menu, go to configure, select PC Configuration, and select port 1
b. In the menu, go to configure, select instrument, and select On
4. Set the instrument to quantitative analysis by going to Acquire mode in the menu, and
selecting Spectrum.
5. Obtain stock solution and dilute
a. Wash a quartz cuvette 3 times with the smallest concentrated solution, and set the
following parameters:
b. Select Configure in the menu, and select Parameters
i. Spectrum Type: excitation
ii. EM Wavelength: 400nm
iii. EX Wavelength Range: 200-900nm
iv. Sensitivity: Low
v. Scan Speed: Fast
vi. Recording Range: -10.00 to 500
vii. EX/EM slit width: 10/10
viii. Response Time: 0.02
6. Click run
7. After this first run, click search (lambda). Set the ranges to the following:
a. EX: 230-450nm
b. EM: 240-650nm
8. Click Search and note the optimal wavelengths for EX and EM.
9. Change the parameters by selecting Configure in the menu, and select Parameters
a. Change the spectrum type to emission
b. Change the excitation wavelength to the optimal wavelength found
c. Be sure the emission wavelength range encompasses the optimal EM wavelength
found
10. Run other samples and record the wavelength and intensities of the largest peak
To view peaks, click Manipulate in the menu and select peak pick. Expand the window to note
peaks.
Data (Fluorescence)
Excitation: 371 nm
Concentration
(ppm)
0.1
1
5
10
Emission: 448 nm
Intensity
Wavelength
(nm)
29.354
448
183.714 450
1014.471 447
1014.49 493
Fluorescence Calibration
y = 104.23x + 140.98
R² = 0.7952
1400
1200
Intensity
1000
800
600
400
200
0
0
2
4
6
8
10
12
Concentration (ppm)
Sample
Tonic Water
Intensity
1014.490
Wavelength (nm)
499
Concentration (ppm)
8.38
Calculations
Sample preparation
Stock solution= 1000 ppm
M1V1=M2V2
(1000 ppm)V1=(10 ppm)(10 mL)
V1= 0.1 mL
Salicylic Acid Concentration
0.212=0.022x - .1062
x= 14.46 ppm
Quinine in Tonic Water Concentration
1014.49=104.23x + 140.98
x= 8.38 ppm
Conclusion
The experiment with the UV-Vis was successful. We created a calibration curve with standards
of SA in hexanes. The R squared value (0.9216) was decent but could have been improved if we had
more time. Since the calibration curve was not great the calculated concentration of SA in the aspirin
sample is not very accurate.
The experiment with fluorescence was not as successful. We had problems with the lack of
detail in the SOP. Our R squared value (0.7952) was not good at all. We did not have enough to create a
new calibration curve. Due to our poor calibration curve the concentration of quinine in the tonic water
is not accurate.