Lab 3: UV-Vis and Fluorescence
Introduction:
UV-Vis is commonly used for analyzing compounds in ultraviolet and visible
regions of the electromagnetic spectrum. It looks at electronic transitions and allows for
the determination of wavelengths and maximum absorbance of compounds. UV-Vis
measures transmittance or absorbance of solutions containing transparent cells.
Fluorescence is used for quantitative determination of large elements that form
vapors and hydrides. To be able to use molecules in fluorescence they must be able to
absorb energy from an excitation source and becomes stabilized at the ground state or
the excitation level before relaxes back down to the ground state. Fluorescence
instruments are very complex and expensive and are not as sensitive as other
possibilities.
Methods:
UV-Vis
1.
2.
3.
4.
Prepare standards (calculations below)
Turn instrument on with switch on left next to power cord
Click on “visionPro” and allow ten minutes for set up
Select the scan icon (the first icon)
 Enter wave length 190-400 nm
 In data mode hit absorbance, and in lamp change select tungsten
5. Click baseline corrections on the bottom of that window and select 100% T
6. Click baseline zero (the 3rd icon in on the second set of icons)
7. Get out quartz cuvet that says UV/Vis on the box
8. Wash cuvet with hexane (three times)
9. Then fill with hexane ¾ of the way
10. Wipe with kim wipe and run as base sample the same prep but click the run icon
to start and watch the lower right of the screen for completion
11. When samples are complete right click on the name and select plot
12. Click graph menu, select track then click on the peak on the graph window to get
wavelength and absorbance
Fluorescence
1. Prepare sample again and make a random sample
2. Open PX-150X program
3. Click configure select PC configuration, port 1
 Configure, instrument, on
4. Click aquire in mode and spectrum
5. Click configure and parameters
 Spectrum type: excitation
 EM wavelength: 400 nm
 EX wavelength range: 200-900 nm
 Sensitivity: low
 Scan speed: fast
 Recording range: -10 to 500
 EX/EM slit width: 10/10
 Response time: 0.02
6. Click run
7. Click the lambda after the first run and set the ranges to
 EX: 230-450 nm
 EM: 240-650 nm
8. Search and then write down the optimal wavelengths
 EX was 343
 EM was 448
9. Select configure and parameters
10. Switch the spectrum to emission
 Put the excision wavelength as the one we found
 Make sure the emission range has the value found in it
11. Run other samples and note peaks (intensity) and wavelengths
Calculations:
UV-Vis
1 𝑚𝑔
0.001 𝑔
1𝑔
100 𝑚𝑔
=
=
=
1𝑔
1,000,000 𝑔 100 𝑚𝐿
1000𝑚𝐿(1 𝑚𝐿) 1000 𝑔
M1V1=M2V2
1000(x)=(40)(50)
=
1000(x)=2000
=2.0mL
1000(x)=(25)(50)
=
1000(x)=1250
=1.25 mL
1000(x)=(10)(50)
=
1000(x)=500
=0.5 mL
1000(x)=(7)(50)
=
1000(x)=350
=0.35 mL
1000(x)=(2)(50)
=
1000(x)=100
=0.10 mL
All at wavelength of 310 nm






2 ppm absorbance= 0.017
7 ppm absorbance= 0.044
10 ppm absorbance= 0.025
25 ppm absorbance= 0.267
40 ppm absorbance= 0.459
Unknown ppm absorbance= 0.335
Unknown ppm calculation
0.335 = 0.01190𝑥 − 0.0234
0.3584 = 0.01190𝑥
30.12 = 𝑥
30.12 𝑝𝑝𝑚
Fluorescence
1 𝑚𝑔
0.001 𝑔
1𝑔
100 𝑚𝑔
=
=
=
1𝑔
1,000,000 𝑔 100 𝑚𝐿
1000𝑚𝐿(1 𝑚𝐿) 1000 𝑔
M1V1=M2V2
1000(x)=(3.5)(50)
1000(x)=(3)(50)
=
=
1000(x)=(2.5)(50)
1000(x)=(2)(50)
1000(x)=150
=
=
1000(x)=175
1000(x)=125
1000(x)=100
=0.175mL
=0.15 mL
=0.125 mL
=0.1 mL
All wavelengths at 447.872 nm





2 ppm intensity= 492.44
2.5 ppm intensity= 634.074
3 ppm intensity= 772.00
3.5 ppm intensity= 850. 593
Tonic water intensity= 538.148
Tonic water ppm calculation
538.148 = 242.2𝑥 + 21.09
517.058 = 242.2𝑥
2.135 = 𝑥
2.135 𝑝𝑝𝑚
Results:
VisonPro software print outs for the UV-Vis can be found on pages 58-68 of the lab
notebook. The concentration curves and R2 values calculated are located on page 70.
The fluorescence instrument did not have a print our capability but the intensitys and
wavelengths can be found on pages 45-47 along with the rest of the write up. The
concentration curve and R2 value calculated can be found on page 71.
Discussion:
As seen on page 70 of the lab notebook, there are two different concentration curves.
The 10 ppm seemed to be an outliar so it was removed to raise the R2 value and give a
better equation of the line. The equation was used in the calculation of the unknown’s
ppm value. Pages 57 through 62 are the original readings with three peaks. Amanda
assisted us and said that we should rerun them with a smaller wavelength range that we
were looking for (single it down to one peak). The wave length for the second set of
data was changed to 280-340 nm.
We had a lot of difficulty using the fluorescence instrument. Originally we used a 5 and
4 ppm value also. These both flat lined and were not good data so they were removed.
Overall these experiments seemed to be for the most part successful. We got good
results and good R2 values.