John Siller
Laboratory 5
UV_Vis Spectroscopy
Introduction
Absorbance of wavelengths in the ultraviolet and visible spectra can be used to determine the
concentration of a particular analyte in solution. Generally the more analyte in a solution corresponds to
more absorption of the electromagnetic radiation and therefore the less transmittance is allowed
through the solution. UV-Vis spectroscopy relies on the principle that π-electrons and non-bonding
electrons can absorb radiation and become excited. This absorption of UV and visible light is used to
determine the concentration of a solution once it is compared to the absorption through a blank
containing none of the analyte. UV-Vis spectroscopy is used to analyze a number of different analytes
from transition metal ions and organic molecules to large biological macromolecules. In this lab the
concentrations of unknown solutions of aspirin containing salicylic acid in hexane were determined
using calibration curves prepared with concentrations of 10 ppm, 20 ppm, 40 ppm, and 50 ppm. In
addition to UV-Vis Spectroscopy this lab also focused on spectrofluoroscopy. When a molecule absorbs
electromagnetic radiation and is excited it can shift down to the lowest vibrational state of the excited
state and then release its energy falling down to the ground state. When this occurs there is the chance
that a photon is released and the sample fluoresces. Measuring the fluorescence of a sample is another
way of quantitative determining the concentration of the analyte. In this lab the concentration of
unknown quinine solutions was determined using a calibration curve with standards of 1 ppm, 2 ppm, 4
ppm, 6ppm, and 8 ppm. The quinine was dissolved and diluted in sulfuric acid and run through our
spectrofluorometer.
Procedure:
UV-Vis
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Turn on instrument
Open VisionPro software and allow startup diagnostics
Select the proper scan
Adjust the wavelength, scanning, and lampsettings
Set the baseline correction
Prepare blanks and samples
Load blanks first
Set the menu to peak pick
Load sample and run using the run icon repeating for multiple samples
Results:
Trial results are listed in lab notebook. Each standard concentration was run three times, and
then the unknown was run three times. Below is the calibration curve calculated by the means of the
three trials at each concentration.
Concentration
Trial 1
Trial 2
Trial 3
Mean
(ppm)
Absorbance
Absorbance
Absorbance
10
0.497
0.492
0.495
0.494667
20
0.862
0.842
0.852
0.852
40
0.998
0.991
0.989
0.992667
50
1.265
1.271
1.260
1.265333
Unknown
1.147
1.136
1.150
1.144
Table 1: Salicylic acid content determined using absorbance on the UV-Vis spectrometer
1.4
Calibration Curve for UV-Vis
1.2
Absorbance
1
0.8
y = 0.0168x + 0.3966
R² = 0.9166
0.6
0.4
0.2
0
0
10
20
30
40
50
60
Concentration (ppm)
Figure 1: Calibration curve of salicylic acid absorption analyzed using the UV-Vis spectrometer
Using the calibration line y=0.0168x+0.3966 and our average absorbance value of 1.144 for our
aspirin unknowns we calculated the unknown as 44.5 ppm. This calibration curve is not as good as it
could have been. This could be due to inaccuracy in our standards or from the use of different cuvettes.
For the analysis on the spectrofluorometer of quinine in tonic water standards at concentrations
of 1, 2, 4, 6, and 8 ppm were prepared. The quinine standards were diluted using sulfuric acid. For each
standard and sample multiple peaks at different wavelengths were observed and recorded. The
averages of these peak intensities are included in the table below. A full list of peak intensities is
recorded in my lab notebook.
341 nm
Concentration
1
2
4
6
8
Trial 1
Trial 2
Trial 3
10.631
10.671
11.256
73.174
74.658
74.027
16.222
14.815
15.264
78.686
75.542
75.116
31.748
29.143
27.572
Mean
10.85267
73.953
15.43367
76.448
29.48767
448 nm
1 280.638 291.422 283.754 285.2713
2 519.088 515.072 515.252 516.4707
4 995.766 993.242 991.648 993.552
6 1015.189 1015.189 1015.189 1015.189
8 1015.189 1015.189 1015.189 1015.189
683 nm
1
2.309
2.395
2.705 2.469667
2
13.003
13.236
13.203 13.14733
4
3.626
3.314
3.195 3.378333
6
12.999
13.013
13.077 13.02967
8
6.632
4.818
6.619
6.023
883 nm
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1
13.714
14.083
13.423
13.74
2
24.464
24.372
24.187
24.341
4
46.235
46.18
46.139 46.18467
6
55.514
55.694
55.56 55.58933
8
84.563
84.516
84.467 84.51533
Table 2: Calibration data for quinine in sulfuric acid
1200
1000
341 nm
800
448 nm
683 nm
600
883 nm
400
Unknown
y = 9.5873x + 4.6073
R² = 0.9792
200
Calibration 883nm
0
0
2
4
6
8
10
Figure 2: Calibration data and curve for quinine in sulfuric acid
This data is rather inconsistent and a good calibration curve can only be made using the peaks
recorded at 883 nm. For some reason the peaks for 448 nm plateaued after 4 ppm and remained
constant for 6 ppm and 8 ppm at 1015.189. The optimal wavelengths for excitation and emission were
supposed to be 343 nm and 448 nm respectively. These data points were not consistent enough to be
used for a calibration curve and therefore I needed to make one using the 883 nm peaks. Using this
curve, y=9.5873x+4.6073, and the average intensity of 25.41 we can calculate the concentration for our
unknown tonic water. The result is a concentration of 3.13 ppm.
Conclusion:
Spectroscopic techniques such as UV-Vis and fluorescence are very useful for determining
quantitative information for a known sample. Concentrations can be determined using calibration
curves prepared with known standards. A wide range of industrial applications use these techniques for
quality assurance and testing. It is important to learn the mechanics behind these instruments and
become familiar with running samples on them. This lab went fairly well with nothing disastrous taking
place, although our results could have been better. Improvement would rely on running the samples
more and troubleshooting any problems that arise. The preparation of calibration standards is the most
important part of the procedure when running these instruments. The quality of these standards
determines the effectiveness of the instrument and the quality of the results. Without good standards
that can be used to create good calibration curves the effectiveness of these instruments is nullified. Our
data could have been better and could be improved by redoing our calibration standards until they
formed a good useable calibration curve, but due to time constrains this was not possible. For such
apparently simple instruments this lab was complex and the errors we received cannot be explained
easily.