UV/VIS, Fluorescence Lab Report
Introduction: Ultraviolet and Visible spectroscopy is the absorption of atoms and molecules in
the UV/VIS spectral region. The absorption in the visible range directly affects the perceived
color of the chemicals involved and by undergoing electronic transitions. The UV region is
scanned from 200-400nm while the visible region is 400-800nm. When comparing spectra of
different compounds, we use Beer’s Law to calculate the absorption values. Sometimes the
equation can be used to measure the molar absorptivity of a specific molecule. Some applications
of the UV/VIS are the quantitative determination of transition metals, organic compounds, and
the biological macromolecules. Fluorescence spectroscopy is almost the opposite of what
UV/VIS is. Fluorescence spectroscopy measures the fluorescence of a sample, through a
fluorometer, where it is the transition of molecules from the excited state to the ground state,
where they are excited at a certain wavelength and emit energy at a different wavelength. Some
applications of fluorescence include the analysis of organic compounds for biochemical,
medical, and chemical research purposes.
Purpose: The purpose of this experiment was to determine the calibration and concentrations of
salicylic acid using ultra violet and visible light radiation and quinine using the fluorescence as
well as the unknown concentrations for pure salicylic acid, aspirin, and tunic water.
Methods: Day 1: For the first day we worked with the UV/VIS. We measured various
concentrations of the salicylic acid dissolved in hexane. Samples were made from a 1000ppm
stock and we made standards of 40 with 4ml, 25 with 2.5ml, 10 with 1ml, and 2 with .2ml ppm
solutions in 100ml volumetric flasks. We also made samples of the salicylic acid in hexane as
well as pure powder salicylic acid as well as aspirin samples. We did three runs of each standard
and samples and recorded the absorbance where each standard had the same wavelength in
common. For us the wavelength was 240 nm and we recorded the absorbance at this wavelength
for each run and created a calibration curve to determine the concentration of unknowns.
Day 2: This day we worked on the Fluorescence spectroscopy and as with the UV/VIS
we made standards of quinine from a 1000ppm stock in sulfuric acid (H2 SO4 ) and used tunic
water as our sample to determine the level of quinine in the water. Our standards were 10 with
5ml, 5 with 2.5ml, 1 with .5ml, and .1ppm with .05ml of quinine in 50ml volumetric flasks with
the H2 SO4 . We did three runs of each standard and sample and recorded the intensity and
wavelength from the largest peak. Our optimal excitation wavelength was 345nm and optimal
emission wavelength was 449nm. We calculated a calibration curve for this also to determine our
unknown concentrations.
Results: We recorded the wavelength and absorbance for the UV/VIS standards and unknowns
and used an equation to determine the unknown concentrations. The average absorbance was
calculated for the three runs to give us more accurate results and the optimal wavelength was
240nm.
Concentration (ppm)
2
10
25
40
Aspirin
Unknown 1
Absorbance
.102
.126
.469
.724
1.436
.127
The calibration equation was y = 55.7017x - .538; the unknown concentration was 6.54ppm and
the aspirin was 79.45ppm. We also calculated the % error for each and the unknown % error was
6.62% and the aspirin was between 74.19 and 84.71% based on our calculated error from the
other sample. The Fluorescence data of quinine is provided below:
Concentration (ppm)
.1
1
5
10
Tunic Water
Intensity
90.6
150.3
40
60.3
14.6
The calibration equation for fluorescence was y = -6.717x +112.37; the tunic water quinine
concentration is 13.8ppm, which falls in our standard concentration range.
Conclusion: Overall both experiments went without complications. However for the UV/VIS
there were some problems with dissolving the salicylic acid in the hexane as well as the aspirin.
So the results probably came out different because not all the light was absorbed since not
everything was dissolved. Some other sources of error could be from not all the standards having
the 240nm peak so the absorbances were not as accurate. Fluorescence worked also but it had its
own problems as well. Some sources of error for this included the inability to accurately make
the .5ml and .05ml solutions of quinine in H2 SO4 because the amount was so small that we had
to “eyeball” the measurements. The intensities for these standards were off from the rest because
of the measurement problems. The concentration of the tunic water was calculated and it turns
out that the concentration of 13.8ppm was within the FDA standards of quinine. On the other
hand it is a lower concentration because the sample tunic water that was used expired in 2008, so
the results weren’t as reliable for any current samples. Also if there was a newer sample used,
there would most likely be a higher concentration of quinine in the tunic water because of the
original manufacturing date for sale.