Brittany Hicks
UV-VIS & Fluorescence Lab- Dr. Foy
Spring 2013
UV-VIS and Fluorescence
Introduction
The ultraviolet-visible spectrophotometer is a chemical instrument that analyzes compounds
that fall under the ultraviolet and visible light spectrum regions of the electromagnetic spectrum. The
goal of this instrument is to analyze compounds through electronic transitions. Wavelength and
maximum absorbance can both be determined. Determination of particular concentrations of the
sample can be explained through Beer’s Law. Analytical analysis for the determination of different
analytes is primarily used in the UV-VIS, in which absorbance and wavelengths are optimal comparisons.
Fluorescence is very similar in technique and analysis of the UV-VIS. The main difference
between the two instruments is that fluorescence uses atomic emission rather than absorption.
Fluorescence uses a beam of light that excites electrons in molecules of certain compounds. These
compounds then emit a wavelength of light. Electromagnetic spectroscopy is used in many different
analysis of medical, chemistry and biology departments.
The purpose of this experiment was to familiarize ourselves with both of these instruments so
that proper analysis could take place. Aspirin was the main sample under analysis. Using the UV-VIS,
aspirin was tested to determine the amount of salicylic acid. Fluorescence will analyze aspirin using
quinine and tonic water to determine amounts. Both known and unknown samples were both ran.
Procedure - Spectrofluorophotometer
 Turn on instrument in the bottom right hand corner, and also the computer
 Open the PX-150x program
 Check lower right corner of screen to see if indications says instrument is on
o If not, go to configure > PC configuration > select the First Portal for the instrument
o Back to Configure menu > Instrument > On
 Go to Acquire mode > Spectrum
 Place a cuvette of H2SO4 in the instrument
o Configure > Parameters > Spectrum type: excitation
o EX wavelength: 400 nm
o EM wavelength: 220-900nm
o Sensitivity: Low
o Scan Speed: Fast
o Recording Range: -10-500 (may need to be adjusted)
o EX/EM slit width: 10/10
o Response time: .02
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o Repeat Scan/Auto File: no setting needed
o Click Ok
Place cuvette of smallest ppm in the instrument
o Click Search λ icon in bottom right of screen
o Set EX to around 230-450 and EM to 230-650
o Click search
o Record optimal wavelengths for EX and EM
Go to Parameters > Spectrum type > Emission
o EX wavelength: optimal wavelength from search
o EM wavelength: range needs to include optimal wavelength
o Sensitivity: Low
o Scan Speed: Fast
o Recording Range: -10-500
o EX/EM slit width: 10/10
o Response time: .02
o Repeat Scan/Auto File: No setting needed
Run a scan of each concentration 3 times, recording the wavelength and intensity of the highest
peak for each trial
Run tonic water dilution as unknown also repeating 3 times and recording wavelength and
intensity of the highest peak
Procedure – UV-VIS
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Turn on UV-Vis
Open Vision Pro Software
Wait for the set up scan to complete
Once that is done, select the scan icon on the toolbar
o Set wavelength to Start:190 Stop:400
o Scan for: Absorbance
o Lamp: Tungsten
Go to Set-up > Baseline tab > 100% T
To blank the instrument fill a clean cuvette ¾ with hexane and place in front slot
o Click the baseline zero icon
When that is complete, remove the blank and put the first sample in
Click the run icon
Run each sample three times only opening the instrument to put new sample in
When finished, close out computer program and turn off instrument
Data and Analysis
Unfortunately chromatograms could not be obtained for this lab from the UV-VIS instrument.
However, all the data was written down in my lab notebook before the program was shut down. A hand
drawn picture of the chromatograph showing how the peaks looked for each concentration as well as
where the unknown sample was pictured in the overall figure. Both the quantitative data and figure of
concentration graph are attached within the lab notebook.
UV-VIS DATA
Concentrations Prepared
10ppm
20ppm
30ppm
40ppm
50ppm
100ppm
Aspirin Sample
Absorbance 1
Absorbance 2
Absorbance 3
0.147
0.157
0.210
0.259
0.273
0.716
0.223
0.136
0.182
Concentration Run 1
wavelength
0.1ppm
466
Run 1
intensity
30.25
Run 2
wavelength
466
Run 2
intensity
28.80
Run 3
wavelength
466
Run 3
intensity
28.60
1.0ppm
466
265.85
466
774.40
466
767.24
5.0ppm
467
1015.69
467
1015.66
467
1015.62
0.171
0.419
0.696
0.789
1.545
0.786
0.277
0.239
0.383
0.451
1.345
0.442
Fluorescence Data
10.0ppm
MAX
MAX
MAX
Blank
360
5.73
360
5.27
360
5.47
Unknown
467
876.41
467
858.84
467
841.98
***Optimal Wavelength… Excitation= 344
Emission= 449
1200
1000
800
600
400
200
0
0
1
2
3
4
5
6
7
This graph shows concentrations verse the intensities received from Fluorescence procedure
Conclusion
Both instruments were used pretty easily, except for the UV-Vis closing out of the
program before chromatograms could be saved as a picture file. All of the data looks good overall in
comparison to results other classmates received on these instruments as well. There are minor
differences in results which could have possibly been to dirty cuvettes being analyzed containing the
samples. More concentrations could have been made up for both of these instruments for further
testing and to see if better results could have been obtained. Our regression looking at the
concentration vs. intensities was not very good which could be fixed by running more samples and
concentrations.