UV-Vis/Fluorescence Lab
Introduction
The UV-vis is used to probe the range of the electromagnetic spectrum from
200nm to 800nm. The energy is absorbed by the bonding electrons of a molecule.
Qualitative data can be obtained but this technique is mainly quantitative. This
experiment will determine the levels of salicylic acid in aged aspirin samples.
Fluorescence is used in the detection of analyte molecules that are found at low
concentrations. Not all molecules are capable of fluorescing so the systems are naturally
selective. This lab uses the standards quinine and sulfuric acid to make a calibration
curve.
Procedure
Day 1: Investigate the absorbance properties of SA by preparing a stock solution of 10mL
SA in hexane 1000ppm. Determine an appropriate wavelength monitor. Create a
calibration curve for the range you expect. Prove the calibration curve by creating an
unknown.
Day 2: Create a new calibration curve while the aspirin sample is dissolving. Filter and
run the aspirin to make sure it falls in the range of the calibration curve.
UV-Vis
Thermo Evolution 600 UV-Vis
 Turn on Instrument (Power switch on the left side near the electrical cord)
 Open the “VisionPro” program on the adjacent computer
 The instrument will perform a startup check to see that everything is in working
order
 Select the Scan icon on the tool bar (names of icons that the cursor is over will
appear in the bottom left corner of the program)
 Four viewing windows should be on screen, in the top center one make the
following adjustments:
 Wavelength: 190nm-400nm
 Scanning for: Absorbance
 Lamp: Tungsten
 Set the Baseline Correction for 100%T
 Prepare Blanks:
 Wash a quartz cuvette with hexane multiple times, fill 3/4th full, and dry with Kim
wipes
 Place one blank in each sample holder
 After setting the Baseline Correction the program will have had a request for
blanks, and a simple approval of the request will start the baseline scan, if denied,
simply press baseline/zero icon
 In the same window from step 5s adjustments; set the results dropdown menu to
peak pick
 Prepare sample/standard the same way as step seven and place in the front holder
and leave blank in the back one
 Press the Run icon (appears to be a person running)
 Run each sample/standard three times without opening the instrument
 Repeat steps 10-12 for remaining sample/standards
 Click on sample name on left and right click. Click plot sample. This puts all
samples on one graph.
 When finished: Close out program, turn off UV-Vis, turn off computer monitor
[LEAVE COMPUTER ITSELF ON]
Fluorescence:
Shimadzu:
 Turn on the instrument (bottom right) and computer
 Open the PX-150x program
 Check lower right corner to see if the indication says the instrument is on.
 Go to Configure > go down to the PC Configuration > make sure the First Portal
is selected for the instrument
 Configure menu > go to the Instrument and make sure that On is selected
 Set the instrument to Quantitative Analysis by going to Acquire Mode > selecting
Spectrum
 With the 1000 ppm stock solution, make a 100 ppm stock solution
 Make 2 ppm, 3 ppm, 4 ppm, 5 ppm standards of quinine in 0.1M H2SO4
 Fill a cuvette with some 2ppm solution, run the instrument under these settings:

Spectrum Type: Excitation

EM Wavelength: 400nm

Ex Wavelength Range: 35o nm to wavelength that contains peak

Sensitivity: Low

Scan Speed: Fast

Recording Range: -10.00 to an amount that shows entire peak

EX/EM Slit Width: 10/10

Response Time: 0.02

Repeat Scan/ Auto File: No setting needed/ disable

Then click Search λ and click Search. Ensure that the default range for the EX
(Excitation) and EM (Emission) are 350 - 600 nm and 240 - 650nm respectively.
Record optimal wavelengths found for EX and EM.

Spectrum Type: Emission

EX Wavelength: The ideal wavelength found in the optimal wavelength search

EM Wavelength Range: Make sure that the optimal EM wavelength is included in
the range

Sensitivity: Low

Scan Speed: Fast

Recording Range: -10.00 (low); 500.00 (high)

EX/EM Slit Width: 10/10

Response Time: 0.02

Repeat Scan/ Auto File: No setting needed/disable

Run a scan of each concentration and record the wavelength and intensity of the
largest peak. Repeat 3 times for each

Make a solution of tonic water by diluting 5mL of tonic water with 95mL of
H2SO4

Run a scan of this solution and record wavelength and intensity of largest peak.
Repeat 3 times
Calculations (UV-vis)
The desired concentrations were made from a stock solution of 1000ppm and the
dilutions were transferred and prepared in a 25mL volumetric flask. The dilutions were
made using a M1V1 = M2V2 equation.
x = 0.25 mL
The calculations of the unknown data were done using the slope of the line equation: y =
0.0181x + 0.2323.
Absorbance of Unknowns Samples at 236nm
Trial 1
0.555
Trial 2
0.546
Trial 3
0.530
Average: 0.543
The average absorbance of the unknown data was 0.543. If you use that number in the
equation of the line that was generated by linear regression analysis, as the y-value, you
will obtain a fitted value for the concentration of the sample.
x = 17.1657ppm
The standard curve showed that there was a high percent correlation given that the rsquared value was 0.978. This shows that there was a 97.8% correlation based on the
linear regression analysis. The equation that we obtained from the data showed to be very
accurate and precise.
Absorbance Values for Standard Concentrations (ppm) Of Salicylic Acid at 240nm
Absorbance
Concentration
10
0.379
10
0.37
10
0.372
20
0.587
20
0.587
20
0.583
30
0.77
30
0.771
30
0.758
40
1.025
40
1.02
40
1.004
50
1.107
50
1.113
50
1.102
60
1.427
60
1.441
60
1.416
80
1.602
80
1.598
80
1.588
Conclusion
We were able to find the unknown concentration to be 17.165ppm. The standard curve
generated used all three samples because we thought that this would give us the best
results. The equation that was generated was adequate because the linear regression/best
fit line showed a high correlation, being 98.7%. This means that there was a strong
relationship.
Results (Fluorescence)
Standard Concentrations of Quinine at 250nm
Concentrations (ppm)
Absorbance
0.1
2.477
1
2.491
1
2.4889
5
3.006
5
3.006
10
3.006
10
3.006
The results showed the equation of the line to be y = 0.058x = 2.5172. The regression
analysis showed that it was not very strong. However, it was good enough to be used for
determination of quinine in an unknown sample.
Absorbance of Quinine if Unknown sample at 508nm
Trial 1
3.006
Trial 2
3.006
Trial 3
3.006
The results showed that the absorbance of quinine in the unknown sample was 3.006,
which was similar to was obtained in the higher concentrations.
Calculations
The desired concentrations were calculated similar to how the standard concentrations
were determined in the UV-Vis experiment. The concentrations were prepared and made
by using the M1V1=M2V2 equation.
x = 2.5mL
The desired standard concentrations were all made using this calculation. The unknown
concentration was found using the equation of the line generated by the calibration curve.
x = 8.4258ppm
The unknown concentration was found to be 8.4258ppm for the unknown sample of
quinine.
Conclusion
We found the unknown concentration of quinine to be 8.4258ppm. This does not seem to
be as accurate, when compared to the UV-vis, because the experiment showed to give
consistent absorbance values from all of the standards after 5ppm. We were not able to
determine if this result was accurate.