Lab 7- UV/VIS and Fluorescence
Intro: The UV/VIS probes the magnetic spectrum from 200nm to 800nm. The energy produced
is absorbed by bonding electrons. The UV/VIS analyzes what energy is absorbed by what
electrons to give quantitative and qualitative data. Primarily, the UV/VIS produces data about
concentration. Fluorescence can be used to analyze molecules in very small concentrations.
When a molecule fluoresces, its electrons are brought up to an excited state and quickly brought
back down, which often produces photons. Not all molecules fluoresce, and those that do are
naturally selective.
Purpose: To learn how to determine concentrations of salicylic acid present in varying samples
using the UV/VIS and fluorescence.
Procedure:
Day one: Investigate the absorbance properties of salicylic acid by preparing a stock
solution in hexane at 1000ppm. Determine an appropriate wavelength for the mirror, and make a
calibration curve of the samples.
Day two: Create a new calibration curve using aspirin. For the fluorescence portion, use
sulfuric acid.
UV/VIS method:
Turn on instrument
Click on vision pro
Start up check runs automatically
Click “scan”
Wavelength 190 nm-400nm. Scanning for absorbance. Lamp: Tungsten.
Baseline set correction for 100%
Place a blank in the slot
Click to approve
Set adjustments window to “peak pick”.
Place sample in slot
Click “run”
Run each sample 3x without opening the instrument
Repeat steps 10-12 until finished running samples
Exit program, and turn off instrument
Fluorescence method:
Turn on computer and instrument
Click “PX-150x”
Click Configure -> PC Configure -> first portal
If box in bottom right is off, click “configure”, “instrument”, then “on”
Click “acquire” and “spectrum”
Fill cuvette ¾ full with least concentrated standard.
Scan
Click “search wavelengths”
Range for excitation and emission 230-450 and 230-650
Click “search”
Run that stuff
UV/VIS data:
Absorbance of Unknowns Samples at 236nm
Trial 1
Trial 2
0.555
0.546
Trial 3
0.530
Average: 0.543
Concentration Vs. Absorbance For
Salicylic Acid At 240nm
1.8
1.6
Absorbance
1.4
1.2
1
0.8
y = 0.0181x + 0.2323
R² = 0.978
0.6
0.4
0.2
0
0
10
20
30
40
50
Concentration
Continued below. Big chart!
60
70
80
90
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
UV/VIS calculations:
Sample concentrations were diluted using M1V1=M2V2 formula.
Ex:
(1000ppm)(x) = (10ppm)(25mL)
x = 0.25 mL
Unknown data was calculated using the slope of the calibration curve and the average of the
unknown sample absorption.
Ex:
0.543 = 0.0181 (x) + 0.2323
x = 17.1657ppm
Fluorescence data:
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
Absorbance of Quinine if Unknown sample at 508nm
Trial 1
3.006
Trial 2
3.006
Trial 3
3.006
Standard Concentration Vs. Absorbance Of
Quinine at Approx. 250nm
3.5
3
y = 0.058x + 2.5172
R² = 0.7581
Absorbanc
2.5
2
Series1
1.5
Linear (Series1)
1
0.5
0
0
2
4
6
8
10
12
Concentration (ppm)
Fluorescence calculations:
All calculations use same procedure and formulas as stated in the UV/VIS portion of the lab.
Ex:
(3.006) = 0.058 x + 2.5172
x = 8.4258ppm
Conclusion:
We determined the unknown SA concentration to be 17.165ppm. On our calibration
curve, there was a 97.8% correlation with the line of best fit and the absorbance at 240nm,
indicating our curve was very accurate. This is good, considering we did not run the 70ppm
sample due to time constraints. The unknown concentration of quinine was 8.4258ppm. the
fluorescence portion of the lab was not as accurate, because our calibration curve was not as
consistent. The 5ppm sample suddenly spiked for unknown reasons. We believe it may be due to
contamination or instrumental error. It may be that the instrument does not measure
concentrations higher than 5ppm, because all absorbance points after this are the same. Next
time, we will further dilute the samples to avoid this error and test our hypothesis.