Lab 12
UV-Vis and Fluorescence
INTRODUCTION
The UV-Vis is used to analyze analytes in the 200nm to 800nm region of the electromagnetic spectrum.
Using certain wavelengths the molecules can absorb energy into it bonding electrons and this
absorbance can yield some qualitative data from the instrument. Thus, the UV-Vis is primarily
instrument that can be used to determine concentration and quantitative data. The UV-Vis can be used
on an aromatics compounds and organic compound that have resonance and conjugation.
PURPOSE
The purpose of this experiment is to be to determine the concentration of Salicylic acid (SA) of an
unknown source. Thus, the first thing to do will be to make standard concentrations of Salicylic Acid and
make a standard calibration curve plotting wavelength and absorbance. Thus, once the calibration curve
is made, the equation of the line will be used to calculate the concentration of the unknown amount of
salicylic acid.
PROCEDURE
The standard concentrations of salicylic acid were made using the desired concentrations as
10ppm – 80ppm.
The instrument was turned on and powered up.
The settings were set to what was indicated in the SOP for UV-Vis. And A blank sample was ran
and parameter adjusted.
Each sample was run at the same setting three times in a row, and the concentrations and
absorbance’s were calculated by the instrument and recorded.
The unknown sample was ran and the concentration and absorbance was recorded by the
instrument. The unknown was prepared by taking a certain amount of taking a couple mL of
solution and diluting it in a 25mL flask and then mixing it up well. Then that sample was
transferred into a volumetric flask where is was then analyzed by UV-Vis.
CALCULATIONS
The desired concentrations were made from a stock solution of 1000ppm and the dilution were
transferred and prepared in a 25mL volumetric flask. The dilutions were made using a M1V1 = M2V2
equation.
(1000𝑝𝑝𝑚)(𝑥) = (10𝑝𝑝𝑚)(25𝑚𝐿)
x = 0.25 mL
This calculation was repeated for all desired ppm concentration from 10ppm-80ppm.
The calculation of the unknown data were done using the slope of the line equation of 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 insert that number into the equation of
the line that was generated by linear regression analysis, as the y-value you will in return obtain a fitted
value for the concentration of the sample.
0.543 = 0.0181 (𝑥) + 0.2323
x = 17.1657ppm
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
60
70
80
90
Concentration
The standard curve showed that there was a high percent correlation given that the r-squared value was
0.978, thus, showing that there was a 97.8% correlation based on the linear regression analysis. Thus,
the equation that we obtained from the data showed to have it is 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
In conclusion we were able to find the unknown concentration to be the 17.165ppm. The standard curve
that we generated used all three results that were used for the when the sample was scanned three
times. I think that this was the best way to go when making the standard curve. The equation we
generated was adequate because the linear regression that was done to make the best fit line showed
that there was a 97.8% correlation and the relatively strong relationship.
We ended up not running the 70ppm standard concentration because we were running out of time and
we already had enough data points to analyze the unknown thus we decided to use the 80ppm as the
end to give a further detection limit for quantifying the unknown concentration. I think that the overall
experiment went well because we were able to create a sufficient calibration curve and determine the
unknown concentration based on the curve, which showed to have a strong linear relationship between
the plotted concentration and absorbance at 240nm.
(Next Page fluorescence Lab)
Lab 12 Part 2
Fluorescence
INTRODUCTION
Fluorescence is used in the detection of analyze molecules found in very low concentrations. Not every
molecule is capable of fluorescence. In order for a molecules to fluoresce, the molecules has to be able
to absorb energy from an excitation source and become stabilized at the ground state of the of the
excitation level before relaxes back down to the ground states. Therefore, molecules that are able to
fluoresce are naturally selective. In this experiment we will be looking at fluorescence of molecules and
determining the concentration of an unknown source.
PURPOSE
The purpose of this experiment is to understand how the Fluorescent instrument works. Understand
how to run samples and analyze data obtained from the instrument. Also, the purpose of this
experiment is to understand analytical ways in which you can manipulate the instrument to find
unknown concentration and other sources of data or information about the molecules or samples
understudy. In this experiment we were given a 100ppm stock solution and then made low
concentrations (ppm) standard of 10ppm, 5ppm, 1ppm, and 0.1ppm. Thus, running the standards on the
instrument to give an absorbance reading at a certain wavelength which was could then translate into a
calibration to find the concentration of quinine in an unknown sample.
PROCEDURE
Sample Preparation
The standard concentrations were made for the stock solution of 100ppm. The standard
concentrations were 10ppm, 5ppm, 1ppm, and 0.1ppm. The desired amount of solution was
taken from the stock solution and diluted with sulfuric acid. The unknown sample was made in
somewhat the same way, however, the sample was not diluted with sulfuric acid but rather it
was put into a curvette in its original form.
Instrument Set-up
The instrument was turned on and set-up. The parameters and setting used were those outlined
in the SOP made for this instrument.
The standard samples were ran and unknown was ran and absorbance and wavelengths were
recorded for each sample.
RESULTS
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)
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 that the equation of the line to be y = 0.058x = 2.5172. The regression analysis
showed that it wasn’t that 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 ff quinine in the unknown sample was 3.006, which was similar
to was obtained din 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.
(100𝑝𝑝𝑚)(𝑥) = (10𝑝𝑝𝑚)(25𝑚𝐿)
x = 2.5mL
The desired standard concentrations were all made using this simple calculation. The unknown
concentration was found using the equation of the line generated by the calibration curve.
(3.006) = 0.058 𝑥 + 2.5172
x = 8.4258ppm
The unknown concentration was found to be 8.4258ppm for the unknown sample of quinine.
CONCLUSION
In conclusion we found the unknown concentration of quinine to be 8.4258ppm. Thus, I think that this
experiment was not as accurate as the previous experiment because the experiment showed to have
consistent absorbance values from all standards after 5ppm. Thus, even the unknown sample had an
absorbance that was the same. Thus, I think that the actual concentration of the unknown is somewhere
above 5ppm. It is relatively still unknown because I think that the instrument is only capable of
determining concentrations and absorbance of 5ppm or lower because this is the point where the
absorbance values started to be the same.
Therefore, I think that the concentration is probably around 8ppm. However, I cannot make a definite
conclusion. Another approach to the method would to dilute the unknown sample of quinine, thus, to
make the sample able to be adequately detectable using the instrument.