Jonathan Perr

Chem 145 AB
Oct. 8, 2016

Experiment 1: Spectroscopic Analysis of Two Analytes

Introduction/Purpose: The purpose of this lab is to measure the absorbance of self-made 30µM
solutions of tartrazine and sunset yellow and TA-prepared solutions of tartrazine and sunset
yellow by using spectrophotometry, calculate the extinction coefficients for each solution, and
assess the similarity of the self-made solutions to the TA-prepared solution. We will also
measure the absorbance of a solution with two analytes and use linear unmixing to determine the
concentrations of the analytes in the solution. This lab teaches students how to mix a solution of
a particular molarity, conduct spectrophotometry, and employ the technique of linear unmixing.

Procedure:
1. Obtain 14mg of sunset yellow and fill a 100mL volumetric flask with deionized water.
2. Add 14mg of sunset yellow.
3. Pipette 10mL of that solution into a separate 100mL volumetric flask.
4. Fill the volumetric flask with 10mL of solution with 90mL of deionized water.
5. Repeat steps 1-2 with 16mg of tartrazine.
6. Follow the procedures outlined in the Lab Manual.

Observations: All I observed was a difference in color in the various solutions. The tartrazine
solutions appeared to have a more yellowish color while the sunset yellow solutions were more
orange in color. The mixed solution had a color between the orange of the sunset yellow and the
tartrazine. The lighter solution, tartrazine, also seemed to have a peak absorbance wavelength at
a greater wavelength than the sunset yellow.

Data:
Table 1. Peak Absorbances of Self-Prepared Solutions
Absorbance Wavelength (nm)
Tartrazine 1.149 421.70
Sunset Yellow 0.855 479.10

Table 2. Absorbances of Self-Prepared Solutions at Recorded Peak Absorbance Wavelengths

Absorbance at 421.70nm Absorbance at 479.10nm
Wavelength Wavelength
Tartrazine 1.143 0.362
Sunset Yellow 0.434 0.841
Table 3. Absorbances of TA-Prepared Solutions at Recorded Peak Absorbance Wavelengths
Absorbance at 421.70nm Absorbance at 479.10nm
Wavelength Wavelength
Tartrazine 0.848 0.268
Sunset Yellow 0.328 0.663
Mixture 0.678 0.406

Tabe 4. Peak Absorbance of TA-Prepared Mixture Containing Two Analytes

Absorbance Wavelength (nm)
Mixture 0.685 429.40

Calculations:
1. Calculate extinction coefficients for TA-prepared solutions

Beer’s Law: A(λ)=ε(λ)cl

Extinction Coefficient for Tartrazine Using Beer’s Law:

Length of path, l: 1.0cm
Concentration, c: 30μM=3.0*10-5M
Absorbance, A(λ): 0.848

0.848= (ε(λ))(3.0×10-5M)(1.0cm)
ε(λ)=28267M-1cm-1
Extinction Coefficient for Sunset Yellow Using Beer’s Law
Length of path, l: 1.0cm
Concentration, c: 30μM=3.0*10-5M
Absorbance, A(λ): 0.663

0.663= (ε(λ))(3.0×10-5M)(1.0cm)
ε(λ)=22100M-1cm-1
2. Calculate the necessary mass of tartrazine and sunset yellow to create two 30μM
solutions

mol
30μM=3.0*10-8
mL

Necessary Mass of Tartrzine:

mol mg
(3.0*10-8 )¿)=1.6*10-2 Tartrazine
mL mL
 mg
(1.6*10-2 Tartrazine)(100mL)=3.2mg Tartrazine
mL

Necessary Mass of Sunset Yellow:

mol mg
(3.0*10-8 )¿)=1.4*10-2 Sunset Yellow
mL mL

mg
(1.4*10-2 Sunset Yellow)(100mL)=1.4mg Sunset Yellow
mL

Procedure for Preparing Solution:

a. Obtain 14mg of sunset yellow and fill a 100mL volumetric flask with deionized
water.
b. Add 14mg of sunset yellow.
c. Pipette 10mL of that solution into a separate 100mL volumetric flask.
d. Fill the volumetric flask with 10mL of solution with 90mL of deionized water.
e. Repeat steps 1-2 with 16mg of tartrazine.

3. Calculate the extinction coefficients for self-prepared solutions and compare to those of
TA solutions

Extinction Coefficient for Tartrazine Using Beer’s Law:

Length of path, l: 1.0cm
Concentration, c: 30μM=3.0*10-5M
Absorbance, A(λ): 1.149

1.149=(ε(λ))(3.0×10-5M)(1.0cm)
ε(λ)=38300M-1cm-1
Extinction Coefficient for Sunset Yellow Using Beer’s Law
Length of path, l: 1.0cm
Concentration, c: 30μM=3.0*10-5M
Absorbance, A(λ): 0.855

0.855=(ε(λ))(3.0×10-5M)(1.0cm)
ε(λ)=28500M-1cm-1

The self-prepared tartrazine solution’s extinction coefficient, 38300M -1cm-1, is 10033M-1cm-1

greater than the TA-prepared tartrazine solution’s extinction coefficient of 28267M -1cm-1.
The self-prepared sunset yellow solution’s extinction coefficient, 28500M-1cm-1, is 6400M-
1
cm-1 greater than the TA-prepared tartrazine solution’s extinction coefficient of 22100M-1cm-
1
. The reason for our solution’s extinction coefficients being greater than those of the TA’s
solutions most likely lies in difficulty measuring the proper mass of the solute. My lab
partner reported that the scale’s mass reading oscillated, making it difficult to get an accurate
measure. This most likely resulted in obtaining too much solute, increasing the concentration
and the extinction coefficient for our solution. This error could explain our discrepancy. One
error that would not explain our discrepancy is that there was still water in the cuvettes from
rinsing that could have further diluted our solution in the cuvette.

4. Shown in Results

5. Use linear unmixing to find the concentrations of tartrazine and sunset yellow in the
mystery solution. Use extinction coefficients from reference solutions. My lab group had
mystery solution number 1.

The total absorbance can be calculated with: Atotal(λa)=ε1(λa)c1l+ ε2(λa)c2l

One can set up a system of linear equations using two sets of wavelengths with
absorbance measures and calculated extinction constants to solve for the unknown
concentrations. Since we already have calculated one set of extinction coefficients, we
only need to calculate one more set.

Create system of equations to solve for c1 and c2

Atotal(λa)=ε1(λa)c1l+ ε2(λa)c2l
Atotal(λb)=ε1(λb)c1l+ ε2(λb)c2l

λa=479.10nm
λb=429.40nm
ε1(λb)=28267M-1cm-1
ε2(λa)=22100M-1cm-1
Atotal(λa)=0.406
Atotal(λb)=0.685

Calculate extinction coefficient of tartrazine at 479.10nm wavelength:

0.268=(ε1(479.10nm))(3.0×10-5M)(1.0cm)
ε1(479.10nm)=8933M-1cm-1

Calculate extinction coefficient of sunset yellow at 421.70nm wavelength:

0.328=(ε2(421.70nm))(3.0×10-5M)(1.0cm)
ε2(421.70nm)=10833M-1cm-1
 Note: We are using the extinction coefficient for sunset yellow at 421.70nm wavelength
to
represent the extinction coefficient of sunset yellow at 429.40nm.

Solve system of linear equations:

0.406=8933M-1cm-1(c1)(1cm)+22100M-1cm-1(c2)(1cm)
0.685=28267M-1cm-1(c1)(1cm)+10833M-1cm-1(c2)(1cm)

Solved with both a computer algebra system and by hand at the very bottom of the last
lab notebook page attached

c1=20μM (tartrazine)
c2=10μM (sunset yellow)

The concentration of tartrazine was found to be 20μM and the concentration of sunset
yellow was found to be 10μM.

6. The advantage of using the peak absorption wavelengths at low absorbances is that it
provides a superior signal to noise ratio. The higher the signal to noise ratio, the greater
the accuracy. Also the peak absorption wavelengths rarely are the same of two solutions,
allowing one to get a set of varied and representative data. Unfortunately, if there the
equipment for acquiring data malfunctions or gets and outlier at the peak absorbance, we
are only using one data point that is flawed, and all of the calculations using that data will
be quite skewed. Also, a different set of wavelengths would also work for linear
unmixing as long as you test each solution at each of the wavelengths.

7. One could use linear unmixing to find the concentrations of the analytes in a mixture with
three analytes. However, one would have to obtain three sets of absorbances by testing at
three different wavelengths (rather than two as we did) and calculate three sets of
extinction coefficients to create a system of three equations. These equations could then
be solved for concentration.
Results:
1. Graph 1: Absorbance vs. Wavelength of Tartrazine, Sunset Yellow, and Mystery
Solutions

Absorbance vs. Wavelength of Tartrazine, Sunset Yellow, and Mystery

Solutions
1.4

1.2

1
Absorbance

0.8

0.6

0.4

0.2

0
0 200 400 600 800 1000 1200 1400

Wavelength (nm)

Mystery Solution Tartrazine Sunset Yellow

The graph above displays the wavelength vs. absorbance of a tartrazine, a sunset yellow, and
a mystery solution.

We found the concentration of the tartrazine in the mystery solution to be 20μM and the
concentration of the sunset yellow in the mystery solution to be 10μM. The self-prepared
tartrazine solution’s extinction coefficient, 38300M-1cm-1, is 10033M-1cm-1 greater than the
TA-prepared tartrazine solution’s extinction coefficient of 28267M -1cm-1. The self-prepared
sunset yellow solution’s extinction coefficient, 28500M-1cm-1, is 6400M-1cm-1 greater than the
TA-prepared tartrazine solution’s extinction coefficient of 22100M-1cm-1.

Discussion:

In this experiment we created one 30μM solution of tartrazine and one 30μM solution of
sunset yellow and tested their peak absorbance wavelengths and recorded their peak
absorbances using spectrophotometry. We also tested the peak absorbances of two TA-
prepared reference solutions of tartrazine and sunset yellow using the wavelengths that
corresponded to absorbance peaks for our own solutions. We ran this same test on a TA-
prepared mixed solution. Finally, we tested the peak absorbance wavelength and measured
the peak absorbance of the mixed solution. We then used Beer’s Law in conjunction with our
knowledge of the concentration and the path length of both our solutions and the reference
solutions to calculate extinction constants for each solution at a 421.70nm wavelength and
471.10nm wavelength. Finally, we used the reference solution extinction values and our data
taken on the full spectrum test of the mixture to create a set of linear equations built from
Beer’s Law that would solve for the concentrations of tartrazine and sunset yellow in the
mixture. Look to the response in question number 10 for an explanation of why the lighter
solution had a peak absorbance at the longer wavelength. This lab was great for teaching us
how to create very dilute solutions and utilize the lab software. We also learned the technique
of linear unmixing and how to conduct spectrophotometry. Many of the calculations were
fairly straightforward, but they were great practice in dimensional analysis. This was also
solid practice in converting between units. I am assuming the data collected seems fairly
accurate as the sum of the concentrations in the mixture was 30μM, which I am guessing was
the TA’s intended sum of the concentrations in the mixture. Additionally, each of the
concentrations came out to a whole number and had a clean 2:1 ratio of tartrazine to sunset
yellow. Unfortunately, it appears as though our mixtures were not very well prepared
because the extinction constants from our solutions were consistently greater than the
corresponding extinction constants of the reference solutions. We also did not save full
absorbance spectrums for the reference solution, forcing us to omit their plots in Graph 1.
Luckily, our data consistently followed the expected trend of the lighter solution having a
peak absorbance wavelength at a greater wavelength.

Questions:

8. The “blank” measurement taken when setting up the spectrophotometer calibrated the
system. Blanking took a measurement of absorbance with just the cuvette and the water,
allowing all other measurements of absorbance to attribute only to the analyte(s) in the
solution rather than the cuvette and the water.
9. The equipment might be limited to a maximum absorbance of about 1.5 because when we
are measuring absorbance we are assuming that every molecule within the solution has an
equal chance of being struck by a photon. However, at high concentrations—which cause
high absorbances like 1.5—more particles block each other out, causing for a lower
measured absorbance than the theoretical absorbance for that solution. This inaccuracy is
why the equipment might be limited to an absorbance of about 1.5.

¿
A=log10( I ∈ I out ¿) A=1.5 and solve for Iin/Iout

At this absorbance, the fraction of light going into the solution is 10 1.5/1 of the light
coming out of the solution and going into the detector. So, 0.0316/1 of the light going in
makes it to the detector. This also makes me believe that it could be difficult for the
detector to accurately measure such low levels of light. If we doubled the path, the
 absorbance would become 3 (solve for Iin/Iout with A=3 rather than A=1.5), and 1/1000
of the light going in would make it to the detector.
10. The colors we see are the colors that are reflected and not absorbed by an object. Objects
display the color opposite of the color they primarily absorb on the color wheel. For
example, a blue object absorbs primarily orange light. So, tartrazine has its peak
absorbance at a more violet wavelength, which is opposite of yellow on the color wheel.
This means tartrazine will be yellow. Similarly, sunset yellow has its peak absorbance at
a red-violet wavelength, which is opposite of orange on the color wheel, meaning sunset
yellow will have a more orange color.