Experiment 3: Spectrophotometric Determination of an Indicator

Harvey Liu
Sam Christensen
9/26/2016
Tavleen Kochar, 241L, 403, 404

"I pledge that I have not used someone elses old or current lab report when writing this
lab report. I pledge that I did not collaborate with any other students and that the report I
submitted here contains my own ideas, thoughts, observations, calculations, data,

conclusions and answers. Lastly, I pledge that the data presented in this report was
collected by my lab partner(s) and myself during my scheduled lab period.
Data & Results:
Table 1: Peaks of Absorbance and their Relative Wavelengths
Wavelength (nm)

Absorbance

Bromothymol Blue pH 5.00

431

0.533

Bromothymol Blue pH 10.00

615, 341

1.176, 0.280 <- in respect to

wavelength

Table 2: Absorbance at 615 nm vs. Theoretical/Actual pH

Theoretical pH

Actual pH

Absorbance at 615 nm (In- max)

5.08

0.012

5.99

0.086

6.98

0.546

7.3

7.27

0.764

7.6

7,58

0.967

8.04

1.105

8.96

1.199

10

9.92

1.233

Table 3: Absorbance vs. Wavelength (nm) at pH 5.00 and 10.00

pH 5.00 Extract
pH 10.00 Extract

Wavelength (nm)
@ HIn Lambda Max (431)
@ In- Lambda Max (615)

Absorbance
0.426
0.345

Table 4: Determination of Molar Absorptivity for pH 5.00 and pH 10.00 of Indicator

Table 5:
Determination of Concentration of Basic Form of Indicator at Different pH Levels

Table 6: Table for Derivative Plot to Find Inflection Point

Chart 1: Plot of pH vs. Absorbance in the 8 bromothymol blue buffer solutions

1.4
1.2
1
0.8
Absorbance

0.6
0.4
0.2
0
4

8
pH

10

11

0.8
0.7
0.6
0.5
dAbs/dPH 0.4
0.3
0.2
0.1
0
5

Average pH

Chart 2: Derivative plot shows inflection point to be at pKa = pH ~7.18, vs

literature value of 7.1 (Augustana)

10

Chart 3: Plot of Absorbance vs. Wavelength for pH 5 & pH 10 indicator

1.4
1.2
1
0.8
Absorbance

pH 5
pH 10

0.6
0.4
0.2
0
300

400

500

600

Wavelength (nm)

700

800

Calculations:
We will be calculating molar absorptivity using Beers Law and the pH solutions of 5.08 and
9.92. Because these two pH values are far outside the pH range of bromothymol blues indicating
potential, we know that the bromothymol form is fully in the acidic and basic forms,
respectively.
Beers Law:
A = bC

where A is absorbance, epsilon is molar absorptivity, b is path length of cuvette,

and where C is concentration of the molecule (241L manual).

Molar absorptivity of the acidic form (concentration divided by 1.5 to account for dilution):
.533
.95
=
=1.579104
5
5.3310
1.5
Molar absorptivity of the basic form (concentration divided by 1.5 to account for dilution):

1.176
.95
=
=3.48410 4
5
5.3310
1.5

Discussion:
Indicators are used as such because they each have the unique property of changing structure that
can cause a color shift when exposed to different environments. In the case of bromothymol blue,
the abstraction of a hydrogen (useful in basic environments) causes the oxygen to have a
negative charge, which is redistributed to the central quaternary carbon in the form of a double
bond. This causes the carbon to break its bond with the attached oxygen, finally distributing the
negative charge onto the previously attached oxygen. In this lab, we take advantage of the color
change to measure the concentrations of the basic form of bromothymol blue via Beers Law and
through spectrophotometry.
Because bromothymol blue has weak acid/base forms, the structural change between its
two forms is in equilibrium. The basic form is blue, while the acidic form is yellow. Whenever
the two exist together in solution, the color shown is somewhere in between yellow and blue,
depending on which side it leans towards. The more basic it is, the darker green it will be, and
the more acidic it is, the lighter yellow-green it will be. When the experiment was performed it
was the closest to perfectly green at around pH 7, so that value can be roughly estimated to be

near the pKa, where the concentration of the basic form is nearly equal to the concentration of
the acidic form.
There is an explanation as to why the basic form of bromothymol blue is blue and why
the acidic form is yellow. Looking at the absorption peeks, it is clear that the basic form at pH
9.92 absorbs the most at 615 nm and that the acidic form at pH 5.08 absorbs the most at 431 nm.
Light works through reflection. The color that is perceived on an object is the light reflected, the
light that is absorbed is the light that is not seen. Therefore, because pH 5.08 absorbs the most
light at or around 431 nm, the color purple, we can expect to see the reflected color, or the
opposite of purple on a color wheel, yellow. The same applies to pH 9.92. It absorbs the most
light at 615 nm, or the color red, so we see the most color reflected on the opposite color on the
color wheel, blue.
The extraction is done in order to test the solubility of the two forms of the bromothymol
blue in water. The form that is the most soluble would theoretically show less change in
absorption, as less of the form would be dissolved in the organic layer. Experimentally, we saw
more decrease in absorption of the basic form, from 1.176 down to .345, compared to 0.533
down to 0.426 in the acidic form. Theoretically, however, the more polar basic form should be
more soluble and should show less decrease in absorption. This is easily explained in an error in
conducting the lab. We had accidentally taken a plastic cuvette, which had reacted with the
organic ethyl acetate layer. We believe that this had some reaction on the basic bromothymol
blue form in solution. By the time we had realized our mistake and had transferred what was left
to a new glass cuvette, we likely had lost a lot of the basic bromothymol blue.
After graphing the derivative plot, we see the slope is near zero at around pH 7.18. This
zero slope in the derivative plot correlates to an inflection point where the change in slope in the
Absorption vs. pH plot begins to decrease after increasing. At this point, pH = pKa because the
concentration of the basic form is nearly the same as the concentration of the acidic form. This is
near the literature value of 7.1 (Augustana University).
End of Lab Questions:
1.
pH= pKa+ log

( [[ ]] )
base
acid

5
x /
.33*10^(-5) x )

5.00=7.18+ log
7

x=3.510

M basic bromothymol blue, 5.30 * 10^(-5) M acidic bromothymol blue

5.30105
=0.994 fraction protonated bromothymol
5.33105

2. I will be using the hypothetical discussed above, as the flawed lab procedure was clearly
changed results.
a. We know that the answer here must be the species without a neutral charge, as the species with
positive or negative charges will be most soluble in aqueous solution. The answer here
will be the B. The B can be protonated by the acidic environment, causing it to mix with
the polar aqueous layer. Additionally, B is a stronger base than the other species, with a
pKb of 6 (14-8), allowing it to protonate more easily. The movement out of the organic
layer will allow more of B to dissolve.
b. We would expect the answer here to be a species with a charge, as the species with non-neutral
charges are more attracted to the polar aqueous layer. The answer here is A-, for a similar
reason as question 2a. The acidic environment can protonate the A- to AH, which with a
neutral charge, will move to the organic layer, freeing up more space for A- to dissolve in
the aqueous layer.
c. We (would have, but could not due to errors) been able to determinate which form was more
soluble in the aqueous phase by seeing the change in absorption. If a lot of the species
dissolved and was thrown away in the organic layer, then we would have seen a big
decrease in the absorption at lambda max for that species.
3. The basic and acidic species are in equilibrium with each other. Where there is a lot of acidic
form, there is little basic form and vice versa. Because of this, we would see a lot higher
absorption at around pH 5 where there is a lot of the acidic species and low absorption at pH 10
where there is little acidic species. The plot of Absorption vs. pH would be almost inverse.
4. The isosbestic point is ~500 nm, where the absorption is identical in both the pH 5 and the
pH10 solutions. The isosbestic point indicates the point on the spectrum where the molar
absorptivity is the same for multiple forms of a molecule. Here, only the formal
concentration matters when finding absorption. Because the two forms absorb the same
amount of light at that wavelength, it does not matter how much of each individual
species exists, only the total amount.
References:
http://faculty.augie.edu/~dew/242-Ka.pdf. Retrieved September 25, 2016.