ACS Chemistry Club

Hillcrest High School Chapter

Spectrophotometry Lab Activity

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NOTE: The following section is pre-lab and covers some of the theory
utilized in the lab. It is not required reading.

Light Absorption
The electromagnetic spectrum is composed of wavelengths, which range from short to
long. In this lab, we will ignore the wavelength extremes at both ends and instead focus
on the wavelengths most familiar to us - visible and near visible light. Human vision is
capable of detecting wavelengths of electromagnetic radiation from about 380 nm-750
nm.

This is called the visible light range. In this lab, you will also examine light at
wavelengths near-infrared, which have a wavelength that is slightly longer than those
observed in the visible light range. When light passes through a substance, some of it is
absorbed due to interactions with the atoms and molecules in the medium. This can be
quantified by defining the term absorbance (A). Absorbance is given by the following
equation:

𝐼0
𝐴 = 𝑙𝑜𝑔( )
𝐼1

where 𝐼0 is the intensity of the light before it enters the substance and 𝐼1 is the intensity
of light after it is transmitted through a substance.

Intensity (𝐼) is defined as the amount of energy transmitted in a unit area in a unit time,
or power (𝑃) per area (𝐴𝑠 ).

𝑃
𝐼=
𝐴𝑠

The absorbance is wavelength dependent and will also vary depending upon the
medium (air, water, glass, etc.) through which light passes. The medium can be
characterized by the molar absorptivity, ε (in m2/mol), by the concentration, 𝑐 (in
mol/m3), and by the length of the medium the light passes through, 𝑏 (in m). The molar
absorptivity has one value for a particular material, e.g., there is one value of ε for salt.
Of course, the medium may consist of various substances; for instance, a solution may
have multiple solutes each with their own absorption coefficient and concentration. The
absorption coefficient and concentration can be combined into a single coefficient, the
____________________________________________________________________________________________________

Adapted From Portland State University’s Spectrophotometry Lab Exercise

http://web.pdx.edu/~ralfw/uploads/1/0/2/6/10260941/pulse_oximetry_laboratory_guide.pdf
 ACS Chemistry Club
Hillcrest High School Chapter

Spectrophotometry Lab Activity

____________________________________________________________________________________________________
absorption coefficient α (in m-1). The dependence of absorbance on these variables is
called Beer’s Law and is given by the following equation:

𝐴 = 𝜀𝑐𝑏 = 𝛼𝑏

The loss in intensity through a medium is known as attenuation.

Figure 1 - Absorption due to light passing through a medium

Spectrophotometer - Bromothymol Blue

Lab Procedure

Background:
Bromothymol blue is a pH indicator that changes color with a change in pH – although it
appears blue in an alkaline solution, its color is green in a neutral solution. In this
experiment, you will utilize bromothymol blue to simulate the color change that occurs
as oxygen binds to hemoglobin and to illustrate how absorption characteristics change
with color. Standard safety precautions must be used when working with any chemical.
Do not ingest and avoid contact with eyes. Wash hands after contact.

Similarly to how we observe hemoglobin as red when bound with an oxygen molecule
(and more purple if it is reduced), CO2 in the form of carbonic acid reacts with the
____________________________________________________________________________________________________

Adapted From Portland State University’s Spectrophotometry Lab Exercise

http://web.pdx.edu/~ralfw/uploads/1/0/2/6/10260941/pulse_oximetry_laboratory_guide.pdf
 ACS Chemistry Club
Hillcrest High School Chapter

Spectrophotometry Lab Activity

____________________________________________________________________________________________________
bromothymol blue molecule so that we observe a change in color from green to a more
yellow color. Over time, the solution reacts with oxygen in the surrounding air and the
pH will change, resulting in another change in color - returning to the initial shade of
green.

Materials:
- Bromothymol Blue IndicatorS
- Spectrophotometer
- Cuvette
- Cup
- Data analysis software(optional)

Purpose Statement: The purpose of this laboratory exercise is to analyze the

difference in absorptivity of Bromothymol blue indicator in different solutions (acidic and
basic conditions). We will determine how the effect of chemical reactions changes the
molecular structure of a compound, resulting in a different absorbance pattern.
Neutral Solution:

1. Fill cuvette with prepared bromothymol blue solution (it should be in its neutral
green phase
2. Using the color wheel, determine the range of optimum wavelength absorbed by
Bromothymol Blue.
3. Adjust the dial on the spectrophotometer to a
wavelength within the optimum range.
4. Place the cuvette in the spectrophotometer as
shown:

5. Record the absorbance in the data table.

6. Repeat steps 3-5 for 4-6 trials, using a wide range of wavelengths within the
optimum spectrum.
7. Optional: Graph the absorbance spectrum by plotting the points for wavelength
and absorbance, as shown, or use a computer program to graph the
wavelength/absorption spectrum.

____________________________________________________________________________________________________

Adapted From Portland State University’s Spectrophotometry Lab Exercise

http://web.pdx.edu/~ralfw/uploads/1/0/2/6/10260941/pulse_oximetry_laboratory_guide.pdf
 ACS Chemistry Club
Hillcrest High School Chapter

Spectrophotometry Lab Activity

____________________________________________________________________________________________________
Acidic Solution:
1. Pour bromothymol blue into a cup and gently blow on it (carefully, so as not to
splatter the solution.) This will result in a reaction between the carbon dioxide
and water which forms carbonic acid, changing the pH of the solution and thus
resulting in a color change of bromothymol blue.
CO2 (g) + H2O(aq) ↔ H2CO3 (aq)
2. Wait 30- 60 seconds to allow time for the reaction to occur and the solution to
change color - note that the color change should be distinct. If you do not detect
a color change, continue to blow until change is observed.
3. Once the color change has happened, transfer the solution into a cuvette (to
avoid spills, transfer solution with a funnel over a sink.) Place cuvette in the
spectrophotometer as shown above.
4. Repeat steps 3-5 to determine the optimum absorption wavelength for the
Bromothymol blue in the acidic condition.

Data Table Wavelength 1 Wavelength 2 Wavelength 3 Wavelength 4 Wavelength 5

Bromothymol
Blue(Neutral)

Bromothymol
Blue(Acidic)

____________________________________________________________________________________________________

Adapted From Portland State University’s Spectrophotometry Lab Exercise

http://web.pdx.edu/~ralfw/uploads/1/0/2/6/10260941/pulse_oximetry_laboratory_guide.pdf
 ACS Chemistry Club
Hillcrest High School Chapter

Spectrophotometry Lab Activity

____________________________________________________________________________________________________
Post-Lab Theory
What occurred in the lab exercise was an acid-base reaction that resulted in a change
in the pH of solution and corresponding change in the structure of Bromothymol blue,
causing absorbance to change which was measured with the spectrophotometer.

Acids/Bases
In simple terms, acids and bases are compounds which can either act as proton (H +)
donors or acceptors; those that donate are labelled acids, and those that accept are
bases. Some compounds are capable of acting as either an acid or a base and are
called amphiprotic, acting a given manner based on the strength of the compound they
are reacting with. Water is an example of this, capable of acting as an acid:
B + H2O ↔ HB+ + OH-
donating a proton and becoming hydroxide, or as a base:
HA + H2O ↔ A- + H3O+

The H2CO3 formed by the reaction with water,

CO2 (g) + H2O(aq) ↔ H2CO3 (aq)
is an acid, able to donate hydrogen ions, which it does with water that acts as a base:
H2CO3 + H2O ↔ HCO3- + H3O+
HCO3- + H2O ↔ CO32- + H3O+
pH
pH is logarithmic measure of the hydrogen concentration in an aqueous solution, which
is present in an aqueous solution as hydronium, H3O+. pH is approximately the negative
of the base 10 logarithm of the molar concentration of H+ in the solution. A lower pH
indicates a more acidic solution. A higher pH indicates a more basic solution.
𝑝𝐻 = −𝑙𝑜𝑔(𝐻+)

The above reactions of carbonic acid with water produce more hydronium, displacing
the equilibrium and thus resulting in a drop in pH.

pH Indicator
A pH indicator is a chemical compound capable of determining the pH of a solution
visually. This is possible because the pH indicator has been experimentally tested to
produce visible color change at certain pH ranges. For example, the pH range of
bromothymol blue is 6.0 - 7.6, meaning that it changes color when the pH of the solution
hits either 6.0, or 7.6.

____________________________________________________________________________________________________

Adapted From Portland State University’s Spectrophotometry Lab Exercise

http://web.pdx.edu/~ralfw/uploads/1/0/2/6/10260941/pulse_oximetry_laboratory_guide.pdf
 ACS Chemistry Club
Hillcrest High School Chapter

Spectrophotometry Lab Activity

____________________________________________________________________________________________________
Bromothymol blue is an acid-base indicator that is yellow at pH values less than 6 and
blue at pH values above 8. Between pH values of 6 and 8, bromothymol blue is some
shade of green. It is more yellow-green near pH 6 and more blue-green near pH 8. At
pH 7 (neutral state), Bromothymol blue is green. Its chemical structure is shown in
Figure 1.

Figure 1: Chemical structure of bromothymol blue (3', 3"-

dibromothymolsulfonephthalein) as it occurs under acidic conditions. Its chemical
formula is HC27H27Br2O5S).

Like all acid-base indicators, bromothymol blue is a weak acid as shown in the following
equilibrium in which HBB represents the protonated (yellow) form of bromothymol blue
and BB- represents the blue conjugate base form.

HBB (yellow) + H2O ↔ H3O+ + BB− (blue)

A neutral pH solution (pH = 7, [H3O+] = [OH-]) will make the indicator be a green color.

____________________________________________________________________________________________________

Adapted From Portland State University’s Spectrophotometry Lab Exercise

http://web.pdx.edu/~ralfw/uploads/1/0/2/6/10260941/pulse_oximetry_laboratory_guide.pdf