Elissia Vecere

2/15/21
CHEM 3560 Section 102
Experiment 6: Spectrophotometry
Introduction
Spectrophotometry is the study of the interaction of electromagnetic radiation with
molecules. The use of spectrophotometry is essential to this lab, along with the basics that
pertain to it. In order for spectrophotometry to be used, the idea of wavelength, the distance
between adjacent peaks in the wave, and the frequency of light, the number of waves passing
through adjacent peaks in the wave, must be understood. The use of this knowledge allows for
students grasp the idea of transmittance and the Beer-Lambert Law (A=elc). Throughout this
experiment, the student learns how to use a spectrophotometer in order to calculate
concentrations, while using Beer’s law. The student also will complete a simple protein assay
while learning how to create standard curves based on the determination of the protein
concentration of the unknown. The protein assay makes use of standard curves and is a very
common use of spectrophotometry.

Objective
The objectives throughout the lab, if completed successfully include having the ability to
zero the spectrophotometer at a variety of wavelengths and measure the absorbencies of
solutions. Along with this, the student should be able to decide when dilutions must be made
and make the appropriate dilutions, along with calculating absorbencies, concentrations, and
extinction coefficients while using Beer’s law. Next, the student should be able to make
standard curved and determine concentrations from the. Finally, the student should be able to
define a reagent blank and decide what reagents must be present in one while also designing
protocols for the creation of a standard curve.

Procedure
To begin the experiment, the student obtained a solution of NADH of unknown
concentration. Next, the student warmed up and zeroed the spectrophotometer at 340 nm.
The student then made a minimal dilution of the NADH to provide enough solution to measure
in the cuvette. The absorbance of the NADH was measured. If the absorbance was greater than
0.8, it was diluted with water and remeasured. These dilutions were recorded in order to assure
the student knew how much NADH was added to how much water. Finally, for part A, the
student used the absorbance and any dilutions made to determine the concentration of the
NADH millimolar (mM).
To begin part B, the student warmed up the spectrophotometer to 595 nm. The student
then set up ten 3 mL cuvettes for the reactions. Next, the student set up a protocol similar to
the table 3.1 found in the lab manual. While using the most accurate pipet available, the
student pipetted the BSA standard into the tubes. The student was very careful in order to
avoid any pipetting error. The student obtained an unknown BSA solution and chose a volume
of 50 microliters, of the unknown to assay and pipet into tube 9. 3 mL of the Bradford reagent
was then added to each tube while inverting each tube immediately after its addition. The
tubes then sat for 10 minutes before the absorbencies were read. Finally, the student made a
plot in order to determine how much BSA could be added without the curve beginning to stray
from linear.
 Elissia Vecere
2/15/21
CHEM 3560 Section 102
Experiment 6: Spectrophotometry

Observations
At the beginning of the lab, the student was assigned unknown D for part A. The student
noticed that the unknown was clear, and the student needed to be extremely thorough in
remembering which cuvette contained the blank reagent and which contained the unknown
NADH solution. The blank reagent was needed in order to zero out the spectrophotometer and
measure the absorbance of unknown D. Since the absorbance level of unknown D was less than
0.8, the student did not need to dilute the solution.
Moving on to part 8, the student was assigned unknown H. The student noticed that
there was less solution in this tube, than in the prior unknown. Approximately 100 microliters
were present in the tube, so the student chose to add 50 microliters to the 9 th cuvette. Finally,
the student noticed that there were some values that needed to be pipetted into each cuvette
that was not directly achievable on the pipets available. Therefore, the student had to complete
multiple pipettes of a smaller value in order to achieve a greater value. For example, to achieve
a 40-microliter pipette of the BSA standard, the student had to either do 2 pipette pull ups of 20
microliters or 4 pipette pull ups of 10 microliters.
 Elissia Vecere
2/15/21
CHEM 3560 Section 102
Experiment 6: Spectrophotometry
Results
(Dilution was not needed because the Absorbance was less than 0.8. The Absorbance of
unknown D was 0.506 therefore 2 and 3 did not need to be completed.
 Elissia Vecere
2/15/21
CHEM 3560 Section 102
Experiment 6: Spectrophotometry
Discussion

Standard Curve Unknown H

1.4
f(x) = 0.0129559763313609 x + 0.108538461538462
1.2
Absorbance (corrected)

1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120
Protein (micrograms)
 Elissia Vecere
2/15/21
CHEM 3560 Section 102
Experiment 6: Spectrophotometry
 Elissia Vecere
2/15/21
CHEM 3560 Section 102
Experiment 6: Spectrophotometry

Summary
Throughout the lab, the student made use of spectrophotometry to calculate
absorbencies of various solutions, with the help of a reagent blank. In part A, the student made
use of deionized water as the reagent blank, and the unknown NADH solution as the interested
absorbency value. Next, in part B, the student went through and created numerous different
solutions with variable BSA volumes, protein unknown values, and the Braford reagent. The
blank cuvette was filled with strictly filled with the Bradford reagent in order to produce an
absorbency of zero. These steps allowed for the student to obtain the skills to decide when
dilutions must be made and how, use Beer’s law for various unknown variables, make standard
curves, and determine the concentration from these standard curved.
 Elissia Vecere
2/15/21
CHEM 3560 Section 102
Experiment 6: Spectrophotometry