UV/Visible Absorption Spectroscopy
Kathleen Nolan and Dylan Catterton
September 4, 2014
Purpose: The purpose of this lab was to understand both the theory for light absorption
by biomolecules and the mechanisms by which the spectrophotometers functions.
Data:
Table 1:The use of Biomate 3 Spectrophotometer to determine the concentration
of an absorbing substance in a solution.
Unknown Sample
A
B
C
D
Absorption @
508nm
1.173 A
0.243 A
0.643 A
1.641 A
Graphical
Concentration
0.6 mM
0.12 mM
0.3 mM
0.9 mM
Regression
Concentration
0.637 mM
0.132 mM
0.349 mM
0.891 mM
Beer’s Law
Concentration
0.639 mM
0.132 mM
0.350 mM
0.894 mM
Figure 1: Standard curve for the absorbance of various concentrations of KMnO4 at 508
nm used to find graphical concentration (Table 1).
Table 2: The use of Biomate 3 Spectrometer and KMnO4 stock solution to create a
standard curve.
Desired
Concentration of
KMnO4 (mM)
0
0.2
0.4
0.6
0.8
1.0
Volume of 1mM
KMnO4
Volume of H2O
Absorbance at
508nm
0.0
0.2
0.4
0.6
0.8
1.0
1 ml
0.8 ml
0.6 ml
0.4 ml
0.2 ml
0 ml
0
.319
.646
.946
1.255
1.568
Figure 2: Absorbance vs.
wavelength graph of 1.0 mM
of KMnO4 and corresponding
data points calculated by the
Biomate 3 Spectrophotometer.
Figure 3: % Transmittance
vs. wavelength graph of 1.0
mM of KMnO4 and
corresponding data points
calculated by the Biomate 3
Spectrophotometer.
Figure 4: Test 1 of
absorbance vs.
wavelength of an LDH
enzyme solution at
340 nm over 5 minutes
and corresponding
data point calculated
by the Biomate 3
Spectrophotometer.
Figure 5: Test 2 of
absorbance vs.
wavelength of an LDH
enzyme solution at
340 nm over 5 minutes
and corresponding
data point calculated
by the Biomate 3
Spectrophotometer.
.
Figure 6: Test 3 of
absorbance vs.
wavelength of an LDH
enzyme solution at 340
nm over 5 minutes and
corresponding data point
calculated by the
Biomate 3
Spectrophotometer.
.
Calculations:
A. Activity One
I. Regression Concentration
Formula: y = 1.8416x (where y is absorbance and x is concentration)
A: 1.173/1.8416 = 0.637
B: 0.243/1.8416 = 0.132
C: 0.643/1.8416 = 0.349
D: 1.641/1.8416 = 0.891
II. Beer’s Law Concentration
Formula: A = ƐL/A (where Ɛ= 1835 M-1*cm-1 and L= 1 cm)
A: (1.173/1835) x 1000 = 0 .582
B: (0.243/1835) x 1000 = 0.9973
C: (0.643/1835) x 1000 = 0.29428
D: (1.641/1835) x 1000 = 0.78147
B. Activity Two
Formula: C1V1=C2V2

(1mM)(x)=(0mM)(2mL)
o x= 0 mL of 1mM KMnO4  2mL of H2O

(1mM)(x)=(0.2mM)(2mL)
o x= .4 mL of 1mM KMnO4  1.6 mL of H2O

(1mM)(x)=(0.4mM)(2mL)
o x= .8 mL of 1mM KMnO4  1.2 mL of H2O

(1mM)(x)=(0.6mM)(2mL)
o x= 1.2 mL of 1mM KMnO4  0.8 mL of H2O

(1mM)(x)=(0.8mM)(2mL)
o x= 16 mL of 1mM KMnO4  0.4 mL of H2O

(1mM)(x)=(1.0mM)(2mL)
o x= 2 mL of 1mM KMnO4  0 mL of H2O
D. Activity Four
Formula: c = A/l and c= (change in A/((6,220 liters/mole*cm)(1cm)))/ 1,000
mL/1 L)(1x109 nanomoles/ 1 mole) where Ɛ= 6,220 M-1*cm-1

(((.193/6,220)/5)/1000) = 3.103 x10-5 (1x109)= 6.206 nanomoles/mL

(((.119/6,220)/5)/1000) = 1.913 x10-5 (1x109)= 3.826 nanomoles/mL

(((.249/6,220)/5)/1000) = 4.003 x10-5 (1x109)= 8.006 nanomoles/mL
Questions:
7. The graph appears to be a decreasing linear line. This is because as the time increases
the absorbance decreases because the reaction in the cuvette is wearing off/no longer
occurring. Since the plot is linear, if the reaction were to continue past five minutes the
absorbance would continue to decrease.
8. Change in absorbance was divided by 6,220 to give moles/liter. This number was then
divided by 5 for the change in time to give us moles/liter*min. This number was then
divided by 1 liter/1000ml and multiplied by 109 nanometer/mole to give us
nmoles/min*mL. The average of the three trials was 6.013 nmoles/min*mL. All three
trails were relatively close with a .200-.400 range meaning our laboratory techniques
were pretty accurate. To decrease variability we would try to eliminate as much human
error as possible and time to get the enzyme mixed and put into the spectrophotometer.
(See Calculations, D. Activity Four)
9. LDH (Lactate dehydrogenese) is present in almost all animal tissues, especially
muscles and deals with lactate fermentation. It catalyzes the reversible oxidation of lactic
acid to pyruvic acid while using NAD+ as the hydrogen/electron acceptor.
Conclusion and Discussion:
The concentrations found from various methods were within close proximity
(Table 1) and unknown samples concentrations were revealed from their absorbance at
508 nm. Concentration dilutions showed that the more diluted the less absorbance (Table
2). For Figure 2 and 3 it was evident that the absorption was highest around the midpoint
of 480 and 580, proving that substances that absorb light usually don’t absorb all
wavelengths and these graphs also showed the inverse relationship between absorbance
and % transmittance. As absorbance went up, transmittance went down and vice versa.
For Figure 4, 5, and 6 it was evident the decrease in absorbance as time goes by. The
more time the enzyme reacts before examined in the spectrophotometer the lower the
starting absorbance. For enzyme reactions, color absorption is immediate and the decline
is steady and relatively quick.
Overall this lab was to help understand the absorption of biomolecules and how
the spectrophotometer works. This lab was also to understand the correlation between
absorbance and wavelength. It was also to learn how to calculate concentration using
different methods and comparing their accuracy.
Literature Cited
Sparace, Salvatore, and Brandon Moore. "UV/Visible Absorption Spectroscopy." Biology
4341 Laboratory Manual: Biological Chemistry Laboratory Techniques.
Clemson: Clemson U, 2014. 11-24. Print.