Experiment 1: Micropipettors, Spectrophotometry, and the Beer-Lambert Law

Mary McAuley and Katarina Kowatsch

Room 428

Tuesday AM

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 1.200 2.

1.000
f(x) = 8.18 x − 0

0.800 f(x) = 0.39 ln(x) + 1.69

Absorbance

0.600

0.400

0.200

0.000
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14
Concentration of 4-nitroaniline in TRIS buffer (mM)

Figure 1. Calibration curve of 4-nitroaniline. 2.50 mM 4-nitroaniline in 50.0 mM TRIS (pH

7.9), 5.0 mM CaCl2. Absorbance measured at 410 nm using Genesys 10S VIS
spectrophotometer.

3.

Table 1. Absorbance and concentration of three 4-nitroaniline dilutions of unknown concentration.

Original
Concentration in
Dilution name Absorbance Dilution factor
concentration of
cuvette (mM)
unknown (mM)
A 0.371 0.0457 50.0 2.29
B 0.556 0.0684 33.3 2.28
C 0.931 0.114 20.0 2.28
Absorbance measured at 410 nm. 4-nitroaniline of unknown concentration #249 in 50.0 mM
TRIS (pH 7.9), 5.0 mM CaCl2 (TRIS buffer).

4. Average original concentration of unknown sample of 4-nitroaniline sample calculated to be

2.28 mM.

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5.
1.800
1.600
1.400
1.200
Absorbance

1.000
0.800
0.600
0.400
0.200
0.000
200 250 300 350 400 450
Wavelength (nm)

Tyrosine Tryptophan Alanine

0.800

0.700
Figure 2. Absorption spectra of tyrosine, tryptophan, and alanine. 1.25 mM tyrosine, 0.100 mM
0.600 tryptophan, 5.00 mM alanine all diluted in 50.0 mM TRIS (pH 7.9), 5.0 mM CaCl2 (TRIS
0.500 buffer). Absorption measured from 245 nm to 400 nm.
Absorbance

0.400

0.300

0.200

0.100

0.000
200 250 300 350 400 450
Wavelength (nm)

DNA BSA

Figure 3. Absorption spectra of DNA and Bovine Serum Albumin (BSA). 1.00 M DNA
solution and 1.00 mg/mL BSA all diluted in 50.0 mM TRIS (pH 7.9), 5.0 mM CaCl2 (TRIS
buffer). Absorption measured from 245 nm to 400 nm.

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 4.000

3.500

3.000

2.500
Absorbance

2.000

1.500

1.000

0.500

0.000
200 250 300 350 400 450
Wavelength (nm)

NADH NAD+

Figure 4. Absorption spectra of NADH and NAD+. 0.250 mM NADH and 0.250 mM NAD+ all
diluted in 50.0 mM TRIS (pH 7.9), 5.0 mM CaCl2 (TRIS buffer). Absorption measured from
245 nm to 400 nm.

Table 2. Molar absorption coefficients of each biomolecules at their wavelength maxima.

Biomolecule sample Molar absorption coefficient (M-1cm-1)
Tyrosine 1.31103
Tryptophan 8.81103
Alanine 3.20
DNA 2.83105
BSA 4.45104
NADH 5.28103
NAD+ 1.48102
The Beer-Lambert Law was used to calculate the molar absorption coefficient using each
biomolecule’s wavelength maxima. All biomolecule samples were diluted in 50.0 mM TRIS (pH
7.9), 5.0 mM CaCl2 (TRIS buffer). A molecular weight of 66 430.3 gmol-1 was used for
calculating the concentration of BSA.2

7. Based upon the data collected, Tryptophan is the amino acid that contributes most to the
overall absorbance of the BSA protein. When comparing the three amino acids absorption
spectra to the BSA absorption spectra, Tryptophan’s is the most closely related in absorbance
values. Tryptophan also has the closest molar absorption coefficient to BSA out of the three
amino acids.

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 References

(1) Nichols, E.R. (2019) Biochemistry I: Biomolecules and an Introduction to Metabolic

Energy Lab Manual.
(2) Sigma Aldrich. https://www.sigmaaldrich.com/technical-
documents/articles/biology/albumin-from-bovine-serum.html (accessed September 23,
2019)