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EXPERIMENTAL QUESTION
Can we use absorbance to
determine concentration and the
identity of an unknown
substance?

Spectrophotomet
er
Used to measure and identify biological
molecules suspended in solution.
Emits light and measures the
percentage of light absorbed by the
solution.
Beer-Lambert Law
Absrobance= lc

HYPOTHESIS
Because some of the energy from
the emitted light is absorbed by the
substance, the concentration and
the identity of the substance can
be determined.

PROPOSED EXPERIMENTAL
METHOD
Every substance has an optimal
wavelength at which it absorbs the most
light. This characteristic can be used to
determine the concentration and identity of
a substance.
Beer-Lambert law allows direct
measurement of the concentration from the
OD which makes it the ideal method for this
experiment.

PROPOSED EXPERIMENTAL
METHOD
Using a spectrophotometer, a standard
curve of known concentrations vs. the
absorbance can be generated.
By comparing the absorbance values of
the unknown samples to a identical point
on the graph, concentration can be
determined.

METHODOLOGY
1) Prepare the standard curve of known
concentrations by diluting from the
0.125 mM stock.
a) The extinction coefficient for Fast Green
FCF is the slope of the graph.
b) Concentration of the unknowns can be
found by comparing the OD readings to a
identical point on the graph.

METHODOLOGY
2) Measure the ODs of the three
unknown samples (A, B and C).
3) Make serial dilutions for each
unknown sample that does not have a
reliable reading (0.1-1).
a) To find the concentration of the original
sample, multiply the concentration of the
diluted sample by its dilution factor.

METHODOLOGY
4) Perform a spectrum analysis for
sample D and E from a
wavelength of 220nm to 360nm.
a) By graphing the absorbance
readings versus wavelength, we can
obtain a spectrum of absorbance for
each sample.
b) Locating the highest peaks on the
graph reveal the optimal
wavelength at which the solution
absorbs light.

METHODOLOGY
5) Use the following formula to
determine the concentration of DNA in
Sample D.
a) DNA concentration = 50 ug/mL x # OD
units

6) Use the following formula to

determine the purity ratio.
a) Ratio= OD/OD
b) Ratio of 1.8 or above indicates no protein
contamination.

0.7

Sample A

0.6
0.5
0.4
ABSORBANCE
0.3
0.2
0.1
0
400

450

500

550

600

650

WAVE LENGTH

Figure 1. Sample A graph measures the

compound spectral absorbance from 400nm to
700nm

700

0.16

Sample D

0.14
0.12
0.1
0.08

ABSORBANCE

0.06
0.04
0.02
0
220

230

240

250

260

270

280

290

300

WAVE LENGTH

Figure 2. Sample D graph measures the compound

spectral absorbance from 220nm to 360 nm.

1.2

Sample E

1
0.8
ABORBANCE
0.6

0.4
0.2
0
255

275

295

315

335

355

WAVE LENGTH

Figure. 3 Sample E graph measures the

compound spectral absorbance from 220nm to
360 nm

Standard Curve
0.5
0.45
0.4
0.35
0.3
0.25
O.D 625nm
0.2
0.15
0.1
0.05
0

f(x) = 0.03x + 0.02

R = 0.98

10

12

Concentration of FCF (mM)

Figure 4. The graph above shows the standard curve of

the known concentrations of Fast Green FCF.

Absorbance Dilution Concentratio

Samples Value Used Factor
n

A
B
C

0.531
0.328
0.870

N/A
1:5
1:5

21.762M
67.213M
178.279M

Table 1. The table above shows the absorbance value used to

obtain the concentration. Also, it shows the dilution factor used
during the serial dilutions.

DISCUSSION
Sample D most likely contains DNA, while Sample E is
most likely protein.
DNA absorbs light optimally at a wavelength of
260nm.
Protein absorbs light optimally at a wavelength of
280nm.
The purity of DNA is determined by the ratio
OD/OD.
Purity of DNA is 1.5, which means its contaminated
with protein.

To give ideal results, more cuvettes of DNA