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Determining the absorbance spectrum of different

liquid samples and verifying Beer-Lambert law using


liquid samples with varying concentration
Ellendale Cruz1, Nikki Rose Esguerra1, John Paul Fuentes2, Ma. Reneeza Galimba1,*, John
Noel Maape3, Marty Lorgino Pulutan1
1
Institute of Mathematical Sciences and Physics, CAS, UPLB
2
Department of Electrical Engineering, CEAT, UPLB
3
Institute of Chemistry, CAS, UPLB
*rengalimba@gmail.com

Abstract
The exercise determined the absorbance spectrum of liquid samples of
different colors, identified the substances that contributed to the absorbance
peaks observed in the absorbance spectrum and lastly, it verified the BeerLambert law using liquid samples with varied concentration. The exercise
used a RedTide and a LabQuest in measuring the absorbance of the different
samples. In using different colored samples, it was observed that the
absorbance peak occurred at the wavelength complementary to the color of
the sample. In using samples with varying concentration, it should have
observed that as the concentration increases, the absorbance of the liquid
should have also increased according to the Beer-Lambert law but this was
not observed in the experiment but there was a trend in wavelength of the
gathered absorbance peak. This was because the light source used was
continuous and there was no monochromator used.

1.Introduction
In the emission spectra, bright lights can be seen in a dark background on the other hand in absorption
spectra, dark bands can be found on bright background. The dark bands are the wavelengths absorbed and it
corresponds to the bright lights seen in the emission spectra but the same could not be said for the emission
spectrum.
The absorbance of a sample and its concentration can be related through the Beer-Lambert law. The BeerLambert law can be written as

A=bc

where A is absorbance, is the molar absorptivity of the sample in M -1 cm-1, b is the cell path length of the
sample in cm, and c is the molar concentration of the sample.
In this experiment the absorbance spectrum of certain liquid is to be determined and the factors that affects
it. Also the Beer- Lambert equation is be verified through varying concentration of different samples.

2. Methodology
The group arranged the setup as illustrated in the figure below. The distances between the light source,
sample, and the RedTide spectrophotometer should be maintained throughout the experiment.

Light Source

Blank/Sample

RedTide

Lab Quest

Figure 1. Setup for determining the absorbance spectrum of a liquid sample

The members of the group then calibrated the spectrophotometer using a blank cuvette. By calibrating the
RedTide using the vial with distilled water, the group is directly subtracting the absorbance of the vial and the
water from the data you will obtain. They then replaced the blank with the sample. They determined the
absorbance spectrum and recorded the wavelength where they observed peak/s. They repeated this step for all
the remaining samples with different colors.

In the next part of the experiment, the group used the same setup as above, and repeated the calibration of the
RedTide. They replaced the blank with the sample and then determined the absorbance spectrum. They recorded
the absorbance value of the peak. They repeated this step for all the remaining samples with different colors.

3. Results and Discussion


When an incident light is directed to a sample, light energy may be absorbed such that the transmitted light
has lower irradiance. Transmittance is the fraction of irradiance of the incident light that passes through the
sample. The negative logarithm of transmittance is the absorbance, a unitless value that is the basis of much of
the spectrophotometric methods.

( PP )

A=log ( T )=log

(1)

Absorbance of a substance in a certain wavelength is measured by passing light of that wavelength (whose
intensity is known or measured) through the sample and measuring the irradiance of the transmitted light.
Absorbance is a function of the amount of absorbing species and the amount of absorption of the species in that
wavelength;hence, the Beer-Lamberts or simply Beers Law is used.

A=bc

(2)

where A is absorbance, is the molar absorptivity of the sample in M -1 cm-1, b is the cell path length of the
sample in cm, and c is the molar concentration of the sample. Spectrometers are calibrated first with the solvent
used so that the absorbance measured are only because of the sample.
The absorptivity of a sample in different wavelengths is governed by the chemical nature of the sample. Parts
of the chemical structure of a sample that renders absorption of the sample at specific wavelengths are called
chromophores. Absorption of a sample is not limited to certain wavelengths but is rather continuous, but there
are certain wavelengths where the sample has maximum absorption. The absorption in the visible region causes
electronic transitions in the molecule, and certain wavelengths that correspond to the energy of these transitions
are most absorbed. For colored compounds, their maximum absorption usually occurs at wavelengths
complementary to the observed color of the compound. For example, the orange dye in the experiment had peak
absorption at 517 nm. Peak absorptions are not also restricted to just one wavelength, as shown on the table
below. The samples were not identified and no comparison was made with literature values of molar
absorptivity.
Table 1. Wavelengths where peak absorbance were observed for different samples.

sample

max
552
649
517
392
477
575
658

blue dye
orange dye

green dye

Absorbance
2.595
2.243
3.229
2.147
2.252
2.071
1.603

2.5
2
1.5
Absorbance
Blue Dye

1
0.5 Orange Dye
0
300

400

Green Dye
500

600

Wavelength, nm

700

Figure 2. Peak absorbance of different dye samples.

Concentration-wise, it is seen on Beers Law equation that on a certain wavelength, as the concentration of
the sample increases, the absorbance on that wavelength also increases (given that the sample holders are of the
same diameter). Experimentally, this was not observed because no monochromator was used and the light
source was continuous. However, it is observed that the peak absorptions occurred with a trending shift in
wavelength. If the peak absorptions of the highest concentration were taken as the fixed wavelengths, the shift in
peak absorption with respect to decreasing concentration would mean decreasing absorbance at the fixed
wavelengths, which still confirms the Beers law equation. The molar concentrations of the sample were not
however given, and also the recording of absorbance was not done in a fixed wavelength so that the molar
absorptivity of the sample blue dye could not be calculated.
Table2. Peak absorptions of the blue dye sample at increasing concentration.

[blue dye]
[1]
[2]
[3]
[4]
[5]

max
607
647
661
567
515
681
521
682
507
689

4
3
Absorbance

2
1
0
400 500 600 700
Wavelength, nm

[1]
[2]
[3]
[4]
[5]

A
2.939
2.611
2.452
2.694
2.325
1.884
2.386
2.333
2.187
2.112

Figure 2. Graph of the peak absorbance of the blue dye sample at different concentrations.

4. Conclusion and Recommendation


With the results, observations and thorough analysis, we can therefore conclude that varying the
concentration of a substance would vary the absorbance of the said absorbing specie that is increasing
concentration yields greater absorptivity. This therefore verifies Beer-Lambert Law written as A=bc which
states that an absorbance of a substance is directly proportional to its molar absorptivity coefficient, path length
and the concentration of the compound in the subjected sample. It is recommended to do the test in a totally dark
and encloses space so as to diminish the stray light that could hit the sensor thus greater accuracy in
measurement.

References
Harris, D.C. (2007). Quantitative Chemical Analysis (7thed.). New York: W.H. Freeman and Company
2. Beer-Lambert Law. Retrieved March 10, 2015 from http://life.nthu.edu.tw/~labcjw/BioPhyChem/
Spectroscopy/beerslaw.htm
1.