INTRODUCTION.
The perception of color derives from the stimulation of photoreceptor cells in the human eye by
electromagnetic radiation (Hunt et al., 2004). He further mentioned that the color categories and
physical specifications of color associated with objects are through the wavelength of light that is
reflected from them and this reflection is governed by the object’s physical properties such as
light absorption and emission spectra.
(Waldam et al., 2002) defined light absorption as a process by which light is absorbed and
converted into energy. He further mentioned that only light of energy that can cause
transmissions from one energy level to another can be absorbed. He further explained absorbance
as a measure of the capacity of a substance to absorb light at a specific wavelength.
Colorimetry is the technique used to determine the concentration of colored compounds in
solution (Pranav et al., 2011). He also mentioned that the colorimeter is a device used to test the
concentration of a solution by measuring its absorbance at a specific wavelength of light.
Spectrophotometry is a procedure that is frequently utilized in biological laboratories. Probably
the most common application in biology of this technique is in the measurement of the
concentration of a compound in solution (Aryal et al., 2018)
A spectrophotometer is a very powerful tool used in both the biological and chemical sciences
yet operates by simply shining a beam of light, filtered to a specific wavelength (or very narrow Commented [ES1]:
range of wavelengths), through a sample and onto a light meter. Some basic properties of the
sample can be determined by the wavelengths and amount of light absorbed by the sample
(Bradford et al., 2020)
1
Spectrophotometers are designed to transmit light of narrow wavelength ranges. A given
compound will not absorb all wavelengths equally-that’s why things are of different colors.
(Some compounds absorb only wavelengths outside of the visible light spectrum, and that’s why
there are colorless solutions like water). Because different compound absorbs light at different
wavelengths, a spectrophotometer can be used to distinguish compounds by analyzing the pattern
of wavelengths absorbed by a given sample (Philips et al., 2015).
Additionally, the amount of light absorbed is directly proportional to the concentration of
absorbing compounds in that sample, so a spectrophotometer can also be used to determine
concentrations of compounds in solution. Finally, because particles in suspension will scatter
light, thus preventing it from reaching the light detector, spectrophotometers may also be used to
estimate the number of cells in suspension (Rockson et al., 2003)
The Beer-Lambert law relates the attenuation of light to the material’s properties through which
the light travels. It is applied to chemical analysis measurements. The Beer-Lambert law, known
by various names such as the Lambert-Beer law, Beer-Lambert–Bouguer law or the Beer’s law
state that for a given material, the sample path length and concentration of the sample are
directly proportional to the absorbance of the light. A= ℇ x L x C, where A is the measure of
Absorbance, ℇ is the absorption coefficient, L is the path length and C is concentration.
AIM: The experiment seeks to determine the wavelength of maximum absorption of different
indicators and shows the relationship between the absorbance and concentration as defined by
the Beer-Lambert’s law.
2
MATERIALS.
UV-Visible light spectrophotometer, 100 mg/l of methylene blue, 100 mg/l of methyl orange,
100 mg/l of unknown mixture of two indicators.
METHODOLOGY.
a. Method for wavelength of maximum absorption
In the first test, exactly 2 ml of 100 mg/L of both methyl orange and methylene blue indicators
were diluted to 20 ml of 10mg/L. This was prepared by pipetting 2 ml of each into two test tubes
and 18 ml of distilled water was added to each of the different test tubes containing the indicators
to get a final volume of 20 ml Also, 2 ml of 100mg/L of the unknown mixture of the two
indicators was diluted to 20 ml of 10 mg/L. The spectrophotometer was zeroed using distilled
water as blank and the absorbance of each sample was determined at wavelengths 400 nm, 420
nm, 440 nm, 460 nm, 470 nm, 480 nm, 490 nm, 520 nm, 540 nm, 565 nm, 580 nm, 620 nm, 660
nm and 700 nm for each of the three samples. Each time the wavelength was varied, the
spectrophotometer was zeroed with the distilled water before the absorbance of the samples was
tested for and the values from the test tabulated.
3
b. Determination and the confirmation of the Beer-Lambert’s law.
For the second part of the test, six clean separate test tubes were setup each for both
methylene blue and methyl orange and labelled 1, 2, 3, 4, 5, 6. Using 10 mg/L of methyl
orange indicator, 5 ml of distilled water was pipetted into test tube 1 without the addition
of the indicator, 1 ml of 4 methyl orange and 4 ml of distilled water was pipetted into test
tube 2, a volume of 2 ml of the methyl orange indicator and 3 ml of distilled water was
pipetted into test tube 3, a volume of 3 ml of methyl orange and 2 ml of distilled water
was pipetted into test tube 4, a volume of 4 ml of the methyl orange indicator and 1 ml of
distilled water was pipetted into test tube 5 and lastly , a volume of 5 ml of the methyl
orange indicator was pipetted without the addition of water to test tube 6. This same
method was repeated for methylene blue indicator. Afterwards, the concentrations of the
various solutions of both the methyl orange and methylene blue was calculated. The
spectrophotometer was again zeroed with distilled water as the blank and the absorbance
of each sample tube at the wavelength of maximum absorbance determined in the
previous test was measured for the methyl orange and methylene blue indicators, and the
absorbance of the sample in each test tube was determined and recorded.
4
RESULTS.
A GRAPH OF ABSORBANCE AGAINST WAVE a. The curve in
LENGHT orange color
represents methyl
2.5 orange indicator
ABSORBANCE (A).
2
b. The curve in
1.5
ash color represents
1 unknown mixture
0.5
0
0 100 200 300 400 500 600 700 800 c. The curve in
WAVELENGTH (nm) the blue color
represent methylene
Series1 Series2 Series3
blue indicator
Figure 1. absorbance of the two indicators and the unknown matures at different wavelength.
The graph represents the absorbance of methylene blue, methyl orange and the unknown
mixtures at different wavelengths, with the peaks showing the wavelength at which the B
absorbance is highest for each indicator and the unknown mixture.
A graph of absorbance against concentration
2
1.8
1.6
jABSORBANCE (A)
1.4
1.2
1
0.8
0.6
0.4
0.2
0 CONCENTRATION mg/L
0 2 4 6 8 10 12
Figure 2. Absorbance of methylene blue indicator at different concentration at its maximum
wavelength of absorption.
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A graph of absorbance against concentration
1
0.9
0.8
ABSORBANCE(A)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10 12
CONCENTRATION(mg/L)
Figure 3. Absorbance of methyl orange indicator at different concentration at its maximum
wavelength of absorption.
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DISCUSSION.
Waldam (2002) explained that absorption of light takes place when matter captures
electromagnetic radiation converting the energy of photons to internal energy and this occurs at
specific wavelengths. At each wavelength there is an amount of delocalized free electrons that
cause or aids in absorbance at that particular wavelength (Skrabel et al., 2005). From Figure 1 at
460nm methyl orange gave the highest peak representing maximum absorption at that
wavelength and at 660nm methylene blue gave the highest peak representing maximum
absorption at that wavelength and this is due to the presence of large amount of delocalized free
electrons at that wavelength as explained by Skrabel (2005).
These wavelengths are considered the maximum wavelengths for each indicator as it has the
highest absorbance which Constable (2006) stated that the maximum wavelength is the
wavelength at which the absorbance is the highest. Because different compounds absorb light at
different wavelengths, a spectrophotometer can be used to distinguish compounds by analyzing
the pattern of wavelengths of absorption by a given sample (Chalmers et al., 2003).
From Figure 1, the unknown sample showed two peaks which represents maximum absorbance
at that wavelength of 470nm and 660nm. This goes to show that the unknown might contain two
different indicators, methyl orange and methylene blue, mixed together since the maximum
absorbance occurred at the wavelengths which corresponds to that of the methyl orange and the
methylene blue from Figure 1. Mixing the two indicators does not affect the absorption spectrum
but rather gave two peaks to show the maximum absorbance at the appropriate maximum
wavelength for each indicator in the mixture. The wavelengths, 460nm for methyl orange and
660nm for methylene blue, at which the absorbance was highest were used for measuring the
absorbance at different concentrations of the indicator because they were the wavelengths at
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which the solutions were most sensitive to concentration changes (Hunt, 2004). Stiles (2001)
mentions that the Beer-Lambert’s Law shows a relationship between the concentration and
absorbance of the solution and this enables the concentration of a solution to be calculated or
measured using the absorbance and vice versa. From Figure 2 and 3 the absorbance of methylene
blue and methyl orange was calculated or measured as the concentration of each indicator was
M,being varied.
According to Prinav (2011), absorbance increases as concentration increases and this can be
observed from Figure 2 and Figure 3. Bohren (1994) mentioned that the Beer-Lambert’s Law
states that there is a linear relationship between concentration and absorbance. A graphical
representation of absorbance versus against concentration produced a linear graph as seen in
Figure 2 and Figure 3 confirming the linear relationship between absorbance and concentration
defined by the Beer-Lambert’s Law.
A plot of absorbance against the concentration of a standard is called a calibration curve and this
2.can be used to calculate the concentration of a sample with unknown concentration by
extrapolating from the absorption axis to the concentration axis to get the concentration of the
unknown sample (Thomas, 2007). From Figure 2, the concentration of methylene blue in the
unknown mixture with absorbance 1.850 at its maximum wavelength of 660 nm is 10.120 mg/L
from the extrapolation from the graph and from Figure 3 the concentration of methyl orange in
the unknown mixture with absorbance 0.914 at its maximum wavelength of 460 nm is
10.027mg/L from the extrapolation from the graph. This value can be considered accurate due to
the linear relationship between the concentration and absorption defined by the Beer-Lambert’s
Law and how the concentration of a solution can be calculated by using its absorbance at
maximum wavelength (Bohren et al.,2007).
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CONCLUSION
Some amount of light incident on a substance is absorbed. Absorption of light intensity occurs at
different wavelengths. Maximum absorption occurs at maximum wavelength. The maximum
wavelength of absorbance of methyl orange is 460nm and that of methylene blue is 660nm. At
maximum wavelength absorbance is directly proportional to concentration as defined by the
Beer-Lambert’s Law. Beer-Lambert’s Law gives a linear relationship between absorbance and
concentration and this enables the concentration of a sample to be calculated or measured if the
absorbance is known
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REFERENCE.
Hunt, w. G. (2004). The Reproduction of Color. In Series in Imaging Science and Technology
(pp. 11 - 12).
Waldam, G. (2002). The Physics of Light, Vision and Color. In Introduction to Light (p. 193).
Mineola: Dover Publications.
Pranav, K. (2011). In Fundamentals of Biophysics and Molecular Biology. (p. 33). New Delhi:
Pathfinder Publishers.
Aryal, S. (2018). Spectrophotometry -Principle, Instrumental, Applications (p. 197). Retrieved
from Microbe note.
Bradford, M. (2003). A rapid and sensitive method for the quantification of microgram quantity
of protein utilizing the principle of protein dye-binding. Analytical biochemistry, pg. 72(1-2):
248-254.
Philips, K. (2015). UV spectrophotometry identifies compounds in pharmaceuticals.
Rockson, S. M. (2003) Qualitative analysis of mass spectra. American Chemistry Society.
Bohren, C. (2007). An introduction with 400 problems. In Fundamentals of Atmospheric
Radiation (p. 214). New York: Wiley - VCH. Bouguer, P. (1972).
Optics essay on the attenuation of light. (16) Paris, France: Claude Jombert. Chalmers, J.
(2003). Handbook of Vibrational Spectroscopy. New York:
Wiley. Constable, E. (2006). Chemistry - an introduction to organic, inorganic and physical
chemistry. (349 - 352) Minnesota: Hill Press.
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Skrabel, M. (2005). Spectroscopy - An interdisciplinary integral description of spectroscopy
from UV to NMR. (p. 146 - 149) Boston: Academic Press.
Stiles, W. S. (2009). Color Science: Concepts and Methods, Qualitative Data and Formulae.
New York: Applied Publishers.
Thomas, M. (2007). The Electric Field Standing Wave Effect in Infrared Trans flection
Spectroscopy (p. 2916 - 2923).
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APPENDIX.
TABLE 1.0
A TABLE OF A WAVELENGTH AND THE DIFFERENT ABSORBANCE OF THE INDICATORS.
The table below shows the absorbances of methyl orange, methylene blue and the mixture of the unknown indicator
at different wavelength.
WAVELENGTHS OF ABSORBANCE OF ABSORBANCE OF ABSORBANCE OF
THE INDICATORS METHYLENE BLUE METHYL ORANGE UNKNOWN MIXTURE
400 0.013 0.565 0.162
420 0.006 0.762 0.200
440 0.026 0.743 0.310
460 0.050 0.870 0.381
470 0.043 0.866 0.331
480 0.064 0.810 0.353
490 0.088 0.700 0.230
520 0.107 0.279 0.090
540 0.161 0.168 0.096
565 0.379 0.090 0.147
580 0.579 0.080 0.219
620 1.272 0.071 0.725
660 2.027 0.064 0.725
700 0.217 0.060 0.021
TABLE 2.0
A TABLE OF THE ABSORBANCES AND THE CONCENTRATIOS OF THE INDICATORS.
This tables gives illustration on how the Beer-Lamber’s law can be determined experimentally. This table gives the
various absorbance read by the spectrophotometer using the wavelengths that showed the highest absorbance in the
first section of the experiment.
Indicator MTHYLENE BLUE
Test tube number 1 2 3 4 5 6
Concentration mg/L 0 2 4 6 8 10
Absorbance 0.000 0.262 0.640 1.078 1.452 1.850
Traced concentration from the graph 0.352 1.735 3.731 6.044 8.018 10.120
mg/L
Indicator METHYL ORANGE
Test tube 1 2 3 4 5 6
Concentration mg/L 0.000 2 4 6 8 10
Absorbance 0.000 0.167 0.363 0.523 0.740 0.914
Traced concentration from the 0.000 2 4 6 8 10
graph mg/L
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