Optical Method of Analysis
Optical Method of Analysis
Optical Method of Analysis
EXPERIMENT NO. 9
OPTICAL METHOD OF ANALYSIS
USE OF BEER’S LAW ON A KMnO4 SOLUTION
Abstract
Introduction
important and versatile tool. Indeed, much of our knowledge of chemical substances comes
As the color of the solution deepens its concentration also increases. This is an
or transmits certain wavelengths of radiant energy but not other wavelengths. The light energy
absorbed or transmitted must match exactly the energy required to cause an electronic
transition (a movement of an electron from one quantum level to another) in the substance
under consideration. Only certain wavelength photons satisfy this energy condition. Thus, the
and important methods of quantitative analysis. They are applicable to many industrial and
clinical problems involving the quantitative determination of compounds that are colored or
An application of this is by the use of Beer-Lambert’s Law. The Beer-Lambert law (also
known as Beer's law) (as it applies to solutions of light-absorbing substances) states that the
absorbance is directly proportional to the path length, l of the sample and its concentration, c:
A = ecl
of the solute, c is the molar concentration (in moles.dm-3), and l, the path length, is measured in
centimeters.
numerous substances, provided that (i) the molecular extinction coefficient e for the subtance is
known at the wavelength at which the measurements are carried out, and (ii), that the path
length of the solution is known accurately. Commonly, cuvettes with a path length of 1 cm are
c = A/e
The Beer-Lambert Law (A = εlc) implies that when concentration is equal to zero (c = 0),
absorbance must also be zero (A = 0). In other words, the calibration line must pass through the
origin.
Law at inappropriate concentrations. The Beer-Lambert Law is strictly applicable only for dilute
solutions. It becomes less and less accurate as the concentration of the solution increases.
Once you have the calibration curve set up, you can measure the absorbance of any
unknown solution at the same wavelength and read off its concentration from the graph or
forming purple crystals with a metallic sheen, soluble in water (intense purple solution),
acetone, and methanol, but decomposed by ethanol; r.d. 2.70; decomposition begins slightly
above 100°C and is complete at 240°C. The compound is prepared by fusing manganese(IV)
oxide with potassium hydroxide to form the manganate and electrolysing the manganate
solution using iron electrodes at about 60°C. An alternative route employs production of sodium
manganate by a similar fusion process, oxidation with chlorine and sulphuric acid, then
Beer-Lambert Law, more commonly known as Beer's Law, states that the optical
absorbance of a chromophore in a transparent solvent varies linearly with both the sample cell
pathlength and the chromophore concentration. Beer's Law is the simple solution to the more
general description of Maxwell's far-field equations describing the interaction of light with
matter. In practice, Beer's Law is accurate enough for a range of chromophores, solvents and
at wavelength λ through a plane parallel slab of material that is normal to the beam. For liquids,
the sample is held in an optically flat, transparent container called a cuvette. Absorbance (Aλ) is
calculated from the ratio of light energy passing through the sample (I0) to the energy that is
Aλ = -log (I/I0)
Aλ = ελbc
In an absorbance experiment, light is attenuated not only by the chromophore, but also
by reflections from the interface between air and the sample, the sample and the cuvette, and
absorbance by the solvent. These factors can be quantified separately, but are often removed
by defining I0 as the light passing through a sample "blank" or "baseline" or reference sample
(for example, a cuvette filled with solvent but zero concentration of the chromophore is used as
the blank).
Many factors can affect the validity of Beer's Law. It is usual to check for the linearity of
Beer's Law for a chromophore by measuring the absorbance of a series of standards. This
"calibration" can also remove errors in the experiment, the equipment, and the batch of
concentration of an analyte in a solution. The experimental approach exploits Beer's Law, which
predicts a linear relationship between the absorbance of the solution and the concentration of
in which the analyte concentration is accurately known. The absorbances of the standard
solutions are measured and used to prepare a calibration curve, which is a graph showing how
the experimental observable (the absorbance in this case) varies with the concentration. For this
experiment, the points on the calibration curve should yield a straight line (Beer's Law). The
slope and intercept of that line provide a relationship between absorbance and concentration:
A = slope c + intercept
The unknown solution is then analyzed. The absorbance of the unknown solution, Au, is
then used with the slope and intercept from the calibration curve to calculate the concentration
Au - intercept
cu =
slope
Because calibration curves are used in reading off the unknown concentrations, their
possible and measure their absorbances carefully. Each standard solution should be prepared in
identically the same fashion, the only difference between them being their concentrations. 8
Methodology
I. Preparation of Spectrophotometer
The spectrophotometer was turned on for 20 minutes before use. It was calibrated
by the use of water and adjusting its wavelength by 460 and its absorbance by 0.
II. Preparation of Solutions with Different Concentration
The 250mL of the assigned concentration (1 x 10-4) was prepared by adding the
computed volume of the distilled water with the standardized KMnO4 solution prepared
small amount of the prepared aliquot solution was transferred. 30mL of the same
solution was diluted with 10mL distilled water and was placed on flask 2. On the third
flask, 10mL of the solution was mixed with 10mL distilled water. 30mL of distilled water
was added to a 10mL solution and was placed on flask 4. And fifth flask was given to the
The absorbance of the five solutions was read with the use of a
spectrophotometer.
After calibrating the spectrophotometer, a cuvette was washed then filled with
the first solution making sure that it reached the half of the circle marker.
The line in the cuvette was placed aligned on the line pointer of the spectrophotometer.
The absorbance of the solution was recorded. The same step was followed with solution
2, 3, 4 and the unknown solution, calibrating the spectrophotometer with water before
A graph of the ratio between the absorbance and the concentration of the
solution was drawn. Using the recorded absorbance of the unknown solution and the
The concentration of every solution was determined by the volume used and the known
concentration of the aliquot solution (1 x 10-4) and the total volume of the desired solution
The concentration of the unknown was identified using the measured absorbance and
the curve generated from the Beer’s Law equation. A graph of the absorbance versus the
concentration of the solution was plotted. Solution 1 with a concentration of 1.0 X 10-4 has an
absorbance of 0.277. In addition, Solution 2 with 7.5 X 10-5 molar concentration reads 0.193
absorbance. Moreover, Solution 3 with 5.0 X 10-5 concentration was found out to have an
absorbance of 0.115. And finally, solution 4 with 2.5 X 10-5 molar concentration has an
absorbance of 0.027.
Using the gathered data and its measured absorbance, the concentration of the
unknown solution was determined by plotting it on the graph. The concentration was known to
have a concentration of 7.66 X 10-5 and an absorbance of 0.198 as seen on Figure 1.0.
Figure 1.0 Graph shows the ratio between the absorbance and the molar
concentration of the four different solution and the unknown.
0.3
0.277
y = 0.0828x - 0.054
0.25 R² = 0.9995
0.198
0.2 0.193
ABSORBANCE
ABSORBANCE VS.
0.15 CONCENTRATION
0.115 UNKNOWN
0.1 SOLUTION
0.05
0.027
0
7.66
2.5 5 7.5 10
CONCENTRATION (10-5M)
Conclusion and Recommendations
When light passes through a colored solution, some of it is absorbed and some are
transmitted. These effects depend upon the transparency of the solution. More light is absorbed
and less is transmitted in opaque solutions while less light is absorbed and more is transmitted
in transparent ones.
Having the gathered data and the computed results, it can be said that the higher the
concentration of the solution, the higher its absorption. This is due to the number of particles in
the solution. If there are many particles present, more light will be absorbed and less will pass
through.
Therefore, the absorbance and wavelength reading should be adjusted to the proper value using
Bibliography
http://www.answers.com/topic/spectrophotometry .
http://employees.oneonta.edu/kotzjc/LAB/Spec_intro.pdf
3. (n.a.). South african structural biology initiative: The beer-lambert law. Retrieved March
http://sbio.uct.ac.za/Sbio/postgrad/modules/GRD/spectrophotometry/beer1.php
http://employees.oneonta.edu/kotzjc/LAB/Spec_intro.pdf
5. (n.a.). Pottasium permanganate. Retrieved March 14, 2011 from
http://www.answers.com/topic/potassium-permanganate
http://www.oceanoptics.com/technical/beerslaw.asp
http://www.chm.davidson.edu/vce/spectrophotometry/UnknownSolution.html
http://employees.oneonta.edu/kotzjc/LAB/Spec_intro.pdf
Appendix
Table 1.0 The computed molarity and measured absorbance of different solutions.
FORMULA: M1 V1 = M2 V2