Reporter: John Kevin G.

San Jose Instructor: Prof Virgilio Agbayani

Section: 2ChEC February 15, 2011
Group No.: 5

EXPERIMENT NO. 9
OPTICAL METHOD OF ANALYSIS
USE OF BEER’S LAW ON A KMnO4 SOLUTION

Abstract

Spectrophotometry is a method of chemical analysis based on the

absorption or attenuation by matter of electromagnetic radiation of a
1
specified wavelength or frequency. Four solutions with different
concentrations were prepared. Absorbance of the four known concentration
was measured and the gathered data was plotted in a graph in the ratio of
absorbance with concentration. As measured from the spectrophotometer, the
absorbance of the unknown solution is 0.198. Through this numbers and the
-5
plotted graph, the solution was found out to have a 7.66 X 10 molar
concentration.

Introduction

A study of the interaction of light or other electromagnetic radiation with matter is an

important and versatile tool. Indeed, much of our knowledge of chemical substances comes

from their specific absorption or emission of light.

As the color of the solution deepens its concentration also increases. This is an

underlying principle of spectrophotometry. The intensity of color is a measure of the amount of

a material in solution. A second principle of spectrophotometry is that every substance absorbs

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

absorption or transmission of specific wavelengths is characteristic for a substance, and a

spectral analysis serves as a “fingerprint” of the compound.

 In recent years, spectrophotometric methods have become the most frequently used

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

that react to form a colored product. 2

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

Where e is the MOLAR EXTINCTION COEFFICIENT (with dimensions of dm3.mol-1cm-1)

of the solute, c is the molar concentration (in moles.dm-3), and l, the path length, is measured in

centimeters.

The Beer-Lambert law is readily applicable to the determination of the concentration of

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

used, then, the molar concentration c is simply: 3

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.

A major source of error in spectrophotometric analysis is applying the Beer-Lambert

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

calculate from the slope. 4

In this experiment, absorbance of KMnO4 will be measured. KMnO4 is a compound

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

treatment with potassium chloride to crystallize the required product.

Potassium manganate(VII) is widely used as an oxidizing agent and as a disinfectant in a

variety of applications, and as an analytical reagent. 5

Review of Related Literature

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

concentrations, and is a widely used relationship in quantitative spectroscopy.

Absorbance is measured in a spectrophotometer by passing a collimated beam of light

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

incident on the sample (I):

Aλ = -log (I/I0)

Beer's Law follows:

Aλ = ελbc

ελ = molar absorptivity or extinction coefficient of the chromophore at wavelength

λ (the optical density of a 1-cm thick sample of a 1 M solution). ελ is a property

of the material and the solvent.

b = sample pathlength in centimeters

c = concentration of the compound in the sample, in molarity (mol L-1)

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

reagents (such as cuvettes of unknown pathlength). 6

One of the most common applications of spectrophotometry is to determine the

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

the analyte (assuming all other experimental parameters do not vary).

 In practice, a series of standard solutions are prepared. A standard solution is a solution

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

of the unknown solution, cu. 7

Au - intercept
cu =
slope

Calibration curve or calibration plot is a plot of absorbance versus concentration for a

series of standard solutions whose concentrations are accurately known.

Because calibration curves are used in reading off the unknown concentrations, their

accuracy is of absolute importance. Therefore, make the standard solutions as accurately as

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

from experiment no. 8.

Four flasks were prepared and labelled as solution 1, 2, 3 and 4. In flask 1, a

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

instructor for the unknown solution.

III. Determination of Absorbance

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

reading the next solution.

IV. Determination of the Unknown Concentration

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

graph, the concentration of the unknown was traced.

Discussion of Data and/or Result

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

(Refer to appendix for computation).

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.

Percentage errors can be acquired through the calibration of the spectrophotometer.

Therefore, the absorbance and wavelength reading should be adjusted to the proper value using

a blank solution. In addition, errors can be obtained by the incorrect preparation of

concentration of the solution to be measured.

Bibliography

1. (n.a.). Spectrophotometry. Retrieved March 14, 2011 from

http://www.answers.com/topic/spectrophotometry .

2. (n.a.). Spectrophotometry: Absorption measurements & their application to quantitative

analysis. Retrieved March 14, 2011 from

http://employees.oneonta.edu/kotzjc/LAB/Spec_intro.pdf

3. (n.a.). South african structural biology initiative: The beer-lambert law. Retrieved March

14, 2011 from

http://sbio.uct.ac.za/Sbio/postgrad/modules/GRD/spectrophotometry/beer1.php

4. (n.a.). Plotting calibration graph. Retrieved Marach 15, 2011 from

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

6. (n.a.). Beer-lambert law. Retrieved March 15, 2011 from

http://www.oceanoptics.com/technical/beerslaw.asp

7. (n.a.). Determination of analyte concentration. Retrieved March 15, 2011 from

http://www.chm.davidson.edu/vce/spectrophotometry/UnknownSolution.html

8. (n.a.). Spectrophotometric analysis. Retrieved March 15, 2011 from

http://employees.oneonta.edu/kotzjc/LAB/Spec_intro.pdf

Appendix

Table 1.0 The computed molarity and measured absorbance of different solutions.

SOLUTION MOLARITY ABSORBANCE

SOLUTION NO. 1 1.0 X 10-4 0.277

SOLUTION NO. 2 7.5 X 10-5 0.193
SOLUTION NO. 3 5.0 X 10-5 0.115
SOLUTION NO. 4 2.5 X 10-5 0.027
UNKNOWN 7.66 X 10-5 0.198

FORMULA: M1  V1 = M2  V2

SOLUTION NO.1: 1 X 10-4

SOLUTION NO.2: 1 X 10-4  30 = X2  40
X2 = 7.5 X 10-5
SOLUTION NO.3: 1 X 10-4  10 = X3  20
X3 = 5.5 X 10-5
SOLUTION NO.4 : 1 X 10-4  10 = X4  40
X4 = 2.5 X 10-5