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Principles of Colorimetric Measurements

Principles of Colorimetric Measurements

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Published by: Dipmalya Basak on Feb 18, 2010
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SPECTROPHOTOMETRY
Absorption Measurements& theirApplication to Quantitative Analysis
A
study of the interaction of light (or other electromagnetic radiation) withmatter is an important and versatile tool for the chemist. Indeed, much of our knowledge of chemical substances comes from their specific absorption oremission of light. In this experiment, we are interested in analytical proceduresbased on the amount of light absorbed (or transmitted) as it passes througha sample.Suppose you look at two solutions of the same substance, one a deepercolor than the other. Your common sense tells you that the darker coloredone is the more concentrated. In other words, as the color of the solutiondeepens,you infer that its concentration also increases. This is an underlyingprinciple of spectrophotometry: the intensity of color is a measure of theamount of a material in solution.A second principle of spectrophotometry is that every substance absorbsor transmits certain wavelengths of radiant energy but not other wavelengths.For example, chlorophyll always absorbs red and violet light, while it trans-mits yellow, green, and blue wavelengths. The transmitted and reflected wave-lengths appear green—the color your eye “sees.” The light energy absorbedor transmitted must match exactly the energy required to cause an electronictransition (a movement of an electron from one quantum level to another) inthe substance under consideration. Only certain wavelength photons satisfythis energy condition. Thus, the absorption or transmission of specific wave-lengths 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 fre-quently used and important methods of quantitative analysis. They areapplicable to many industrial and clinical problems involving the quantitativedetermination of compounds that are colored or that react to form a coloredproduct.
LIGHT AND THE PERCEPTION OF COLOR
Light is a form of electromagnetic radiation. When it falls on a substance,three things can happen:the light can be reflected by the substanceit can be absorbed by the substance• certain wavelengths can be absorbed and the remainder transmitted orreflectedSince reflection of light is of minimal interest in spectrophotometry,we willignore it and turn to the absorbance and transmittance of light.The color we see in a sample of solution is due to the selective absorption
Chemistry 111 Lab: Intro to Spectrophotometry Page E-1Revised: June 2005
 
of certain wavelengths of visible light and transmittance of the remainingwavelengths. If a sample absorbs all wavelengths in the visible region of thespectrum, it will appear black; if it absorbs none of them, it will appear whiteor colorless. We see the various colors when particular wavelengths of radiantenergy strike our eyes. For example, the wavelength we perceive as green is0.0000195 inches or, expressed more scientifically, 495 nanometers.Suppose we shine a beam of white light at a substance that absorbs bluelight. Since the blue component of the white light gets absorbed by the sub-stance, the light that is transmitted is mostly yellow, the complementary colorof blue. This yellow light reaches our eyes, and we “see” the substance as ayellow colored substance. The table below gives pairs of complementary col-ors and the corresponding wavelength ranges.
Wavelength (nm) Color Absorbed Color Observed 
400 violet yellow-green435 blue yellow495 green purple560 yellow blue650 orange greenish blue800 red bluish greenYou should remember, of course, that the visible range is only a very smallpart of the electromagnetic spectrum. Ultraviolet and infrared spectrophoto-metric methods are suitable for many colorless substances that absorb stronglyin the UV or IR spectral regions.
TRANSMITTANCE, ABSORBANCE, ANDTHE BEER-LAMBERT LAW
We define
transmittance
as the ratio of the amount of light transmitted to theamount of light that initially fell on the surface.
 P 
 o 
Sampl incidentligh transmittedligh P 
 
 Transmittance =PP
0
=intensity of transmitted lightintensity of incident light
Absorbance
is defined as the negative logarithm of the transmittance, andyou will note that absorbance and transmittance bear an inverse relationship.
Absorbance = - log T = - log P/Po
Going back to our example of chlorophyll, if you have two colored solu-tions, you may deduce that the darker colored green solution appears darkerbecause it absorbs more of the light falling on it. Because the darker solutionis also the more concentrated one, you can also say that the more concentratedone absorbs more of the light. That is,
the absorbance increases as concentra-
Page E-2 Chemistry 111 Lab: Intro to SpectrophotometryRevised: June 2005
 
tion increases.
Next, suppose that there are two test tubes, both containing the same solu-tion at the same concentration. The only difference is that one of the test tubesis thicker than the other.
 P 
 o 
 P 
 o 
 P P shortpat longpat
We shine light of the same intensity (Po) on both containers. In the first casethe light has to travel through only a short distance, whereas in the secondcase it has to pass through a much longer length of the sample. We mightdeduce that in the second case more of the light will be absorbed or cut off,since the path length is longer. In other words, absorbance increases as path-length increases.The two observations described above (those dealing with the relationshipbetween absorbance and concentration and absorbance and path length) con-stitute the
BEER-LAMBERT LAW
.Beer-Lambert LawAbsorbance
path length (
l
) • concentrationA =
ε
l
• cwhereA is a dimensionless number.
ε
the proportionality constant, is called the molar extinction coefficient ormolar absorptivity. It is a constant for a given substance, provided the tem-perature and wavelength are constant. It has units of liter/mol • cm.
l
and c have the usual units of length (cm) and concentration (mol/liter).The quantitative measurement of light absorption as a function of wave-length can establish both the identity and the concentration of a substance insolution. The spectrophotometer is an instrument that separates electromag-netic radiation into its component wavelengths and selectively measures theintensity of radiation after passing through a sample. In this laboratory assign-ment you will use what is affectionately known as a “Spec 20” or Spectronic20. Its operation is described below.
SPECTROPHOTOMETRIC ANALYSIS
This section of the introductory material outlines a general approach forspectrophotometric analysis, first finding the absorption spectrum of “fingerprints” of a substance and then determining its concentration.
1. Plotting Absorption Spectra
Recall that the extinction coefficient for any given substance is a constant onlyso long as the wavelength of light is constant. You will see that the absorbancechanges with wavelength.
Chemistry 111 Lab: Intro to Spectrophotometry Page E-3Revised: June 2005

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