Objectives

This tutorial aims to familiarize you with the following principles of colourimetry:

• Why use colourimetry?
• How does colourimetry work?
• Proper experimental technique

Why Use Colourimetry?

Colourimetry can yield a wealth of information on coloured solutions. It is a quick and non-destructive method that can identify solutes in a solution and very accurately determine their concentrations. Computer technology has automated the somewhat tedious calculations required for colorimetric analysis and now allows colourimetry experiments to be performed within the span of a few minutes from start to finish. In short, a colorimetric analysis is straightforward, relatively foolproof, and highly informative.

How Does Colourimetry Work?

Colourimetry is a form of spectroscopy, an analysis that measures how atoms or molecules respond when exposed to electromagnetic radiation of a certain wavelength, and therefore, of a certain energy. In a way, colourimetry is the most familiar kind of spectroscopy, because the wavelengths used are from the visible light region of the electromagnetic spectrum.

In colourimetry, a light wave of a certain wavelength and intensity is shined at a solution (this is called incident light). The intensity of the light exiting the sample (transmitted light) is measured on the other side of the sample. By comparing the incident intensity to the transmitted intensity, the absorbance, A, can be determined for that wavelength of light. More precisely, A = -log(I/I₀), where I is the transmitted intensity and I₀ is the incident intensity.

A vast majority of the light that has not been transmitted through a translucent sample is absorbed by the sample (a negligible fraction of the energy is lost to scattering). Therefore, a substance that transmits most of the light at a particular wavelength will have a low absorbance at that wavelength. These measurements are repeated at many different wavelengths of light from the visible region of the spectrum.

An absorbance spectrum is created by plotting absorbance versus the light wavelength. For example, a red-coloured sample will transmit large proportions of light at wavelengths near the red range and exhibit a low absorbance at that wavelength. However, it will absorb more (and transmit less) at all other wavelengths. Shown below to illustrate this property is a spectrum of FD & C Red 40, one of the food dyes that you will be analyzing during the experiment. Note the markedly low absorbance at the higher, red-coloured wavelengths on the right.

These spectra are characteristic of a particular chemical substance, and can be used to identify unknown solutions by comparison to the spectra of known solutions.

Question 1

At the top of the table displayed below is an absorbance spectrum of an unknown substance. Below the unknown spectrum are the absorbance spectra of four candidate substances that may be the unknown. Identify the unknown using the shape of its absorbance spectrum.

The colour of a dye molecule depends on its structure, particularly on the grouping of certain atoms called "chromophores". Different chromophores absorb light at different wavelengths, giving rise to a variety of colours. Sometimes two dyes may contain the same chromophore and differ only in groups of atoms attached to that chromophore. The colours of these dyes are very much alike and difficult to tell apart visually. However, the minor structural differences are sufficient to produce subtle differences in the absorbance spectra of these dyes. Consider the structures of two dyes, Blue #1 and Green #3, provided below. Can you tell what the only difference is in the structure of these two dyes?

Since similar dyes have similar spectra, you have to carefully compare the spectrum of your unknown to the spectra of known dyes with similar colour. In the laboratory, the program called ColourMixer displays spectra of three dyes on the computer screen at the same time, making the identification process quick and accurate.

In the spectrum below, is the Unknown dye Blue #1 or Green #3?

Notice that the highest bar appears at 620nm in the spectra of both Blue #1 and Green #3. Therefore, the identification is best achieved by comparing the relative heights of bars on both sides of that bar. This would be the bars at 605 and 644 nm in the spectra of the Blue #1 and Green #3.

Once an absorbance spectrum of a particular substance is available, and the identity of the substance has been established, its concentration in solution can also be measured by colourimetry. This analysis is based on Beer’s Law, which in simple terms relates the colour intensity of a solution to its concentration. More precisely, Beer’s Law states that A = l c, where A is the absorbance of the sample, is a substance- and wavelength-specific coefficient, l is the length the light travels through the sample, and c is the sample’s concentration. The box below summarizes the relationship of incident and transmitted light intensities, absorbance, and the concentration of a substance in solution.

First, the wavelength of maximal absorbance is chosen from the substance’s absorbance spectrum. This is called the analytical wavelength and is the wavelength at which Beer’s Law analysis is done.

Question 2

For each of the substances shown below, locate and state the analytical wavelength. What colour does each wavelength correspond to?

A series of solutions of the substance with known concentrations are prepared. The absorbance of these solutions at the analytical wavelength is measured in sequence. When the measured absorbance values are plotted versus the solution concentrations, a straight line can be drawn to connect the points. This is because A = l c, and and l are the same for each sample. Thus A varies linearly with c. This plot is called a calibration graph. Shown below is a calibration graph for FD & C Blue #2.

Next, the absorbance of the sample of unknown concentration is measured. This absorbance value corresponds to a concentration on the calibration graph: this is the concentration of the unknown. In the graph below, the absorbance of a sample of FD & C Blue #2 of unknown concentration is measured at the analytical wavelength and placed on the graph (look for the X mark).

A computer calculates the unknown's relative concentration from the graph by fitting it into the equation of the red line. The unknown's concentration turns out to be 0.499, or 49.9% of the concentration of the standard sample marked 1.0.

Proper Experimental Technique

Before acquiring any absorption or transmission spectra of your samples, calibrate the colorimeter by taking a reading with a clean cuvet filled with deionized water. The colourimetry software will set the absorbance of this “blank” to zero: for the blank, I=I₀.

Before doing any Beer’s Law calculations on a solute of unknown concentration, you must first identify the solute. This is done by matching its absorbance spectrum to that of a known chemical substance. Beer’s Law holds only for absorbance values below 2, so there is a good chance that you will need to dilute your substance in order to obtain meaningful measurements. The spectrum of undiluted blackcurrant Powerade, shown below, is an example of when this is the case. Note the astronomical absorbance values!

Question 3

Beer's Law holds for absorbance values below 2. What minimum value of I/I₀ does that represent? (What is the minimum relative intensity of the transmitted light compared to the incident light?)

When performing your dilution, remember that quantitative accuracy will be required for subsequent Beer’s Law calculations, even though it is not necessary to identify the substance. Thus, it is wise to keep careful track of your dilutions for future Beer’s Law calculations.

Observe the spectrum of your (diluted) unknown, and locate the highest peak. This is the analytical wavelength. If the spectrum has two or more high peaks separated by a valley, this may mean that your sample contains more than one dye. In our blackcurrant Powerade example, after diluting the beverage tenfold and reacquiring the spectrum, we have good reason to suspect that this is the case.

Blackcurrant Powerade, Diluted Tenfold Note two potential analytical wavelengths, at 502 and 609 nm. This indicates that more than one dye may be present in the beverage being analyzed.

A quick run of paper chromatography will tell you if this is the case. If so, use column chromatography to separate the dyes and perform the above analysis on each one. Remember: you cannot do Beer’s Law calculations on column eluates! The concentration of your eluted products has nothing to do with their concentration in the sample that is loaded on the column! Therefore, the eluted products can only be used to identify the constituents of the sample, and not to determine their concentrations.

To identify the unknown solutes, compare their individual absorbance spectra to those of known “suspect” dyes. If the shapes of the spectra match perfectly, you’ve successfully identified your unknowns. Shown below are absorbance spectra of red and blue coloured fractions from blackcurrant Powerade that have been separated by column chromatography. Remember that these fractions are not suitable for Beer's Law analysis, since their concentrations in relation to the original sample cannot be precisely known.

Blackcurrant Powerade Red Fraction Note analytical wavelength of 502 nm and compare to spectrum of diluted blackcurrant Powerade.		Blackcurrant Powerade Blue Fraction Note analytical wavelength of 621 nm and compare to spectrum of diluted blackcurrant Powerade.

Once your unknown has been identified and its analytical wavelength(s) established, you can determine the concentration of the dye(s) in your unknown. The dyes do not need to be separated for this analysis as long as their analytical wavelengths are sufficiently different. As long as this is the case, it is perfectly acceptable to perform Beer's Law calculations on the mixture at each dye's analytical wavelength.

For each analytical wavelength (and dye), do the following:

Prepare a set of samples of the dye of known concentrations. This is best done by taking a stock solution of the dye and diluting it to, say, 0.2, 0.4, 0.6 and 0.8 of the initial concentration. Be careful when making your dilutions! The accuracy of your technique here will affect the accuracy of the Beer’s Law calibration graph and of your calculated unknown concentration.

Using the colourimetry software interface for making a calibration graph, acquire measurements of each of your dilutions and of the undiluted stock solution at their analytical wavelength. Then, acquire the absorbance of your (precisely diluted) unknown at that wavelength. The computer interface will automatically calculate its concentration relative to the stock solution from the equation of the calibration graph. With knowledge of the stock solution’s actual concentration, the unknown’s concentration is easily calculated.

Summary

This tutorial on colourimetry has presented the following topics:

1. The advantages of colourimetry as an analytical technique.
2. The laws of physics and chemistry underlying colourimetry.
3. Experimental technique for identifiying solutes and measuring concentrations by colorimetric analysis.