COLORIMETRIC ANALYSIS

1.(a) Define colorimetry

Colorimetry is one of most widely used method for determining the concentration of biochemical compounds is which makes use of the property that when white light passes through a colored solution, some wavelengths are absorbed more than others. A colorimeter is the instrument designed to determine the concentration of a solution by analyzing its color intensity.

(b)Define each of the following terms as used in colorimetry

(i)Absorbance

(ii) molar absorbance

(iii) monochrometer

(iv) blank

Explain the principle of colorimetry

Many compounds are not themselves colored but can be made to absorb light in the visible region by reaction with suitable reagents. These reactions are fairly specific and, in most cases, very sensitive. So that quantities of material in the region of mol/L concentrations can be measured.

The depth of the color is proportional to the concentration of the compound being measured, while the amount of light is proportional to the intensity of the color and hence the concentration.

The earliest colorimeters relied on the human eye to match the color of a solution with that of one of a series of colored discs. The results obtained were too subjective and not particularly accurate.

Different chemical substances absorb varying frequencies of the visible spectrum. Colorimeters rely on the principle that the absorbance of a substance is proportional to its concentration, i.e., a more concentrated solution gives a higher absorbance reading.

A quantitative reading for the concentration of a substance can be found by making up a series of solutions of known concentration of the chemical understudy, and plotting a graph of absorbance against concentration. By reading off the absorbance of the specimen substance on the graph, a value for its concentration is found.

A colorimeter is generally any tool that characterizes color samples to provide an objective measure of color characteristics.

In chemistry, the colorimeter is an apparatus that allows the absorbance of a solution at a particular frequency (color) of visual light to be determined. Colorimeters hence make it possible to determine the concentration of a known solute since it is proportional to the absorbance.

Describe the components of a colorimeter

The essential parts of a colorimeter are:

a light source, which is usually an ordinary filament lamp;
a lenswith an aperture that can be adjusted;
a set of filtersin different colors;
a cuvettethat holds the working solution;
a detector(usually – photoresistor or photocell), which measures the light that has passed through the solution;
a meterto display the output from the detector.

Filter in the colorimeter is used to select the color of light which the solute absorbs the most to maximize the accuracy of the experiment. Note that the color of the absorbed light is the ‘opposite’ of the color of the specimen, so a blue filter would be appropriate for an orange substance. Sensors measure the amount of light that has passed through the solution, compared to the amount entering, and a display reads the amount absorbed.

Cuvettes. Solutions to be measured are put into a small square test tube-like vessel called a cuvette, which is available in various grades and made of plastic or glass. Note that all samples must be put only into a cuvette and never directly into the colorimeter itself or damage will certainly result – the colorimeter labeling makes this very clear for the benefit of new users. Each cuvette varies slightly optically, so when the procedure involves changing the concentration of a solution best practice is to use the same cuvette for the complete experiment. Most cuvettes have only two optical faces, which are very clear, the other two usually being duller or sometimes embossed to make them easier to grip.

How does a colorimeter work?

White light from a tungsten lamp passes through a slit, then a condenser lens, to give a parallel beam which falls on the solution under investigation contained in an absorption cell or cuvette. The cell is made of glass with the sides facing the beam cut parallel to each other.
Beyond the absorption cell is the filter, which is selected to allow maximum transmission of the color absorbed. If a blue solution is under examination, then red is absorbed and a red filter is selected. NOTE: The color of the filter is complementary to the solution.
The light then falls onto a photocell, which generates an electrical current in direct proportion to the intensity of light falling on it.
This small electrical signal is increased by the amplifier, which passes to a galvanometer of digital readout to give absorbance reading directly.
Discribe how to operate a colorimeter

Switch on the instrument at least 5 minutes before use to allow it to stabilize.
Select the most appropriate filter for the analysis and insert it in the light path (Griffin) or dial it in with the selector (Jenway).
Place the reagent blank solution (or water) in the cuvette and zero the instrument (either using the ëset zeroí control or the ëcalibrateí switch. Make sure the clear faces of the cuvette are in the light path .
Place the sample in the colorimeter and read the absorbance of the solution. If the absorbance is “over range” (usually > 2.0) then the sample must be diluted to yield a value within the limits of the instrument.
At intervals, recheck the reagent blank to ensure that there is no drift in the zero value.

Distinguish between visual colorimetry and photoelectric colorimetry

The filters are used to isolate a part of the visible light spectrum that is absorbed maximally by the sample. Different colourimeters use different sets of filters but typical wavelengths passed are red filter: 630-750nm, green filter: 510-570nm and blue filter: 360-480nm. Although you will normally be told which filter to use you should consider and understand the reason for this choice.

a . State Beer -Lambert’s Law

Beer-Lambert Law (also known as Beer’s Law) states that there is a linear relationship between the absorbance and the concentration of a sample.

b.State three causes for deviation from Beer -Lambert’s Law

Beer-Lambert law only holds true in the following scenarios;

The light passing through the sample must be monochromatic (of a single wavelength.
The solution must be homogeneous. If not, at high concentrations, the molecules may aggregate and this would lead to incorrect readings.
The solution must not have a molecule that emits fluorescence when excited. If it does, then this can lead to erroneous readings.
The temperature of the solution must not change, as the molar extinction coefficient depends on it.
. Name two types of filters used in colorimeter

The filters are used to isolate a part of the visible light spectrum that is absorbed maximally by the sample.

Different colourimeters use different sets of filters but typical wavelengths passed are red filter: 630-750nm, green filter: 510-570nm and blue filter: 360-480nm.

State any three causes of deviation to Beer-Lamberts

These deviations are due to:

chemical reasons arising when the absorbing compound, dissociates, associates, or reacts with a solvent to produce a product having a different absorption spectrum,
the presence of stray radiation, and
the polychromatic radiation.

Convert 0.78 absorbance units into percentage transmittance

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