Colorimetry Guide Feb. 25, 2015

Introduction

Many biochemical experiments involve the measurements of compound or group of compounds present in a complex mixture.

The most widely used method for determining the concentration of biochemical compounds is colorimetry, which makes use of the property that when white light passes through a colored solution, some wavelength are absorbed more than others. Many compounds are not themselves colored but can be made to absorb light in 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 big advantage of is that complete isolation of compound is not necessary and the constituents of a complex mixture such as blood can be determined after little treatment.

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.

Colorimeter - is the instrument designed to determine the concentration of a solution by analyzing its color intensity.


Colorimeter’s Construction & Function


Construction.

The essential parts of a colorimeter are:

  • a light source, which is usually an ordinary filament lamp,
  • an aperture which can be adjusted and a lens,
  • a set of filters in different colors,
  • a cuvette that holds the working solution,
  • a detector (usually - photoresistor or photocell) which measures the light which has passed through the solution,
  • a meter to display the output from the detector.

Filter in the colorimeter is used to select the color of light which the solute absorbs the most, in order to maximize the accuracy of the experiment. Note that the colour of the absorbed light is the 'opposite' of the colour of the specimen, so a blue filter would be appropriate for an orange substance. Sensors measure the amount of light which 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 are available in a number of grades and made of plastic or glass. Note that it is crucial that all samples are only put into a cuvette and never directly into the colorimeter itself or damage will certainly result - the colorimeter labelling 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.

Function.

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.

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.

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 under study, 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.

Beer’s Law.

In general, absorbance is important because of its direct relationship with concentration according to Beer’s law. Many experiments in chemistry and biology are based on this concept. To obtain a Beer’s law curve, several standards (solutions of known concentration) are prepared and their absorbance values are determined using a Colorimeter.

A graph of absorbance vs. concentration is then plotted. A solution of unknown concentration is placed in the colorimeter and its absorbance measured. When the absorbance of this solution is interpolated on the Beer’s law curve, its concentration is determined on the horizontal axis. Alternatively, its concentration may be found using the slope of the Beer’s law curve.

How does a colorimeter work?

  1. 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.

  2. 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.

  3. The light then falls on to a photocell which generates an electrical current in direct proportion to the intensity of light falling on it.

  4. This small electrical signal is increased by the amplifier which passes to a galvanometer of digital readout to give absorbance reading directly.

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