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COLORIMETRY- Simplified
Introduction
A colorimeter is a device used in colorimetry. The word generally refers to the device that measures the absorbance of particular wavelengths of light by a specific solution. This device is most commonly used to determine the concentration of a known solute in a given solution by the application of the Beer-Lambert law.
Principle of colorimetry
Colored solutions have the property of absorbing light of definite wavelengths. The amount of light absorbed or transmitted by a colored solution is in accordance with the Beer-Lambert law.
Beer’s law- The intensity of the color is directly proportional to the concentration of the colored particles in the solution.
Lambert’s law- The amount of light absorbed by a colored solution depends on the length of the column or the depth of the liquid through which the light passes.
Equations
When a monochromatic light with an original intensity ‘Io’, passes through a solution that can absorb radiant energy, Is will be less than the Io.
Some of the radiant energy is reflected back by the cell containing the solution, or absorbed by the cell wall or the solvent.
The amount of radiation absorbed may be measured in a number of ways:
- By measuring transmittance
- by measuring absorbance
1. By measuring transmittance- The transmittance (T) is defined as-
T= Is/ Io
The ratio is expressed as a percentage, thus
% T= 100 x Is/ Io
As the concentration of the compound increases, less light is transmitted.
%T varies inversely and logarithmically with the concentration.
2) By measuring absorbance- Absorbance measurement is convenient than transmittance.
Absorbance (A) or optical density is directly proportional to the concentration.
The relationship between absorbance and transmittance can be expressed as –
A = – log Is/ Io
= – log T
= log 1/T
To convert T to % T,
A = log 1/T x 100/100
= log (100)/%T
= log 100-log %T
= 2-log %T
Thus,
A = 2-log %T
In other words, absorbance (Optical density) and Transmittance (T) are reciprocally related.
So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite (Figure-1)
Figure 1: Transmittance and absorbance are reciprocally related.
Lambert -Beer’s law
The mathematical expression at a given wavelength can be represented as follows-
OD = A = Ʃcl
Since,
OD = – log Is/ Io
(Absorption has no units since it is a ratio)
Thus:
– log Is/ Io = cl
or Is/ Io = e Ʃcl
Where =Ʃ is a Constant- It is the molar extinction coefficient( Molar absorptivity) with units of L mol-1 cm-1
C = Concentration of the colored substance, expressed in mol L-1
l = is the path length of the sample – that is, the path length of the cuvette in which the sample is contained.
e- the base of the natural logarithm.
Since Is/ Io is known as transmittance (T)
Thus,
T= e Ʃcl
Taking logarithm:
-log 10T =Ʃcl
As per the equation:
-log T= A
Hence
A = OD = Ʃcl
Since the thickness of the layer of solution is constant in the instrument, optical density is proportional to the concentration.
When optical density is plotted against concentration “c”, a straight line passing through the origin should be obtained, because the absorbance is directly proportional to the concentration. (Figure-2)
Figure 2: Relationship of absorbance and concentration of a solute in a solution
The concentration of an unknown solution can be readily determined by measurement of its absorbance and interpolation of its concentration from the graph of the standards.
When % T is plotted versus concentration, a curvilinear relationship is obtained.
The linear relationship between concentration and absorbance is both simple and straightforward, which is why it is preferred to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T.
Calculation of unknown concentration in the test sample
Since there is a linear relationship between absorbance and concentration, it is possible to calculate the unknown concentration of a substance in the test sample by a simple proportional equation-
The absorbance of unknown Concentration of unknown
———————————- = ————————————–
The absorbance of standard Concentration of Standard
Absorption of unknown
The concentration of unknown = ——————————— x Concentration of Standard
Absorption of standard
Thus,
The concentration of Unknown (Test sample T)
OD of Test
= ————————– x Concentration of Standard
OD of standard
Some of the incident energy may be reflected by the cell containing the solution or absorbed by the cell wall or the solvent. To eliminate these factors and to consider the absorption by the compound, a blank solution or a reference solution having everything but the compound to be measured is used.
Thus the concentration of unknown can be expressed as-
The concentration of unknown (Test sample T)
OD of Test – OD of Blank
= —————————————— x Concentration of Standard
OD of standard – OD of Blank
Deviations from Beer’s law are observed when a very large concentration of an unknown substance is measured or when the incident light is not monochromatic light.
Components of a photo colorimeter
1) Light source
The light source is usually a tungsten lamp for wavelength in the visible range (320-700 nm) and a deuterium or hydrogen lamp for ultraviolet light (below 350 nm). Hydrogen lamp is usually preferred to UV range.
2) Monochromators
This is for the selection of a sufficiently narrow waveband. The monochromator consists of an entrance slit to exclude unwanted, followed by absorption or interference filters, prisms or diffraction grating for wavelength selection. (Figure-3)
Figure 3: Components of a colorimeter.
The interference filters consist of a thin layer of magnesium fluoride crystals with a semitransparent coating of silver on each side. The interference filters have a bandpass of 5-8 nm. The bandpass is defined as the width of the spectrum that will be isolated by a monochromator. The choice of filter depends upon the final color of the solution formed.
| Wavelength (nm) | Filter used/Color absorbed | Color of solution |
| 350-430 | Violet | Yellow Blue |
| 430-475 | Blue | Yellow |
| 475- 495 | Green-blue | Orange |
| 495-505 | Blue-green | Red |
| 505-555 | Green | Purple |
| 555-575 | Yellow-green | Violet |
| 575-600 | Yellow | Blue |
| 600=650 | Orange | Green-blue |
| 650-700 | Red | Blue-green |
3) Lens
Instruments using filters as wavelength selectors require lenses to focus correctly the light from the source through the filter and cuvette to the detector. In the ultraviolet range, quartz or fused silica is essential because the glass does not transmit light efficiently at a wavelength shorter than 340 nm.
An exit slit at the end of the monochromator allows only a narrow fraction of the spectrum of reaching the sample cuvette.
4) Sample cuvette
For accurate and precise reading, cuvette must be transparent, clean, devoid of any scratches. The optical path of the cuvette is always 1 cm. Glass cuvettes are used for reading in the visible light range while quartz or fused silica cuvettes are used for UV range.
5) Photosensitive detectors
These detectors contain a light-sensitive surface that releases electrons in number proportional to the intensity of light on it, converting light energy into electrical energy. Different detectors used are-
a) Barrier layer cells
b) Photosensitive tubes
c) Photomultiplier tubes
d) Photoconductive cells
6) Readout devices- The detector response can be measured by any of the following readout devices-
a) Galvanometer
b) Ammeter
c) Recorder
d) Digital readout.
The signal may be transmitted to a computer or print out device. Most modern instruments are of the direct-reading type where the amplified detector signal operates a galvanometer.
SPECTROPHOTOMERTY
In contrast to a colorimeter, a spectrophotometer is an instrument that is capable of splitting the incident radiation into a spectrum producing radiation of the defined wavelength and subsequently measuring the intensity of that radiation. In place of colored filters, a diffraction grating or glass prism or quartz prism is fitted, which disperses the white light into a continuous spectrum (figure-4). The total wavelength at which measurements can be made include the visible spectrum (400-750 nm) and extends to each side into the near ultraviolet (360-400nm) and near infra-red (750-1000nm). It is thus possible to eliminate substances which are more or less colorless in the visible region, but which absorb light and are therefore colored, in the ultraviolet or infrared region.
Figure 4: Components of a spectrophotometer. First, a collimator (lens) transmits a straight beam of light (photons) that passes through a monochromator (prism) to split it into several component wavelengths (spectrum). Then a wavelength selector (slit) transmits only the desired wavelength.