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# Colorimeter

A form of energy is produced by oscillating electric and magnetic disturbance is known as electromagnetic radiation. These radiations can travel through a vacuum or matter due to movement of electrically charged particles. These radiations move perpendicular to both electric and magnetic fields which are perpendicular to each other. Electromagnetic radiation can travel through vacuum with the speed of light 2.99792458 x 108 m s-1. The distance of either crests or troughs of waves is called as wavelength of waves. It is characterized by the Greek symbol Î».

It measures the magnitude of oscillation and represents the energy and height of the wave. Higher energy corresponds to larger amplitude whereas lower amplitude relates to lower energy. The relation between wavelength and speed of light can be given c= Î»Î½

Here
c =Speed of light
Î» =Wavelength
Î½ =Frequency

So we can say that frequency and wavelength are inversely proportional to each other. Here frequency can be defined as the number of cycles per second. The unit of frequency is sec-1 or Hertz (Hz). The relation between frequency and energy can be express as:
E=hÎ½

Here
E =Energy
h =Planck's constant (h= 6.62607 x 10-34 J)
Î½= Frequency.

Different types of electromagnetic radiations have different wavelength and frequency values. The arrangement of electromagnetic radiations on the basis of their wavelength and frequency is called as electromagnetic spectrum. Since wavelength and frequency are inversely related, so as the wavelength increases, the frequency decreases and vice-versa.  Different parts of electromagnetic spectrum are; radio waves, microwaves, infrared waves, visible light, ultraviolet radiation, X-rays, and gamma rays. Out of these, only visible light spectrum is visible to human eyes.

Radio waves are high wavelength waves approximately 103 m. These waves are transmitted by radio broadcasts and TV broadcasts. Due to highest wavelength, these radiations have the lowest energy levels and that is the reason, they are used in remote sensing, in radar systems etc. Just after radio waves, there are microwaves in electromagnetic spectrum which can be used in remote sensing, to broadcast information through space, in Doppler radars to predict weather forecasts and warm food in microwave ovens. The wavelength of microwaves can be measured in centimeters.
Next part of electromagnetic radiations is Infrared radiations which are used in remote sensing as infrared sensors. On the basis of wavelength, they can be divided as near IR, IR and far IR.

After IR region, there is visible region which can see with an unaided human eye. It includes a range of different colors with a particular wavelength. Different colors of visible region with their wavelength values are listed below.

 Color Region Wavelength (nm) Violet 380-435 Blue 435-500 Cyan 500-520 Green 520-565 Yellow 565-590 Orange 590-625 Red 625-740

When we pass the visible radiation through a prism, the dispersion of radiations occurs and light waves splits into seven colors.

After visible radiations, there are ultraviolet, X-Rays, and Gamma Rays in electromagnetic spectrum. UV radiation effects on the skin from the sun and also leads to cancer whereas X-rays are used to produce medical images of internal solid parts of human body. Gamma Rays are important part of chemotherapy. They are shortest waves with approximately 10-12 m in wavelength.

 Electromagnetic radiations Frequency Wavelengths Uses Radio waves 30 gigahertz (GHz) 10 millimeters For communications including voice, data and entertainment media Microwaves 30 terahertz (THz) 10 mm  to 100 micrometers For high-bandwidth communications, radar and as a heat source for microwave ovens and industrial applications Infra red 30 THz up to about 400 THz 100 Î¼m  to 740 nanometers Used in IR spectroscopy to determine the structure of chemical compounds Visible light 400 THz to 800 THz 740 nm  to 380 nm Visible to most human eyes Ultraviolet 8 Ã— 1014 to 3 Ã— 1016 Hz 380 nm  to about 10 nm Numerous medical and industrial applications X-rays 3 Ã— 1016 to about 1018 Hz 10 nm to 0.1 nm Applications in medical field Gamma-rays greater than about 1018 Hz less than 100 pm Applications in medical field

## Colorimeter Definition

A light sensitive device which is used to measure the transmittance and absorbance of light passing through a liquid sample is called as colorimeter. It measures the intensity or concentration of the color which develops due to presence of specific reagent into a solution. Colorimeter can be two types; color densitometers and color photometers. Color densitometers measure the density of primary colors whereas color photometers measure the color reflection and transmission.  We know that the spectrophotometer works Beer-Lambert’s Laws. Beer’s Law states that when monochromatic light passes through the colored solution the amount of light transmitted decreases with increase in concentration of the colored substance. The mathematical relation can be written as;

It = Ioe-KC

Lambert’s Law states that the amount of light transmitted decreases with increase in thickness of the colored solution.
It = Ioe-kt

Both laws can be written as IE/Io = e-KCT and also called as Beer-Lambert’s law.

Here;
•    IE = intensity of emerging light
•    Io = intensity of incident light
•    e = base of neutral logarithm
•    K = a constant
•    C = concentration
•    T = thickness of the solution

## Colorimeter Diagram

There are mainly three components in the colorimeter; a light source, a cuvette containing the sample solution, and a photocell which is used to detect the light passed through the solution. This device contains either colored filters or specific LEDs which can generate color. The output from a colorimeter can be displayed by an analog meter and reading can come either in terms of transmittance or absorbance. Some of these devices contain a voltage regulator to protect the instrument from fluctuations in mains voltage. They are available in different sizes and useful for laboratory testing. Letâ€™s discuss different parts of colorimeter in brief.

Light source- In colorimeter, the light source is usually an ordinary filament lamp.
• Aperture- It is a slit between light source and condenser lens which can be adjusted.
• A lens- A condenser lens is used through which the light beam is passed.
• Set of filters-There is a set of filters with different colors which can filter a certain wavelength of light and remaining light can passed through it. Filter is used to select the color of light that absorbs by solute present in the solution. The color of light absorbed is the 'opposite' of the colour of the specimen. In other words if the substance is orange in color, a blue filter would be appropriate.
• Cuvette- It is a small square test tube like vessel that used to hold the working solution. Cuvettes are slightly optically and are made of plastic or glass.
• Detector- It is a photoresistor or photocell that measures the light passed through the solution.
• Meter- it displays the output from the detector in terms of absorbance or transmittance. Here sensors measure the amount of light passed through the solution and compare it with the incident light.

## Principle of Colorimetry

Colorimetry is frequently used in biochemical investigations as it involves the quantitative estimation of colors. So if we want to measure the quantity of substance in a mixture, we can use colorimeter in which the substance allows to bind with color forming chromogens which can be detected with colorimeter. The color of solution causes absorption of light and by measuring the absorbance and transmittance we can determine the quantity of substance.

The instrument will comprise of light source, a filter which selects the desired wavelength, cuvette chamber that allows to  pass the light and presence of sample solution can change the intensity of light. A photosensitive detector converts the light into electrical signals and galvanometer is used to measure the electrical signal.

Like other spectrophotometer, colorimeter is also based on two fundamental laws of absorption; Lambert’s law and Beer’s law. These laws play important role in colorimetric estima¬tion. When monochromatic light passes through a solution of constant concentration, the absorption by the solution is directly proportional to the length of the solution. This is called as Lambert’s law.

log10 Io/I = K1I

Beer’s law gives the relation between concentration and absorption. It states that when monochromatic light passes through a solution of constant length, the absorption is directly proportional to the concentration of the solution.

log10 Io/I = K2I
here:
• Io = Intensity of incident light
• I = Intensity of transmitted light
• l = Length of absorbing solution
• c = Concentration of solution
• K1 and K2 = Constants
Hence transmittance (T) can be written as;
T = I/Io
Or         A =   log10 (I/T) = log10 (Io/I)

Here A is absorbance which is also called as optical density (OD). As the concentration changes for sample solution, variation in color can be observed that is the basis of colorimetric analysis.

## How Does a Colorimeter Work?

The unknown substance can be measured quantitatively with the reaction of substance and appropriate reagent that form a colored substance in appropriate proportion. The observed intensity of color can be compared with the standard reaction mixture of known amount of substance. Let’s discuss the steps for operating the photoelectric colorimeter. First choose the appropriate glass filter and insert it in the device. Fill cuvette with blank solution and place it in the cuvette slot. Switch on the device and allow it to warm up for 4 – 5 minutes with zero optical density. Now take the unknown solution in another cuvette and record the optical density. We can use standard solutions with different concentrations and record the optical density as S1, S2, S3, S4 and S5. Plot the curve between concentrations of standard solution and the optical density.

We can determine the concentration of unknown solution with the help of this calibrated curve. So overall a colorimeter allows the absorbance of a solution at a particular frequency of visual light. It determines the concentration of a known solute which is proportional to the absorbance.