Spectrophotometry Instrumental Method of Analysis

Principle of Spectrophotometry

Spectrophotometry is based on the electromagnetic radiation in the visible, ultraviolet and infrared ranges.

According to the Quantum theory; the energy states of an atom or molecule are defined and change from one state to another, therefore, they would require a definite amount of energy. If this energy is supplied from an external source of radiation, the exact quantity of energy required to bring about a change from one given state to another will be provided by photons of one particular frequency, which may be selectively absorbed. The study of the frequencies of the photons which are absorbed will therefore provide information about the nature of the material. Also, the number of photons absorbed may provide information about the number of atoms and molecules of the material present in a particular state. This hence provides us with a method to have a qualitative and quantitative analysis of a substance.

Molecules possess 3 types of internal energy, namely:

• Electronic
• Vibrational
• Rotational

When a molecule absorbs radiant energy, it can increase its internal energy in a variety of ways. The various molecular energy states are quantized and the amount of energy necessary to cause any change in any of the above states would generally correspond to specific regions of the electromagnetic spectrum. Electronic transitions correspond to the ultraviolet and visible regions, the vibrational transitions to the near infrared and infrared regions and rotational transitions to the infrared and far-infrared regions. The method of analysis based on the absorption of radiation of a substance is known as absorption spectroscopy.

Spectrophotometer

A Spectrophotometer is used for measurement of the amount of photons (the intensity of light) absorbed, transmitted or reflected through a sample. There are two kinds of Spectrophotometers:

• Single beam
• Double beam

Components that make up a Spectrophotometer

A light source

It is a tungsten lamp filled with low pressure iodine or bromine to increase the life of the lamp. This source of light is good for radiation in the visible, the near infrared, and near ultraviolet regions of the electromagnetic spectrum. For work in the infrared region, a tungsten lamp may be used, however, due to high absorption of the glass envelope and the presence of unwanted emission in the visible range, tungsten lamps are not preferred. In such cases, Nernst filaments or other similar type are preferred. They are operated at lower temperatures and still radiate sufficient energy.

High pressure hydrogen lamps or deuterium lamps are used if radiation in the ultra violet is required. Deuterium discharge lamps have a longer life as compared to hydrogen lamps and the intensity of the radiation is also thrice that of the hydrogen lamp. Xenon arc lamps or high pressure mercury vapour lamps provide high levels of ultraviolet radiation. All these lamps become extremely hot and require an auxiliary cooling system while in use.

Monochromator

This is an optical system which provides a better isolation of spectral energy than the optical filters, and hence preferred when it is required to isolate narrow bands of radiant energy. Monochromator may incorporate a small glass of Quartz prism or Diffraction gratings system as the dispersing media. The radiation from a light source is passed either directly or by means of a lens or mirror into the narrow slit of the Monochromator and is allowed to fall on the dispersing medium, where it gets isolated. When white light passes through a prism it is refracted. Shorter wavelengths bend more than longer wavelengths and the desired narrow band spectrum can be isolated.

Diffraction gratings consist of closely-spaced parallel lines on a glass substrate or a polished aluminium or aluminium copper alloy. Light rays bend around the sharp edges of the parallel lines and the wavefronts formed reinforce if they are in phase, or cancel if they are out of phase. A narrow spectrum of radiation is obtained.

Adjustable aperture

It admits light of desired wavelength on the cuvette.

Cuvette

It is also known as an absorption cell. It holds the sample i.e. solute and solvent. For measurements in the visible range, cuvettes are cylindrical glass tubes. For measurement below 340 nm, rectangular or square cuvettes of Silica or Quartz are used as they are free of optical aberration.

Detector

It is a Photoresistor whose resistance changes with the incident light. The current passing through it can be calibrated to read transmitted light. Other detectors are photodiode arrays or photomultiplier tubes.

A Read out device

There are 2 types of read out devices: The direct reading or the null deflection system. In direct reading, there is a linear relationship between current in milliamps and percentage transmittance.

Related: Types of Spectrophotometers

Colorimeters

The Colorimeter measures the absorbance of different wavelengths of light in a solution to determine the concentration of a solute. Different substances absorb different wavelengths of light and the absorption of light increases with increase in the concentration of the solute according to the Beer Lambert’s Law:

I_r = I_o10^-alc

Where,

I_o = radiant power arriving at the cuvette

I_r = radiant power leaving the cuvette

a = absorptivity of the sample (extinction coefficient)

l = length of the path through the sample

c = concentration of the absorbing substance

The Colorimeter consists of a light source which is usually a filament lamp which emits light of wavelengths ranging from 400 nm to 700 nm. The light passes through an adjustable aperture and a set of filters for filtering different colour frequencies. Generally a Tristimulus absorption filter is used. The desired frequency of light passes through a solution which is placed in a glass or plastic cuvette. The output light is measured by a detector which can be a Photodiode, Photoresistor or Photomultiplier. The analog value of light absorbed is converted to a digital value and displayed on a digital meter.

The output is shown as transmittance on a linear scale from 0-100% or as absorbance on a logarithmic scale from 0 to infinity. The useful range is from 0 to 2, but the reliable range is 0 to 1, since beyond this range; results become unreliable due to scattering of light. Zero adjustment is done by setting the base value or zero value of a reference substance. The absorbances of other substances are recorded relative to the initial zero.

Spectrophotometer vs. Colorimeter: What are the differences?

• Spectrophotometer measures the amount of light passing through a sample while Colorimeter measures the absorbance of light.
• Spectrophotometer uses a wide range of wavelengths in the Ultraviolet, Visible and Infrared regions whereas Colorimeter uses fixed wavelengths in the visible range only.
• Spectrophotometer can be used in the identification and quantification studies of inorganic or organic compounds while Colorimeter can be used to determine the concentration of an individual compound based on the amount of absorbance.
• The Spectrophotometer is used for high precision analysis and accurate colour management in laboratories and research & development applications while Colorimeters find common use in the production and inspection applications for colour difference measurements.

You can also read: Introduction to Medical Diagnostics

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Author: John Mulindi

John Mulindi has a background in Instrumentation, and he writes on various topics ranging from Technical, Business to Internet marketing fields. He likes reading, watching football, writing and taking on adventure walks in free time. View all posts by John Mulindi