Spectrophotometry is quantitative study. It is one of the most useful tools that offers high degree of precision, sensitivity, and accuracy.
In addition, it is inexpensive method for measurement and analysis of variety of substances.
Spectrophotometer is photometer that can measures intensity of the light as its function.
This technique has various applications from scientific research to forensic examinations.
- Fluorescence Spectroscopy is a technique to detect molecules by analyzing fluorescence emitted from sample, which is generally an organic compound.
- Mass Spectrometry is used for the identification of compounds by their mass-to-charge ratio.
- Nuclear Magnetic Resonance (NMR) spectroscopy is most powerful tool. It available for determining the structure of organic compounds.
NMR relies on the behavior of atomic nuclei as a small magnet and their alignment with an external magnetic field.
General Principle
Spectrophotometry is carried out by an instrument, spectrophotometer for the quantitative measurement of reflection or transmission properties of a material as a function of wavelength.
It deals with visible light, near ultraviolet, and infrared region of the electromagnetic spectrum offering high degree of precision, sensitivity, and accuracy.
These techniques have long been employed as tool for probing structure and intramolecular interactions of biomolecules.
Spectrophotometers
Spectrophotometers are classified on the basis of types of measurement techniques involved, wavelengths at which they work, a way how they acquire spectrum, and sources of variation in the intensity.
They are used for the quantitative measurement of reflection or/and transmission properties of substance.
Important features of spectrophotometers include measurements of spectral bandwidth and linear range of absorption as well as amount of light that a sample absorbs.
The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching detector.
Electromagnetic radiations are electromagnetic waves travelling at a speed of 3 × 108 ms-1. These radiations are called quanta or photons.
Atoms occupy lowest energy levels at ground state, commensurate with laws of quantum mechanics. When excited by absorption of discrete amount of energy, they occupy higher energy levels.
These excited electrons return to ground state emitting radiations of specific wavelength, which in turn gives rise to spectroscopic phenomenon.
Various changes in the energy levels of electrons occur due to absorption or emission of the distinct quanta of radiations called absorption and emission spectra, respectively.
The frequency of the wave is proportional to the energy of the particle. Charged particles act as energy transporters as photons are emitted and absorbed by them. The energy per photon is given as,
E = hv
Where, E= the energy of radiation absorbed or emitted by the particle (E = E1 – E2),
h = Planck’s constant = 6.63 × 10-34 Js
v = frequency of radiation in hertz = c/λ
E1 = the energy of electron in the original level
E2 = the energy of electron in the final level
c = speed of light = 3 × 108 m s-1
λ = wavelength of radiation (usually measured in cm, μm, or nm)
Electromagnetic spectrum is a range of all possible frequencies of electromagnetic radiations emitted or absorbed by particular object. EM waves are described by frequency, wavelength, or photon energy.
f = C/λ or f = E/h or E = hc/λ
Absorption or emission of ultraviolet or visible light by a molecule depends on the electron transitions between molecular orbital energy levels similar to absorption or emission of electromagnetic radiation by an atom.
It is determined by electron transitions between different energy levels in an atom and differential energies for those transitions.
Electronic and fluorescence spectra arises due to change in the energy levels of outer electrons of an atom, vibration-rotation spectra (Figure1) are formed by changes in the vibration levels of electrons, while electron spin and nuclear magnetic resonance spectra are formed due to change in spins of electrons and nuclei, respectively in a magnetic field.
Figure 1 Vibrational and rotational energy level of an atom
In spectroscopy, transmittance is fraction of incident light at a specified wavelength that passes through a sample and absorbance is the fraction of light absorbed by a sample at a specified wavelength.
T = I/Io
Where, T = transmittance
I = intensity of the incident radiation
Io = intensity of the transmitted radiation
The extent of radiation absorption is referred to as absorbance (A) or extinction (E), which is equal to the logarithm of the reciprocal of the transmittance, as shown below.
A = E = log 1/T = log Io /I
The Beer-Lambert law states that the extinction is proportional to concentration of the absorbing substance and to the thickness of the layer, i.e.
A = E = ελcd
Where,
ελ = molar extinction coefficient for absorbing material at wavelength λ (dm3 mol-1 cm-1)
c = concentration of the absorbing solution (molar)
d = light path in the absorbing material (cm)
There are few circumstances when the Beer-Lambert law is not valid such as, ionization or polymerization of specimen at higher concentrations, coagulation of specimen, and susceptibility of the instruments to stray radiations.
Spectrophotometer is formed by combining two instruments, spectrometer for producing light of different wavelengths, and photometer for measuring the intensity of light.
Instruments are arranged so that liquid in a cuvette can be placed between spectrometer beam and photometer. The amount of light passing through the tube is measured by the photometer.
Photometer delivers a voltage signal to a display device, which is generally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes.
