Spectrophotometry - Principles

We will be discussing some of the biophysical techniques in some posts following from now. In this post, we will have a look at some of the basic principles of spectrophotometry.

Electromagnetic Radiation (EM Radiation)

We will have to brush-up a little knowledge about Physics and Chemistry before going into the details of spectrophotometry to make things easy. Recall what you studied regarding light in your graduation. Briefly, we will revise here. The beam of light consists of a stream of photons or electromagnetic wave-form disturbance. Basically, the term 'electromagnetic' is a precise description of radiation such that the radiation is made up of an electrical and magnetic wave which are in phase and perpendicular to each other and to the direction of propagation as can be seen in the diagram above.

If matter is exposed to electromagnetic radiations (for example, infra-red rays), as can be seen in the adjacent figure, the radiation can be absorbed, transmitted, reflected, scattered or undergo photoluminescence (which can include a number of effects like fluorescence, phosphorescence etc. about which we will discuss in our future posts). Electromagnetic radiation is produced by events at molecular, atomic or nuclear level. A little understanding of Chemistry is needed here to understand the events that give rise to electromagnetic radiations such as the oscillations of nuclei and electrons in electrical or magnetic fields, molecular bending and vibration, excitation of orbital electrons, ejection of an inner orbital electron and rearrangement of the other electrons and nuclear break-up. Each of these events differ in terms of energy that is involved and thus, the radiation that they will emit will have different wavelengths. Thus, a complete spectrum of electromagnetic radiation is produced. Such a spectrum is shown in the form of the table below.

Spectrophotometry:

Now, coming to what is spectrophotometry. The spectrophotometry takes the advantage of dual nature of light namely:
              A particle nature which gives rise to photoelectric effect.
              A wave nature which gives rise to visible spectrum of light.

A spectrophotometer consists of two words as ‘spectrometer’ for producing light of selected wavelength and ‘photometer’ for measuring the intensity of light. Thus, a spectrophotometer is an instrument which is used to measure the amount of light (electromagnetic radiation) that a sample absorbs. The instruments are so arranged such that the sample can be placed between the spectrometer beam and photometer thereby measuring an unknown analyte concentration. In other words, it operates by passing a beam of light through a sample and measuring the intensity of the light reaching the detector.

Points to remember:

• The distance of one cycle is the wavelength (λ).
• The frequency, ν, is the number of cycles passing a fixed point per unit time.
• λ = c/ν (where c – velocity of light, 3x10⁸ms^-1)
• The shorter the wavelength, the higher the energy, E=hν

Now, the question arises, how does spectrophotometer know the concentration of unknown sample just by measuring the amount of light it absorbs. Here, the "laws of absorption" will play a crucial role and will clear all the doubts.

Laws of absorption:

Beer-Lambert Law (or Lambert-Beer Law or Beer’s law) states that there is a linear relationship between the absorbance and concentration of a sample. For this reason, Beer's law can only be applied when there is a linear relationship. The simple equation is:

                               A=εbc

where,

A - Absorbance (no unit)
ε - Molar absorptivity (Unit: Lmol^-1cm^-1) 

b - Path length of the sample (i.e.; the path length of the cuvette in which the sample is contained; unit: cm)
c - The concentration of the compound in the solution (Unit: mol L^-1)

Experimental measurements are usually made in  terms of transmittance which is:

                             T = I/Io                 

where,
I – Intensity of light after it passes though the sample.
Io – Initial intensity of the light.

The relation between A and T is:

 A = -log T = -log(I/Io)

The spectrophotometer displays either the % transmission or absorbance. Thus, unknown concentration of an analyte (sample) can be determined by measuring the amount of light the sample absorbs by applying Beer’s law. If the absorptivity coefficient is not known, then the unknown concentration can be determined by using a working curve of absorbance versus the concentrations derived from the standards. The graph on the left will make it clear.

Instrumentation and Mechanism:

Here, we will just see the outline of the instrumentation of the spectrophotometer. The basic structure of spectrophotometer is illustrated in the figure below. 

As we have seen above, the working of spectrophotometer is described as two instruments namely, 'spectrometer' and 'photometer'. Hence, here I will describe the role of each to make things easy to understand.
Spectrometer: It consists of a light source which produces a desired range of wavelength of light. Then there is a collimator which is a lens that will transmit a straight beam of light (photons). This beam of light will then pass through a monochromator which can be a prism or a grating that will spilt the light into several component wavelengths which will give rise to a spectrum. Then the slit will transmit only the desired wavelength (as in the figure, the desired wavelength being transmitted is in the range of yellow color).

Photometer: After the desired range of wavelength of light passes through the solution of a sample in cuvette, the detector or photocell detects the amount of photons that is absorbed and then sends a signal to galvanometer or a digital display.

I hope the basics and principles of spectrophotometer are clear to you.

In the next few posts, we will have a look at the different types of spectrophotometry like that of UV-visible spectrophotometry, IR spectrophotometry, fluorimetry.
Any doubts are welcome!