Wilson and Walker's Principles and Techniques of Biochemistry and Molecular Biology

INTRODUCTION

Spectroscopic techniques are based on the interaction of light with matter and probe certain features of a sample to learn about its consistency or structure. Light is electromagnetic radiation, a phenomenon exhibiting different energies. When light interacts with matter, it can emerge with other characteristics and properties, and therefore different molecular features of matter can be probed. Conceptually, matter can either annihilate (absorb) or create (emit) light (discussed in this chapter), or it can scatter light (discussed in the next chapter, Section 14.3.5). An understanding of the properties of electromagnetic radiation and its interaction with matter leads to an appreciation of the variety of types of spectrum and different spectroscopic techniques, and their applications to the solution of biological problems.

In this section, we introduce the basic principles of interaction of electromagnetic radiation with matter and then examine absorption and emission of light by matter and the corresponding molecular spectroscopic techniques of absorption (Sections 13.2, 13.3, 13.4) or emission (Sections 13.5, 13.6). Atomic spectroscopy (Section 13.7) is based on the same principles, but uses light of higher energy.

The applications considered use visible or UV light to probe consistency and conformational structure of biological molecules. Usually, these methods are the first analytical procedures used by a biochemical scientist, and are routinely employed in a variety of experimental approaches (e.g. enzyme kinetics, Section 13.8).

Properties of Electromagnetic Radiation

Electromagnetic radiation (Figure 13.1) is composed of an electric (E) and a perpendicular magnetic vector (M), each one oscillating in plane at right angles to the direction of propagation, resulting in a sine-like waveform of each, the E and the M vectors. The wavelength ƛ is the spatial distance between two consecutive peaks (one cycle) in the sinusoidal waveform and is measured in submultiples of metre, usually in nanometres (nm). The maximum length of the vector is called the amplitude. The frequency ν of the electromagnetic radiation is the number of oscillations made by the wave within the time frame of 1 s. It therefore has the units of 1 s −1 = 1 Hz. The frequency is related to the wavelength via the speed of light in vacuo, c = 2.998 × 10 8 m s −1 by