SPECTROSCOPY

1. Introduction

A variety of the instruments that are commonly used to analyze food materials are based on spectroscopy, e.g., UV-visible, fluorescence, atomic, infrared and nuclear magnetic resonance spectroscopies. These instruments utilize interactions between electromagnetic radiation and matter to provide information about food properties, e.g., molecular composition, structure, dynamics and interactions. An appreciation of the operating principles of these instruments depends on an understanding of the distribution of energy within atoms and molecules, of the characteristics of electromagnetic radiation, and of the interaction of electromagnetic radiation with atoms and molecules.

2. Distribution of Energy in Atoms and Molecules

Atoms and molecules can only exist in a limited number of discrete energy levels: they cannot have energies between these levels, i.e., their energy levels are quantized. Each molecular species has a unique set of energy levels that depends on its unique atomic structure (electrons, protons, neutrons) and molecular structure (type and arrangement of atoms and bonds). The lowest of these energy levels is referred to as the ground state, while higher levels are referred to as excited states. The potential energy of an atom or molecule is usually defined relative to the ground state (which is arbitrarily taken to have zero energy). The potential energy of a molecule is made-up of contributions from a number of different sources: electronic, vibrational, rotational, translation and nuclear.

· Electronic Energy Levels. Electrons in an atom are arranged into a number of different shells and sub-shells. An electron can move from one of these sub-shell levels to another by absorbing or emitting radiation of an appropriate energy. The system is then said to have undergone an electronic transition. Electronic transitions may involve electrons that are in inner shells (higher energy) or outer shells (lower energy) of atoms.

· Vibrational Energy Levels. Molecules (but not atoms) can vibrate in a number of different modes, e.g., the atoms can compress or stretch along the axis of a bond, or they can bend symmetrically or asymmetrically. Each of these vibrations occurs at a characteristic frequency (energy) which depends on the mass of the atoms and the strength of the bonds involved.

· Rotational Energy Levels. Molecules often contain chemical groups that are capable of rotating around certain bonds at fixed frequencies (and therefore energies). Each group has a specific number of frequencies at which it rotates and therefore has a specific number of quantized rotational energy levels. The rotation frequency is determined by the mass of the atoms involved and their distance from the axis of rotation.

· Nuclear Energy Levels. The nuclei of certain atoms have a property known as spin. A (charged) spinning nucleus generates a small magnetic field and can be thought of as being a small magnet. Normally, this magnet can be orientated in any direction, but in the presence of an external magnetic field it can only align itself either with or against the field, i.e., it is quantized. Transitions between the different energy levels within the nuclei can be made to occur by applying radiation of a specific energy to the sample.

· Translational Energy Levels. Atoms and molecules are in continual translational motion because of the thermal energy of the system. Translational energy levels are quantized, however, the differences between the energy levels are so small that the molecules act as though the energy is distributed continuously.

3. Characteristics of Electromagnetic Waves

Electromagnetic waves may be thought of as particles of energy (photons) that move through space with wave-like properties, i.e., they exhibit wave-particle duality. They consist of oscillating electric and magnetic fields that are perpendicular to one another, and to the direction of propagation. The sinusoidal variation in the amplitude of the electric vector of the wave can be plotted as a function of time (at a fixed position within a material) or as a function of distance (at a fixed point in time). A monochromatic (single wavelength) electromagnetic wave that propagates through a vacuum can be described completely by its frequency, wavelength and amplitude (or parameters derived from these):

• The frequency (v) of a wave is the number of cycles per second (Hz = s^-1).
• The period (T) of a wave is the time taken to complete a cycle: T = 1/v.
• The wavelength (l) is the distance between successive maxima of a wave.
• The wave number () is the number of cycles per unit distance (=1/l).
• The amplitude (A) of a wave is the maximum magnitude of the electric vector.
• The intensity (I) of a wave is proportional to the square of the amplitude. It is the amount of energy passing through a given area per second. Increasing the intensity of an electromagnetic wave increases the number of quanta passing a given area per second, not the energy of each individual quantum.
• The velocity (c) of an electromagnetic wave is the distance traveled per second: c = vl. The velocity of an electromagnetic wave travelling through a vacuum is c = 3 x 10⁸ m s^-1. The velocity of an electromagnetic wave travelling through a material is always less than that in a vacuum. The refractive index of a material is equal to c_vacuum/c_material.
• The energy (E) of the photons in an electromagnetic wave is related to the frequency of the wave:

E = hv = h/T = hc/l = hc

where, h = Planks constant (6.6262 x 10^-34 J s). These expressions can be used to relate the energy of an electromagnetic wave to its frequency, period, wavelength or wave number. This relationship indicates that monochromatic radiation (i.e., radiation of a single frequency) contains photons that all have the same energy.

The electromagnetic spectrum consists of radiation that ranges in wavelength from 10^-12 m (high energy) to 10⁴ m (low energy). The physical principles and mathematical description of radiation across the whole of the electromagnetic spectrum is the same, however, it is convenient to divide it into a number of different regions depending on the origin of the waves, i.e., cosmic rays, gamma rays, x-rays, ultraviolet, visible, infrared, microwaves, and radio waves.

4. Interaction of Radiation with Matter

Spectroscopic techniques utilize the fact that atoms and molecules have a discrete set of energy levels and that transitions can only occur between them. When an electromagnetic wave propagates through a material the atoms or molecules can absorb energy and move to an excited state if the photons in the wave have energies that are exactly equal to the difference between two energy levels (DE = hv). Alternatively, if an excited atom or molecule emits energy in the form of radiation the waves emitted must have energies that are exactly equal to the difference between two energy levels (DE = hv). The energy of the photons in different regions of the electromagnetic spectrum corresponds to different types of energetic transition that can occur in atoms and molecules, e.g., electronic, rotational, vibrational, translational, nuclear transitions. Electromagnetic radiation can therefore be used to probe different molecular characteristics of matter. The atomic or molecular origin of the transitions that occur between different energy levels in matter, the region of the electromagnetic spectrum that these transitions correspond to, and the spectroscopic techniques that can be used to measure these transitions are summarized below:

Transition Region of e/m spectrum Spectroscopy technique

Electronic (1000 kJ mol^-1) UV-Visible UV, Visible, Atomic, Fluorescence

Vibrational (10 kJ mol^-1) Near and Mid Infrared Infrared

Rotational (0.1 kJ mol^-1) Far Infrared, Microwaves Infrared, microwave

Nuclear (10-6 kJ mol^-1) Radio waves Nuclear magnetic resonance (NMR)

The difference between electronic energy levels is greater than between vibrational energy levels, which is greater than between rotational energy levels. Thus higher energy radiation (shorter wavelength) is needed to cause transitions between electronic levels than between vibrational or rotational levels. In practice, a molecule can be thought of as having a number of different electronic energy levels, with rotational and vibrational energy levels superimposed on them.

Absorption

Absorption is the process by which energy is transferred from an electromagnetic wave to an atom or molecule and causes it to move to an excited state. Absorption can only occur when an atom or molecule absorbs a photon of light that has an energy which exactly corresponds to the difference between two energy levels, i.e., it must be quantized. At room temperature the ground state of atoms and molecules is usually the one which is most highly populated and so transitions usually occur from the ground state to higher energy levels. At higher temperatures, more of the higher energy levels are occupied and so, transitions between higher energy levels may also become important.

If an atom or molecule is subjected to electromagnetic radiation of different wavelengths (energies) it will only absorb photons at those wavelengths which correspond to exact differences between two different energy levels within the material. A plot of the fraction of photons absorbed at a particular wavelength versus the energy of the photons at that wavelength is called an absorption spectrum. Conventionally, the axes of absorption spectra are specified in terms of easily measurable quantities: x-axis ® transmittance or absorbance (rather than fraction of photons absorbed); y-axis ® wavelength, frequency or wave number (rather than photon energy).

Emission

Emission of radiation is the reverse of absorption, occurring when energy from an atom or molecule is released in the form of a photon of radiation. When a molecule is raised to an excited state it will only exist in this state for a very short time before relaxing back to the ground state. This is because it will always try to move to its lowest energy state. There are two important relaxation processes through which an excited molecule can dissipate its energy:

• Non-radiative decay. This is the most common way that an excited molecule loses its energy. Energy is dissipated in a number of small (quantized) steps due to transfer of energy from the exited molecule to surrounding molecules in the form of kinetic energy (heat). Nevertheless, the heat generated is usually so small that it has little effect on the overall temperature of the system.
• Radiative decay. In some cases an atom or molecule loses its energy in the form of a photon (emission). This is the case in atomic emission spectroscopy.

Sometimes both of these processes occur together. In fluorescence spectroscopy, a molecule absorbs electromagnetic radiation, which causes it to move into an excited state. It then returns to the ground state by dissipating some of its energy in the form of non-radiative decay and the rest in the form of a photon of radiation. The photon emitted is therefore of lower energy (longer wavelength) than the incident wave. Usually, an electron decays to the lowest energy level in the excited electronic state, and then returns to the ground state.

5. Measurement Modes

The design of an analytical instrument based on spectroscopy depends on the nature of the energetic transitions involved (e.g., electronic, vibration, rotation, translation, nuclear), the nature of the radiative process involved (e.g., absorption, emission, fluorescence) and the nature of the food matrix (e.g., absorbing or non-absorbing). These factors determine the wavelength (frequency) of electromagnetic radiation used, the way that the electromagnetic radiation is generated and the way that the electromagnetic radiation is detected. Some commonly used designs are highlighted below:

· Emission. The sample being analyzed is energetically stimulated (e.g., by heating or application of radiation) and the amount of electromagnetic radiation produced by the sample is measured at different wavelengths, e.g., atomic emission spectroscopy, NMR, fluorescence.

· Transmission. An electromagnetic wave generated by the analytical instrument is propagated directly through the sample and the reduction in its amplitude due to interaction with the sample is measured at different wavelengths, e.g., atomic absorption spectroscopy, infrared transmission measurements, UV-visible spectrophotometery.

· Reflection. An electromagnetic wave generated by the analytical instrument is reflected from the surface of the sample and the reduction in its amplitude due to interaction with the sample is measured at different wavelengths, e.g., infrared reflection measurements, color measurements.

6. Spectroscopic Analysis

Quantitative Analysis

One of the most important applications of the interaction between electromagnetic radiation and matter is the determination of the concentration of certain components in foods. This application relies on there being a relationship between the amount of radiation absorbed by a material and the concentration of the components present. The power (P) of an electromagnetic wave exiting a solution is less than the power entering the solution (P₀), because solute molecules absorb some of the energy. The amount of energy absorbed is usually expressed in terms of either the transmittance or the absorbance. The transmittance is simply the ratio of the exiting and incoming radiation: T = P/P₀ , and is often expressed as a percentage %T = (P/P₀) ´ 100. Unfortunately, T or %T are not proportional to the concentration of the absorbing species and so another parameter, known as the absorbance A, has been defined which is proportional to the concentration: A = -log (P/P₀) = -log T. In dilute solutions the absorbance is proportional to the concentration of the absorbing species, which is extremely convenient for quantitative analysis of concentration. The relationship between the absorbance of a solution and its concentration is known as Beer's Law.

A = abc

Here a is a constant called the absorptivity which depends on the molecular properties of the absorbing species and the wavelength of the radiation, b is the pathlength of the sample and c is the concentration of the sample.

Qualitative Analysis

Spectroscopy techniques can also be used to provide valuable information about the type, structure and environment of molecules present in food materials.

• Atomic or Molecular Type. Each type of atom or molecule has a unique set of energy levels and therefore a unique electromagnetic spectrum. By measuring the electromagnetic spectrum of a material and identifying the magnitude and position of the absorption or emission peaks it is often possible to determine the type of atoms or molecules present.
• Molecular Structure. Certain kinds of molecular groups have characteristic absorption or emission peaks in specific regions of the electromagnetic spectrum. For example, certain kinds of molecular groups give absorption peaks at specific wavelengths in an infrared spectrum, while the number, type and organization of atoms and bonds within a molecule leads to characteristic absorption peaks in a NMR spectrum. It is therefore possible to obtain important information about the structure of molecules by measuring their electromagnetic spectra.
• Molecular Environment. Spectroscopy techniques can also be used to provide information about the molecular environment of atoms and molecules within a sample. The absorption or emission of energy between two energy levels within a specific atom or molecule is influenced by the presence of other atoms and molecules in their immediate vicinity. Consequently, if the molecular environment of an atom or molecule within a sample is altered, then its absorption or emission spectra may change. Spectroscopy can therefore be used to monitor physicochemical changes that result in an alteration in the molecular environment of atoms and molecules, e.g., protein unfolding, solubilization, or aggregation.

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