INFRARED SPECTROSCOPY

energy increments (Es) of lower value than those involved in electronic transitions correspond to frequencies in the infrared part of the spectrum (i.e. = 103105 nm;). Vibrational energy levels are quantized in the same way as electronic energy levels and, if infrared light of a frequency corresponding exactly to the E between two vibrational energy levels is absorbed by a molecule; it may undergo a vibrational transition.

Figure Non-electronic transitions. (a) Vibrational transitions involve alternating between a number of allowed bond lengths and bond angles, of differing energy. The energy increment between vibrational energy levels is denoted by E. (b) Rotational transitions. Atoms bonded together can rotate relative to each other. The different rotation states have distinct energies and the energy increment between them is denoted by E. the various vibrational energy levels are mainly due to differences in bond length and angle which are possible due to stretching and bending of bonds. 1 Physical Basis of Infrared Spectroscopy When atoms come together to form a covalent bond, they undergo an electronic rearrangement which involves two competing sets of forces. The positively-charged nuclei of the two atoms tend to repel each other (electrostatic repulsion) while the nucleus of each atom is attracted to the negatively-charged electrons of the other (electrostatic attraction). The mean distance settled on between the atoms (i.e. the bond length) is a reflection of a point of balance between these competing attractive and repulsive forces and, in general, will be characteristic for a particular

chemical bond. For example, in polypeptides CC bonds are 1.52 A while NC bonds are 1.45A . It is possible to plot the energy of a molecule as a function of the interatom distance, r, in a Morse diagram (Figure 3.41).

Most molecules in a population at rest are at the minimum of this diagram, but individual bond lengths can vary by0.5 A and bond angles by 5 at room temperature.We can think of each chemical bond, therefore as existing in a particular vibrational energy level characterized by a particular bond length, bond angle and electron density.

However, if radiation of an appropriate frequency is passed through the sample, it is possible for the molecule to undergo a transition to a higher vibrational energy level by absorption of radiation. As described in Section 3.2.2, vibrational energy levels are quantized in the same way as electronic energy levels although the E values associated with vibrational transitions are much smaller than those associated with electronic transitions. A vibrational transition from the ground state to the first excited state due to absorption of infrared light is called a fundamental absorption and the frequency, , associated with this is called the fundamental frequency. Whilst other transitions are also possible (e.g. from the ground to the second or third excited state), they occur much less frequently and represent weak absorbance. An infrared spectrum consists of a plot of absorbance versus frequency or wavenumber (1/). In comparison with absorbance spectra in the ultraviolet/visible range, infrared spectra of small molecules consist of narrowlines rather than broad peaks (Figure 3.42). However, because of the large number and variety of bonds in macromolecules, infrared spectra of proteins and DNA consist of a small number of broad peaks. Water gives a strong infrared spectrum because of its high absorption coefficient in the infrared range and high concentration (55 M). For this reason, infrared spectroscopy may be carried out either on nonaqueous samples prepared as dry films or on samples dissolved in alternative solvents such as D2O or chloroform. It is also possible to subtract the spectrum due to the water solvent by difference spectroscopy (Figure 3.15) but this requires high concentrations of protein or DNA (520%). These conditions are generally unsuitable for the study of biomacromolecules in their native state and this limits the practical application of simple infrared spectroscopy to their study. However, the technique has become standard for the study of low molecular mass biomolecules and yields structural information complementary to that available from other methods such as mass spectrometry (Chapter 4), CD (Section 3.5) and NMR(Section 3.7). Study of biomacromolecules by infrared spectroscopy requires more elaborate techniques which are described below (Sections 3.6.4 and 3.6.5, respectively). Equipment Used in Infrared Spectroscopy The overall design of an infrared spectrophotometer is very similar to that of the ultraviolet/visible spectrophotometer shown in Figure 3.9. The main differences are in the technology used for diffracting light and for detection of transmitted light. The monochromator used in an infrared spectrophotometer is usually a diffraction grating. It will be recalled that either a prism or a diffraction grating may be used in the ultraviolet-visible spectrophotometer. The detector component of an infrared spectrophotometer is a thermocouple rather than a photocell normally used in a ultraviolet-visible spectrophotometer. This basic infrared spectrophotometer

suitable for collecting spectra from small molecules is called a dispersive or grating infrared spectrophotometer. Uses of Infrared Spectroscopy in Structure Determination In principle, any chemical bond of a given type (e.g. CH) might be expected to have a fundamental absorption identical to that of any other bond of the same type. In practice, the chemical environment of the bond has an effect on the precise frequency of absorption, since this may alter the bonds electron density. Studies of infrared absorption from a large number of molecules of known chemical structure suggest that absorbance of infrared light near particular frequencies are characteristic for specific chemical groups (Table 3.5). These group frequencies allowus to determine aspects of the molecular structure of small molecules from their infrared absorbance pattern alone. It should be noted that many of the chemical groups detectable by infrared spectroscopy give little or no absorbance in the ultraviolet or visible parts of the spectrum. A further advantage of infrared spectroscopy is the variety of sample forms which can be analysed. It is possible to obtain infrared spectra of solutions, films or powders, and of crystalline samples. While dispersive infrared spectroscopy has limited applicability to the study of biopolymers generally, it has found some applications in structural studies on DNA. The formation of hydrogen bonds and tautomerization between C=O (keto) and COH (enol) forms of nucleotide bases have detectable effects on infrared spectra.

Ref Wiley.Physical.Biochemistry.Principles.And.Applications.2nd.Edition.