INTRODUCTION I 



not destroyed by making such measurements, and may be used later for other tests. 

 Often in recent years the combination of chromatography with spectrophotometry has 

 made possible the complete fractionation of a very small quantity of some natural mix- 

 tures and the unequivocal identification of each component. 



Three types of absorption spectra are commonly distinguished, infrared, visible, 

 and ultraviolet. Absorption of radiation in the infrared region depends on the vibration 

 and rotation of atoms in the molecule. Rotational spectra have been little studied, and 

 for practical purposes the infrared spectra which are normally measured are entirely 

 due to vibration. Different atomic groups can vibrate only at specific frequencies and 

 absorb radiation of just these frequencies. These absorption bonds appear in the region 

 of wavelengths from 2 - 100 microns, but most instruments cover this range only up to 

 about 25|U. From about 2-8fi the absorption bonds observed are highly characteristic 

 certain atomic groups and therefore give good indications as to what functional groups 

 are present. For example, hydroxyl groups absorb at about 2. 8ju, carbonyl groups at 

 5. Sji, nitrile groups at 4. 4jj,, etc. The region above 8/j is referred to as the "finger- 

 print region" since it is unique for the molecule as a whole rather than for specific groups. 

 Frequently the positions of infrared absorption bands are expressed as wavenumbers 

 rather than wavelengths. The wavenumber is the reciprocal of the wavelength expressed 

 in centimeters (1 cm. = 10, 000|U ). The identity of the infrared spectrum of a pure, un- 

 known compound with that of a known sample may be taken (with rare exceptions) as proof 

 that the two compounds are identical. 



Ultraviolet and visible absorption spectra depend not on the vibration of atoms in 

 a molecule but on the fact that certain loosely-held electrons may be raised to higher 

 energy levels by absorbing radiation of specific wavelengths. For this reason ultraviolet 

 and visible spectra may be lumped together as electronic spectra. There is no theoretical 

 difference between them. Since loosely-held electrons are required for absorption to 

 occur, molecules with unsaturated bonds are the ones which absorb in this region. How- 

 ever, specific absorption bands do not indicate specific functional groups as in infrared 

 spectra, rather they are more characteristic of the molecule as a whole. They may often 

 permit decisions to be made as to the class of compounds involved (e. g. an anthocyanin 

 vs. a naphthoquinone) but give little indication as to details of structure. The common 

 commercial instruments permit spectral measurements to be made over the ranges 200 - 

 400 m/j (ultraviolet) and 400 - 750 m|U (visible). Observations in the near infrared (750 - 

 2000 m/j.) can be made but are seldom useful. While electronic spectra are not as impor- 

 tant as infrared for identification or structural determination of a molecule, they have 



other important advantages usually smaller amounts of material are required; it is 



easier to determine the quantity of a substance which is present; and many solvents are 

 available which do not absorb in the ultraviolet - visible regions. 



There is no space here for a more thorough description of spectrophotometric 

 theory and procedures. General discussions of both types of spectra may be found in the 

 articles of Miller (22) and Glover (23) or the book of Oster and Pollister (24). Infrared 

 spectroscopy is the subject of several excellent books (25, 26); and the infrared spectra 

 of natural products are discussed by Cole (27). Comprehensive catalogs of spectral data 

 are also available (28-34). 



With the advent of commercial instruments the application of nuclear magnetic 

 resonance spectra to the characterization of natural products will undoubtedly increase. 

 As yet, however, only a few plant constituents have had their structures elucidated by 

 this technique; and since a brief explanation is impossible, we wish merely to call atten- 

 tion to the method. Nuclear magnetic resonance (NMR) spectra are dependent on the ab- 

 sorption of radio frequency signals on exposure of certain atomic nuclei to a radio fre- 

 quency field and a strong magnetic field. Of the so-called magnetic nuclei which respond 

 in this way almost the only one of interest in natural products is hydrogen. Therefore 



