6 INTRODUCTION 



work of Paech and Tracey describes quantitative procedures for a large number of plant 

 constituents. Hamilton (9) has reviewed recent developments in biochemical analysis. 



Over the years many simple color tests and spot reactions have been developed to 

 indicate the presence of particular compounds or classes of compounds. Some of these 

 tests have proven themselves to be consistently specific and sensitive. Others, unfor- 

 tunately, have resulted in false conclusions which still persist in the literature. The 

 most useful spot tests and color reactions have been described, with their limitations in 

 the appropriate chapters. A complete coverage of such methods may be found in the 

 works of Feigl (10). 



Chromatographic methods are now the preferred way of characterizing compounds 

 or mixtures. By column chromatography, as mentioned under "Isolation", mixtures are 

 separated into fractions which may then be characterized by their physical and chemical 

 properties. The quantities involved may be large enough to permit several tests to be 

 performed on each fraction, and it is these tests rather than the chromatographic separa- 

 tion itself which are most important for characterization. In chromatography on paper 

 sheets the quantities of material involved are usually much smaller (e.g. 1-100 micro- 

 grams), only a few tests may be applied to the separated compounds, and the way that 

 they migrate in various solvent systems offers important information for characterizing 

 them. In thin layer chromatography, adsorbants with plaster of Paris or another binder 

 are spread on glass strips then used much in the same way as paper sheets. Separations 

 are much faster than on paper, however, and often better resolution is attained (11). 

 Gas chromatography permits fine separation of minute amounts of material and rapid 

 determination of the number of components in a mixture. Tentative identification of com- 

 pounds can be made by comparison of their retention times on the column with the times 

 of known materials. Final identification must, however, depend on isolation of a frac- 

 tion and its characterization by other techniques. Even compounds not ordinarily con- 

 sidered to be volatile may be separated by high temperature gas chromatography (12). 



There are several excellent books on chromatography. The newcomer to this field 

 is particularly referred to those by Williams (13) and Smith (14) and an article by Johnson 

 (14a). Other works (5, 15, 16, 17) offer extensive and detailed reviews of methods which 

 have been used for different classes of compounds. The special application of chroma- 

 tography to analysis of plants is described by Linskens (18) and by Thompson et al. , (19). 

 Heftmann (20) has reviewed recent developments in chromatography. In the chapters 

 which follow we have selected solvent mixtures and detection reagents which seem gen- 

 erally applicable to a class of compounds. Reference to the above books will usually re- 

 veal several other procedures that may be used once some indication is available regard- 

 ing the nature of the compounds to be characterized. The Rf value is a physical constant 

 for each compound and is defined as the distance from the starting point to which the com- 

 pound has migrated divided by the distance the solvent has migrated. Rf values vary with 

 temperature, direction of paper grain, amount of material applied to the paper, etc. 

 They therefore can not be relied upon without question for identification. Known com- 

 pounds should be run for comparison alongside of unknowns. In addition to specific detec- 

 tion reagents useful to indicate various types of compounds on paper chromatograms, 

 there are a few general reagents which detect almost any organic compound. Alkaline 

 silver nitrate is a common one of these, and its use is described in detail by Smith (14). 

 Other such general reagents are iodine vapor (21) and alkaline potassium permanganate 

 solution. 



One of the most important and widely used techniques for characterization of or- 

 ganic compounds is the measurement of absorption spectra by the use of photoelectric 

 spectrophotometers. The light absorption spectrum of a molecule is one of its most 

 distinctive properties, and excellent instruments are available which permit determina- 

 tion of this spectrum with very small quantities of material. In addition the material is 



