ISOLATION AXl) IDENTIFICATION OF ANTIBIOTICS 



45 



It can also be carried out moi'e than once on 

 the same sheet of paper (two-dimensional 

 chromatography) . I'aper chromatography 

 can be used to compare the Ivf vahie of one 

 antibiotic with that of anothei- or to compare 

 the decomposition products or the deriva- 

 tives of one antibiotic with those of another. 

 The antibiotic can be located on the paper 

 chromatogram biologically (bioautogram) 

 or by chemical color reactions. Laboratories 

 actively engaged in the searcii for new anti- 

 biotics have developed a number of solvent 

 systems which permit separation of most of 

 the known antibiotics into small subgroups. 

 Despite the fact that such paper chroma- 

 tographic systems play an important role in 

 the characterization of antibiotics, it should 

 not be taken for granted that two substances 

 are identical if they have identical Rf values 

 in a lumiber of solvent systems. Neomycin B 

 and neomycin C, for example, were resolved 

 by paper chromatography only after being 

 acetylated (Pan and Dutcher, 11).")()). 



Electrophoresis 



Similar antibiotics may, in certain cases, 

 be separated from one another by submission 

 to a difference in electric potential in a 

 buffered environment. For the isolation and 

 characterization of antibiotics, electropho- 

 resis is carried out either in agar or on paper. 



Co un tercurre n t Distrib 1 1 Hon 



Countercurrent distribution is another 

 method that can be used for both purification 

 and characterization of antibiotics. Similar 

 in principle to partition chromatography, 

 this method permits separation of substances 

 as the result of differences in partition coeffi- 

 cients betw^eeii two immiscible solvent sys- 

 tems. More laborious than chromatography, 

 the method recjuires costly instruments. 

 Countercurrent distril)ution is often used as 

 a criterion of purity, especially for anti- 

 l)iotics that cannot be crystallized. 



Elementary A ncilysis 



The elementary analysis of a pure prepara- 

 tion of antibiotic is, of course, necessary for 

 the determination of its empirical formula. 

 An elementary analysis of crude prepara- 

 tions of antibiotics can also be most useful for 

 characterization. Its usefulness is limited, 

 however, primarily to the negative results 

 obtained: such analyses can show, for 

 example, that a substance crjutains no 

 halogens, no nitrogen, or no sulfur. 



Ehijsical ( 'onstants 



Once a new or unknown antil)i()tic has 

 been obtained in a pure form it can then be 

 characterized on the basis of its physical 

 constants — such as density, refractive index, 

 molecular weight, melting point, and optical 

 rotation — and the physical constants of its 

 salts and derivatives. 



Eventually, the substance is fully charac- 

 terized when its exact chemical structiu'e is 

 known in all its spatial intricacies. 



Classification of Antibiotics of Actino- 

 niycetes 



The antibiotics of actinomycetes represent 

 a great variety of chemical compounds. They 

 vary greatly in their physical properties, 

 including stability to heat and to oxidation, 

 and in their solubility in water and organic 

 solvents. They vary also in their chemical 

 composition, in their antimicrobial activities, 

 and in their toxicity to animals. These differ- 

 ences are frec^uently not only c[ualitative but 

 also (luantitative in nature. Hence, it is 

 difhcult to classify them. However, various 

 systems for the classification of antibiotics 

 have been proposed. 



Several criteria ha\'e been used in estab- 

 lishing such systems. These are based upon 

 (1) the nature of the organisms that form 

 the antibiotics, (2) the specific antimicrobial 

 spectra or selective action of the antibiotics 

 upon different groups of microorganisms, (3) 



