PRINCIPLES OF TRACER METHODOLOGY 17 



This means that many methods may now be used which in the past 

 showed some advantages but were discarded because of poor or uncertain 

 recoveries. Likewise many procedures may be shortened because it will 

 not be necessary to accomplish complete collection of precipitates and 

 complete electroplating or to attain quantitative transfers from one vessel 

 to another throughout a complex determination. The advantage of the 

 isotope-dilution method used in this way will depend upon the added 

 burden of the isotope incorporation and measurement, as compared with 

 the saving in the elimination of the necessity for quantitative recoveries. 



The fact that the isotope-dilution method does not require quantitative 

 isolation of the substance to be measured is of particular interest in sys- 

 tems where purity and recovery are mutually exclusive. The assays for 

 amino acids in a protein hj'-drolysate or for individual fatty acids fall into 

 this category because there are so many chemically similar substances 

 present in each case. Assays for these materials are performed in the 

 same manner as described above for elements. However, they require 

 the availability of the individual substance with an appropriate label. 

 These methods have been especially effective with stable isotopes, and 

 general equations have been reported (61). 



Modifications of Isotope Dilution. The dilution methods so far 

 described involve the addition of the isotopically labeled substance to the 

 analytical sample. If the unknown in the sample can be produced in 

 labeled form, then the procedure can be carried out according to the same 

 principles and methods by adding a known amount of the unlabeled sub- 

 stance. Two advantages of this inverse method over the direct method are 

 that smaller amounts of the unknown can be determined and that it is 

 not necessary to have available the labeled diluting substance. A con- 

 venient form of the equation is 



^ {M){SA) 



SA* - SA ^^ ^^ 



where M* = unknown mass of labeled substance in sample 



M ^ known mass of normal substance added to sample 

 SA* = measured specific activity of substance isolated from orig- 

 inal sample 

 SA = measured specific activity of substance isolated from mix- 

 ture of sample and added normal substance 

 This procedure may be illustrated as follows: Assume that a plant has 

 been treated with a radioisotope known to become incorporated in an 

 amino acid A, for which an analysis is desired. Many other amino acids 

 besides A could become labeled, but this will not interfere, for presumably 

 A can be isolated and highly purified, since yield can be sacrificed. Let 

 us suppose that 1 mg of the labeled amino acid A is isolated from 10 g of 



