QUANTITATIVE ESTIMATION 57 



ment, as in the case of the casein in milk, no more accurate and 

 generally reliable method of procedure has yet been devised. 

 Unfortunately in many, if not in the majority of, instances it is 

 the fulfilment of this preliminary condition of complete separa- 

 tion from nitrogenous contaminations which presents the most 

 serious difficulties. For instance the most valuable reagent for 

 accomplishing the separation of various members of the globulin 

 group from one another and from other proteins is ammonium 

 sulphate, but the coagulum which is produced by this reagent, 

 necessarily employed in high concentration, is heavily contami- 

 nated by the ammonium salt and must be freed therefrom by 

 very prolonged dialysis before it can be utilized for a determination 

 of nitrogen. Prolonged dialysis, on the other hand, is not only 

 time-consuming but also involves the possibility of serious errors 

 arising from autohydrolysis or even hydrolysis due to enzymes 

 or to bacterial contamination, due to the protracted period during 

 which the labile protein must be exposed to the action of water. 

 Any alternative method of freeing the protein from contamina- 

 tion by ammonium salts, such as resolution followed by recoagu- 

 lation with some other reagent, is attended with many difficulties 

 and uncertainties attributable in large proportion to the modi- 

 fications of the actions of coagulating agents which are brought 

 about by the saline contamination which we desire to remove. 



The high combining-weight of the proteins attributable to the 

 mass of their molecules, is, as we have seen, the chief obstacle 

 to the application of chemical methods in their quantitative 

 estimation. In the application of physical methods for this pur- 

 pose, however, the bulk of the protein molecule, far from being 

 a disadvantage, is frequently a positive advantage and may indeed 

 enable us to employ physical methods of estimation not ordi- 

 narily applicable to the smaller molecules of the majority of 

 inorganic or the simpler classes of organic substances, for the 

 great majority of the physical properties of a substance are 

 determined by the mass or volume of its molecules and those 

 properties which are magnified by increasing mass of the molecule 

 are of course displayed exceptionally prominently by the proteins. 



It appears extremely probable therefore that in the future 

 we will come to rely more and more upon physical methods for 

 the estimation of the characteristically bulky molecules of col- 

 loidal substances. Many attempts have been made to estimate 



