60 CHEMICAL STATICS 



ether. Correction must be made for this change of volume in 

 the estimate of protein-content. 



In order to obtain the casein-content of the milk, the casein 

 from a fresh portion of milk is precipitated in accordance with the 

 "official" method by the addition of acetic acid, and the opacity 

 of the suspension obtained in an aliquot part of the filtrate, by 

 adding four volumes of 3 per cent sulphosalicylic acid solution, 

 is determined nephelometrically. This determination yields an 

 estimate of the "globulin and albumin" fraction which, sub- 

 tracted from the "total proteins" estimated above, yields the 

 amount of casein precipitable by the "official" method. 



A modification of this method of protein estimation has been 

 utilized by Pfeiffer, Kober and Field (10) in the estimation of 

 the globulins and total proteins in cerebrospinal fluid. 



It will be seen that the chief difficulty attaching to the nephelo- 

 metric method arises from the necessity of achieving constant 

 conditions of coagulation, a difficulty which is especially serious 

 when the coagulating process has to be superadded to the pro- 

 cedures incident to the separation from one another of the vari- 

 ous proteins in a mixture in which it is desired to determine 

 each protein separately. This difficulty does not attach to the 

 refractometric method described below, in which, once the sepa- 

 ration of the various protein fractions has been achieved, no 

 further procedures other than dilution, etc., are necessitated 

 before a determination can be made. On the other hand, the 

 refractometric method does not permit the extreme accuracy 

 which may be attained with the nephelometric method under 

 favorable conditions. 



3. The Refractometric Method of Estimating Proteins. — 

 This method, originally employed by Reiss (11) (12) (13) (14) 

 (15) (16) for the estimation of the total proteins in blood-serum, 

 has been modified by the author so as to render it applicable to 

 the separate determination of the non-proteins, globulins, albu- 

 mins and total proteins in a little over 1.5 cc. of blood-serum (24). 

 The following are the details of the method: 



(a) Estimation of the non-proteins. 



Glass tubes 25 cm. long, having an inside diameter of 5 mm. and 

 walls about 1 mm. thick, are sealed at one end. It is well to blow 

 gently into the tube while the sealed end is still soft, thus making 

 the contour of the bottom of the tube hemispherical and dimin- 



