364 METHODS OF ANALYSIS [Chap. 



a refrigerator. (Solid pepsin preparations may often be extracted with hydro- 

 chloric acid of appropriate strength and prepared for assay in the same manner.) 



(b) Dilute comparison solution of the sample. — Add 1 cc. of N/10 hydrochloric 

 acid to 9 cc. of the dilute solution of the sample. 



(C) Dilute inactive solution of the sample. — Immerse a stoppered glass vessel, con- 

 taining 45 cc. of the dilute solution of the sample and 5 cc. of N/10 hydrochloric 

 acid, in boiling water for 15 minutes and filter. 



(d) Standard solution containing 0.5 mg. of active U. S. P. pepsin per cc. — Im- 

 merse a stoppered test tube containing 18 cc. of the dilute solution of the sample 

 in boiling water for 10 minutes and, after cooling, add 2 cc. of the standard pepsin 

 solution, containing 5 mg. of pepsin per cc, and filter if necessary. 



If the solutions to be tested are not clear, filter through hardened filters. If, 

 however, they cannot thus be clarified, make check comparison tubes containing 

 the same amounts of the preparation made up in the same way with 2 cc. of water 

 in place of the ricin solution used in the determination. 



40 DETERMINATION. 



To each of 15 tubes, add from a burette 2 cc. of the ricin solution and 0.5 cc. of 

 N/10 hydrochloric acid, heat to 37.5°C. and add the following quantities of the 

 solutions: 



To the first 5 tubes, add 0.00-1.00 cc. of the dilute comparison solution of the 

 sample in 0.25 cc. increments, and 1.00-0.00 cc. of the dilute inactive solution of 

 the sample in 0.25 cc. decrements. To the next 5 tubes, add 1.00-0.00 cc. of the 

 dilute inactive solution of the sample in 0.25 cc. decrements and 0.00-1.00 cc. of the 

 standard solution containing 0.5 mg. of active U. S. P. pepsin per cc. in 0.25 cc. 

 increments. To the last 5 tubes, add 1.00-0.00 cc. of N/10 hydrochloric acid in 

 0.25 cc. decrements and 0.00-1.00 cc. of the standard pepsin solution containing 0.5 

 mg. of pepsin per cc. in 0.25 cc. increments. 



By comparing any tube of the first group of 5 with the tubes in the remaining 

 groups the degree of proteolytic activity of the dilute comparison solution of the 

 sample may be matched against known amounts of standard pepsin both in ordi- 

 nary acid medimn, last group of 5, and in the same medium as the sample itself, 

 second group. 



Introduce the acid and the dilute inactive solution of the sample into the tubes 

 first and then pour in the solutions to be tested as rapidly as possible from gradu- 

 ated pipettes, noting the total time consumed in the process after adding the pepsin. 



After the addition of the solution to be tested, again immerse the test tubes in 

 the 37.5°C. bath, preferably arranged in corresponding order in a partitioned square 

 or oblong wire rack, such as is used in bacteriological work. Shake and examine the 

 tubes from time to time for 1-2 hours, noting the time when the digestion begins 

 and ends. In case of very weak solutions they may be allowed to digest over- 

 night. 



If the rate of digestion is the same in each group, the dilute comparison solution 

 of the sample contains exactly 0.5 mg. of pepsin per cc. If the rate is more rapid 

 in the first group than in the others, it is stronger, the comparative strength being 

 closely indicated by the time of action in the tube containing less of the solution. 

 If the rate of clearing is more rapid in the last group than in the second, some in- 

 terfering substance is present and must be removed by dialysis, or by evaporation 

 in vacuo at a low temperature until, upon re-examination and further dilution or 

 concentration, the rate of digestion is identical or nearly so in each series. 



Smaller quantities of pepsin may be determined in the same way by comparing 



