318 PHYSIOLOGICAL CHEMISTRY 



(b) Plasma Bicarbonate (Titration Method) Van Slyke, Stillman 

 and Cullen. 1 Principle. Plasma is treated with an excess of standard 

 acid which is titrated back with standard alkali under carefully stand- 

 ardized conditions. 



Procedure. In drawing and centrifugating the blood the precautions outlined 

 by Van Slyke and Cullen for preventing loss or accumulation of CO 2 and consequent 

 change in the distribution of bicarbonate between corpuscles and plasma, are to be 

 observed. Oxalate plasma is used. Up to the beginning of the analysis, the blood 

 and plasma are handled exactly as described for the CO 2 method. Pipette 2 c.c, 

 of plasma into a 150-200 c.c. round-bottomed flask and add 5 c.c. of 0.02 N HC1 

 (this is about 2 c.c. in excess of the bicarbonate normally present). Shake the 

 flask vigorously with a rotary motion so that the solution is whirled in a thin layer 

 about the inner wall. One minute of this treatment is sufficient completely to 

 remove the-CO 2 set free by the acid. Pour the solution as completely as possible 

 into a 50 c.c. Erlenmeyer flask and rinse the walls of the larger flask with 20 c.c. 

 of H 2 O measured within i c.c. in a cylinder, using a third for each of 3 washings. 

 Add 0.3 c.c. of o.i per cent solution of neutral red in 50 per cent alcohol. Run in 

 0.02 N carbonate- free NaOH from a burette (preferably but not necessarily a 

 "microburette") until the color of the solution matches that of 29 c.c. of a standard 

 phosphate solution of p ff y.4 2 contained in a similar 50 c.c. flask. In place of 

 neutral red, 0.3 c.c. of a 0.04 per cent solution of phenolsulphonephthalein may be 

 used and gives an end point slightly more easy to distinguish. When it is used, 

 however, the standard phosphate solution must be of p# 7.2 instead of 7.4. With 

 both indicators, a peculiar phenomenon occurs as the end point is approached. 

 Each drop appears to change the color past the end point, but within a few seconds 

 the color shifts back and it is seen that at least another drop is needed. Con- 

 sequently, the final color comparison should not be made until at least 30 seconds 

 after the last drop of 0.02 N NaOH has been added. It is better, particularly with 

 neutral red, to overrun the end point by a drop, rather than stop short of it when 

 in doubt. In calculating the results the c.c. of 0.02 N NaOH used in the titration 

 is subtracted from the c.c. required to neutralize to the same indicator 5 c.c. of the 

 0.02 N HC1 used. The number is approximately 5 but usually varies from it 

 slightly because of difference in factors of acid and alkali and because of the calibra- 

 tion error of the 5 c.c. pipette used in measuring the acid. The maximum accuracy 

 is obtained by performing a preliminary titration on 5 c.c. of the acid plus 20 c.c. of 

 distilled water, using the same pipette, indicator, and end point as in the plasma 

 titration. The titration result represents c.c. of o.oi M NaHCO 3 per c.c. of plasma 

 and it is transformed into terms of molecular concentration of NaHCOa by merely 

 dividing by 100. If the NaHCOs molecular concentration is multiplied by 2240 or 

 the number of c.c. of 0.02 N HC1 used in the titration by 22.4, the volume per cent 

 of bicarbonate CO 2 in the plasma is obtained and the results can thus be compared 



1 Van Slyke, Stillman and Cullen: Jour. Biol. Chem., 38, 167, 1919. 



2 To obtain the standard solutions of p#7.2 and 7.4 proceed as follows: for p#7.2 mix 

 50 c.c. of 0.2 M KH 2 PO 4 (27.23 g. per L.) and 35 c.c. of 0.2 N NaOH and dilute to 200 c.c.; 

 for p# 7.4, mitf 50 c.c. of 0.2 M KH 2 PO 4 and 39.5 c.c. of 0.2 N NaOH and dilute to 200 c.c. 

 To prepare the solutions by Sorenson's method, dissolve 6.89 g. Na 2 HPO 4 and 247 g. of 

 KH 2 PO 4 in H 2 O and dilute to i liter for p#y.2; for P#y.4 the amounts are 7.72 g. and 1.67 

 g., respectively (if Na 2 HPO 4 -2H 2 O is used instead of the anhydrous salt, the amounts are 

 increased to 8.66 and 9.67 g.). Both salts prepared especially for this purpose may be 

 obtained from Merck & Co. 



