V O L U M E 23, NO. 12, D E C E M B E R 1 9 5 1 3.5 to 5.0 (8). Within these limits dithizone, normally a bright emerald green, rapidly turns pink in the presence of zinc ions. The pH value of the solution must be carefully controlled during both titration of the unknown zinc solution and standardization of the ferrocyanide solution. As both dithizone and its metallic derivatives are very soluble in chloiinated organic solvents such as carhon tetrarhloride, but practically insoluble in water, localizatiori of the color in droplets intensifies the color change at the pnd point. This a l l o ~ sgreater color contrast than is possiblr u h e n the indicator is dispersed throughout the entire solution. .Is the time required for the color change from green t o pink tieponds upon the concentration of the zinc solution, it is advisable to niake a preliminary test hg adding 1 drop of the zinc solution to 50 ml. of the phthalate buffer solution, placing 1 drop of the indicator solution in the mixture. and noting the tiinc required for the color change. If the time is more than 30 seconds, either a more dilute solution of the indiraior or a more coiic*rntmtetl zinc solution should he used. All cations nhich form insoluble feriocyanides in the presence of hydrochloric acid interfere with the titration and may be removed hy the Waring procedure (4, 7), which consists of preripittition of (sopper with aluniinuin etrips follon-rd b! prwipitn-
1877 tioii of zinc sulfide in formic arid solution. The separatrri zinc sulfide is then dissolved in h;\-drochlorir acid. This procidurc, was not necessary in t,hepresent ivork. :is iron and aluminum ~ i . r e the only interfering cations in the spli:11wite ores and the!- \vim: wsily removed by double previpitatioii with ammoninni t i 3 ~ tlroside. .inion interference prcwiite no prohkrn. LITERATURE CITED
(-'oiie, V..€ I . , atid Cady, L. C., J . =Lm.Chem. Soc., 49, 35ti ( I ! U i ) .
('owling, H.. and Miller, E. J., IND. E m . HEM.. A N . ~ L .E. n . , 13, 145 (1941).
Hibbard, P. L., Ibid., 9, 127 ( l 9 3 T ) . lIahiii, E. G., "Quantitative Analysis," 4th ed., York. SIcGraw-Hill Book ('o., 1932.
p . 2 7 0 , Srw
I h i d . , y. 524. XIehlig, J . P., and Clauss, J. K.. Chemist-dnulyst, 34, 52 (1!>42). \Taring. K. G., J . Am. Cheni. Soc., 2 6 , 4 (1904); 29, 262 (1907). \\-ichmaiin, 13. J., IND.ENG.h m r . , ANAI..ED., 11, 68 (IOHS). \Villoughhy, C. E., Ibid., 7, 33 (1935). Ziiehlke, (;. W.,Eastnian Kodak Co., Rochester, S . Y..Misc. Pub. 4, I (1949). KLCEIVLDJ u n e 26, 1Y.51. .Ibstract.ed froin a thesis submitted b y A , P. ( 2 i i i l l to the Graduate Sohoul of Oregon State College in partial fulfillment of tile reqiiireinentn f o r the degree of master of science.
Microdetermination of Carbon Dioxide Liberated by Acidification or Oxidation DAVID SELIGSOK' AND HARRlET SELIGSON George S. C o s tfedirnl Research I n o l i t i r t e , Unioersity of Pennsylvania, Philndelphin. Pu.
1IICR.OXETHOD for the determination of (,arbon diox-
A4
idv, which utilizes it8sseparation by diffusion and deterniination 1)y titration, has been developed. The procedure is ruitable for free carbon dioxide, carbonates, or re:~dilyoxidized compounds such as oxalic acid. Jt is accurate for amounts ranging from 0.25 t o 4 micromoles ( 3 to 24 mirrogrtims of caFhoii or 0.005 tro 0.1 nil. of carbon dioxide gas). The STOPPER WELL method has been used for the determination of the carbon dioxide con-VIAL tent of Mood, and of Borne organic acids that are stoichiometrically oxidized. This is a modificat,ion of t'he microdiffurion niethod of Conway Figure 1. M a ( 1 ) with Rome added advantage?. gram of Diffusion (;ell The diffusion vessel is simpler. cheaper, and easier to clean anti Vial is 15 m m . in handlrs lws carbon dioxide. I t i p diameter and 25 mm. high, fitted with easy to perform large numbers of tlrwhite penicillin bottle stopper, white t,erniiiiiitionr with arcuracy. color providing back-
a -
TOTAL CARBON DIOXIDE IN B L 0 0 1 ) OR OTHER FLUIDS
round for titration. ell i n stopper holds 0.1 ml. of 0.05 N BdOHh. Diffused COi i i determined by titrating ~ x c ~ s s Ra(OH)2 reniaining (see Figure. 2)
%'
Reagents. i5tanciartl carbonate solutiori, 0.05 S. 1,actii; acid, 6 K . Barium hvdroxide. 0.05 N . coiltairiiiig 0.02(; thymol blue. Standard sohition of hydrochloric acid, 0.05 .\-. Apparatus. Diffusion vials and white rubber stopperfi (k'igurc1). Thwe rubber stoppers, which fit the vials, are comniercially available L ~ Fpenicillin bottle stoppers (S-29 from T. C. TVheatoii Co., hIillville, S. J.). Two Scholander ( 3 )microburets assembled for titration as shown in Figure 2. The micrometer is divided into 25 units. each of which is divided into 100 parts. In tht. buret,s used, each unit delivered 0.0316 ml., which in terms of 0.05 S hydrochloric acid is equal to 1.58 equivalents of carbon dioxide. Stirrer to contain the rubber stop er with the w-ell in the upright position (Figure 2 ) . The speed o f t h e motor is adjusted to about 200 r.p.m. Rocker, which consists of an 8-inch platform 1
Present addresa, Army Medical Center, mashinaton 12, D. C.
moved through an angleof 10" to 15" liy R I I eccentric wheel. 'l'hi* rate of pulsation is about 150 per minute. Procedure. Six drops of 6 S lactic acid are introduced into t i vial, and 3.00 uiiits (0.0948 nil. i i i ttie burets used) of bariuiii hydroxide froni a microburet, arr placed in the well of a r u h h r stopper n-hich is used to cap a via1 (;ift,erinversion of the stopper its contents do iiot spill). To achieve uniform delivery, the til) of the buret is t'ouched to the ceiiter of the fluid in the well aiirl gently removed. In this manner accurate and precise delivei'y of the barium hydroxide is achieved. The capped vial is neighed to the nearest 0.1 mg. One or more drops of sample from a filict,ipped dropper are int'roduced into each vial, which is opened ant1 closed quickly. Vials with standard carbonate solution for checking the niet,hod are similarly prepared. Each vial is reweigheil to determine the m i g h t of the sample. The volume is calculatetl by dividing the weight by the specific gravity of ttie sample. The standard carhoiiate is assumed to h a w ii specific gravity of 1.00 and blood a specific gravit!. o f 1.05. Blood may h i , MICRO dropped into the vial diBURET rectly from the syringe and needle used for venipuiit~WELL ture. This procedure M : I ~ used to avoid the chaiigrs i n the carbon dioxide coti-
1
Figure 2.
Jlicro t it ra t io 11
Assembly Rubber stopper containing Ba(0H)z within well is placed on
No. 2 rubber stopper mounted
on motor (about 200 r.p.m.). Tip of Scholander microburet containing 0.05 N HCl is placed i n well of rubber stopper. so that during titration contents of spinning stopper are thoroughly mixed
L
-
The vials are roched yrtitly for 40 minutes, a t nliich time diffusion is complete. The stoppers are removed, inverted, a n d placed on the stirrer for titration of the excess barium hydroxide. The buret containing the standard 0.05 S hydrochloric acid is adjusted so that its tip acts R S the stirring rod inside the well of the rubber stopper, as shown in Figure 2.
ANALYTICAL CHEMISTRY
1878 Table I.
Determination of Carbon Dioxide in Blood
COz Content, Millimoles per Liter Microdiffusiona Van Slykeb 1 19.9 19.4 2 19.3 19.6 3 39.2 38.8 4 23.7 23.8 hiean6 of a triplicate a n d b duplicate determinations. Determinations by \’an Slyke (2) manometric method were performed by Richard Singer, Department of Physiological Chemistry.
Sample KO.
The diffused carbon dioxide is apparent as a film of white barium carbonate on the blue barium hydroxide solution. The titration is carried to a yellow-gray color. Blanks are run simultaneously to determine the titer of the barium hydroxide. A stream of carbon dioxide-free air is blown gently over surface of the barium hydroxide during the titration to reduce contamination by the carbon dioxide in room air. The calculations are rn follows:
[MI. of HCI (blank)
- ml. of
HCI (sample)] 0.0500 X lo00 = M. of GO2 in sample
vials are rocked for 30 minutes and the excess barium hydroxide is measured by titration with standard hydrochloric acid as described above. The calculation is made as above. Results. The stoichiometric osidation a t room temperature of compounds like oxalate takes several minutes, even when catalyzed by osmic acid. Figure 3, B , shows a time-diffusion curve for carbon dioxide obtained by oxidizing oxalate with ceric sulfate containing osmic acid. Complete oxidation and diffusion are achieved in 60 minutes. Without the catalyst (Figure 3, C) carbon dioxide is completely recovered in 120 minutes. Table I1 gives typical recovery data for carbon dioxide obtained by this procedure from oxalate, alloxanate, and oxomale nate DISCUSSIOS
The shape and size of the diffusion cell were chosen because of the availability of the vials and rubber stoppers. Under the circumstances of use they are adequate, easy to clean, cheap, and small. Variation of size and shape, which according to Connay ( 1 ) affects the rate of diffusion, was not studied. This method can be used for free carbon dioxide, carbonates, or carbon dioxide liberated by stoichiometric oxidations. The method was originally designed to mrasure alloxanic acid as it was formed from alloxan ( 4 ) .
loo.
+
Table 11. Determination of Carbon Dioxide from Known Amounts of Organic Acids
90.
Substance Sodium oxalate
w 80. 0 K 70.
Sodiunib alloxanate
\SO.
SodiumC oromalonate
w
0
COa Theoretical, re. 4.00 3.00 4.00 2.00 4.00 2.40 3.00
Recovery,
%
99 101 100 100 101 101 100
a Each value is mean of triplicate determinations. b Alloxanic acid is stoichiometrically oxidized by cenc sulfate, one mole yielding 2 eq. of COz (4). 6 Oxomalonic acid is stoichiometrically oxidized b y ceric sulfate, 1 mole yielding 6 eq. of Cog (4).
* 50. 40 0
COz Observeda, pe. 3.96 3.02 4.00 2.00 4.05 2.42 3.00
30 40 50 60 MINUTES Figure 3. Time-Diffusion Curves IO
20
70
A t 25” C for recovery of COz A . Fr&n 1.00 pM NalCOs after acidification B . From 0.50 U M oxalate oxidized in presence of osmic acid catalyst From 0.75 p M oxalate without catallst Trine C: measured from addition of oxidant, immediately followed by rocking vials
Results. Using the above procedure, a timediffusion curve with standard sodium carbonate was prepared (Figure 3, A ) . Complete diffusion was obtained in less than 40 minutes. The carbon dioxide content of blood was measured by this procedure and by the Van Slyke manometric procedure ( 2 , 5 )(Table I). CARBON DIOXIDE RELEASED BY OXIDATION WITH CERIC SULFATE
Reagents. Sulfuric acid, 12 N . Sodium oxalate, 0.001 M . Ceric sulfate, 0.5 N , in 3 iV sulfuric acid containing osmic acid to 0.005M , in a dropping bottle. Apparatus. Vacuum desiccator and carbon dioxide trap. Procedure. The sample to be oxidized, which in this case is 0.001 M oxalate, is measured (0.5 ml.) into a vial containing 2 drops of 12 N sulfuric acid. The vial is capped with a marble and placed in a vacuum desiccator. The air is evacuated with a water aspirator and then is replaced with carbon dioxide-free air through a carbon dioxide trap. The process is repeated to eliminate free carbon dioxide from the samples. The vials are removed from the desiccator, treated with a drop*ofceric sulfrqte solution, and capped with a rubber stopper containing 3.00 units of barium hydroxide, Blanks are prepared and run simultaneously. After standing a t room temperature for 30 minutes, the
The reported data for knoxm compounds show the recovery of carbon dioxide to be within ~t1.2’3~.The working range is 0.25 to 4 millimoles of carbon dioxide, but can be extended above or below this range by changing the concentrations of barium hydroxide and standard hydrochloric acid. Fifty or more analyses were performed by one analyst in less than an 8-hour day. The incorporation of the indicator (thymol blue or phenolphthalein) into the barium hydroxide proved satisfactory. Thymol blue alone proved stable for 6 months or more, whereas phenolphthalein faded. The end point with thymol blue is readily detected, the first definite end point being used. Delay in defining the end point leads to solution of barium carbonate and low results. The amounts of carbon dioxide determined are smaller and the diffusion cells are simpler and smaller than in the method of Conway (1). Smaller quantities can be measured than by the manometric method ( d , 5 ) , and the procedure is simpler and more rapid. LITERATURE CITED
(1) Conway, E. J., “Micro-DiffusionAnalysis and Volumetric Error,” 2nd ed., p. 189, London, Crosby, Lockwood & Son, 1947. ( 2 ) Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemistry, Methods,” Baltimore, Williams & Wilkina Co.,
1932.
(3) Scholander, P. F., Science, 95, 177 (1942). (4) Seligson, D., and Seligson, H., J . BioZ. Chem., 190, 647 (1951). ( 5 ) Shock, N. W., and Hastings, -4.B., J. BWZ. Chem., 104, 566
(1934). RECEIVED M a y 18,1951.
