660 A N A L Y T I C A L C H E M I S T R Y

Kegelea, G., J . Am. C'hem. Soc., 69, 1302 (19471, Kege1e-j. G.. and Gosting, L. .J.. I M . , 69, 2516 (19471. Kegeles, G.. and Gutter, F. J . , I h i d . , 73, 3539 (19511.

Longsworth, L. G.. J . Am. ('hem. Sor., 61, 529 (1939). Longsworth, L. G. Ib id . , 69, 2510 (1947). Loitgsworth, L. G.. Rec. Sei. Instruments, 21, 524 (1950); . ~ s . \ L .

Perlmann. G. E., and Longs~vorth, L. G., .J. d m . ( ' h e m . Soc. , 72,

Philpot. J. St. L., Suture, 141, 283 (1938). Philpot, J. St. L. . and Cook, G. H. , Research, 1, 231 (1948). Rayleigh. Lord, Proc. Ro,y. Soc. (London). 59, 203 (1896). Sober, H. .I., and Kegeles, G., Federation Proc., 10, 299 (1981). Soher, H. .I.. Kegelej, G., and Gutter, F. J., .Ibstracts of 117th

lfeetitig, . ~ ~ \ I E R I C . L N CHmite.Li. SOCIETY, April 1950; .I. A m . ( 'hem. .%IC.. in pres .

(1 949).

Longsfforth. L. G., IND. EX(?. C H E M LI,. ED., 18, 219 (1946).

CHEM., 23, 346 (1981).

2719 (1948).

Solier, H. A , , Kegeles, G. , and Glitter. 1;. . J . ,

teiit. I\-. H.. and Moore. tcim. K. G. , L-. S . Patent ~ ~ I I ~ W I I . H., d r t u C'hem.

, .J . Bioi. Chem., 176, 337 (1948). 495,297 (.Jan. 24, 1950). m d . . 3, 1170 (1949,; 4, 399 (1950):

5, 7 2 (1951). S v e n ~ w i i . Harry. Kolioirl-Z.. 87. 181 (19.'39) : 90. 111 (1940).

volvw only the cell t,hicknc chromatic light employed.

ind the ~v:ive length of rhe mono-

ACKSOWLEDGMENT The authors are indebted to L. R. C

Dr J3roske of the Instrunient Design and Research Section, n'a- tioixil Iristitut,es of Health, for ashistance in the design and for the construction of many of the machined parts. They v-ish to tli:unk the .4rts Section for dran-ing Figures 2, 3, 4, and 5 .

The investigators wish to espress t,heir great appreciation to l.:mil Maier, Pyrocell Nfg. Co., Xew Tork, X. Y., for construct- ing thr glass prism cell and donating it to them.

LITERATURE CITED

(1) ;\dams, L. H., J . -4m. Chem. Soc., 37, 1181 (1915) ( 2 ) Baratow, 0. E., Paper 49-4-2, presented before In~trument

Society of America. St. Loiii3, 1949. (:% Claesson, s., zirkic Kpmi ?fi?md. ( h i . , 23A, No. 1 (1916). ( 4 ~ Coulson, C. A., Cox, J. T., Ogstoii, -1. G.. aiid Philpot, J. St. L

Proe. ~ o y . SOC. (~oi tc ioi i ) , .AICJZ, 382 (1948). ~

(5) Dutton, H. J., J . f h u s . C'hem., 48, 179 (1944). ((ii Gosting, L. J . , Hanion, E. M., Kcpeles. G. . and Morris, R I . S.,

(7 , Gosting, L. J., and ?Jori.i-. &I. S., .J. A m . C'hcm. Soc.. 71, 1998

(81 Gouy, G. L., Compt. w / ~ d . , 90, 307 (1880). (9) Gutter, F. J., and Kegele., G. , t o he publialierl.

(10) Hagdahl, L., Acta C'hem. S'caitd.. 2, 574 (1948). (11) Holman, R. T., and Hagdahl. L.. als.\~. C H m f . , 23, 794 (1951). (12; ,Jenkins, F. A, and White. H . E.. "Futidamental~ of Physical

Chap. VI, Yew York. XIcGrair--Hill Book Co., 1937. (1:Cj Jones, H. E., Aahma11. I,. E., mid Stalily, I:. E. , - 1 s ~ ~ . CHEX,

Res. Sei. Instrunicrzts, 20, 209 (1949).

(1949).

21, 1470 (19491.

, , . _ , ,

~veii>-oi~, Harry, personal eommunicatioti anti Swedkh Pat-

'Tltoma~. G. R.. O'Kon&i, C. T.. aiid Hiird. C. D.. .Is \I,. etit .11>~>lic.atioii 1621 150.

CHEII.. 22, 1221 (1950).

Separation o f Biosynthetic Organic Acids by Partition Chromatography

E. t.'. I'lIiRES, E. H . .1IOSB4CII, 1;. 'J . DEUISOK, JR. , 4 Y D S. F. CARSON, with the technical assistance of AI. \ . LONG AhD B. 4. GWIX

Biology Division. Ouk Ridge .Vational Laboratory, Oak Ridge. Tenn.

S I l l P O R T . ~ S T prerequihite in radioactive tracer studies of A organic acids in various biological s>-stems is the separation of chcmically and isotopically pure coinpounds from the reaction mixture. Although satisfactory methods have lieen described for the isolation of some of the intermediates of the tricarboxylic acid c>-cle ( 5 ) and a number of other acids (8) 11)- partition chromatog- raph>-, several pairs of acids ~ve re ohtsined which could not be separated to a satisfactory degree of purit>- on the chloroform- butanol chromatograms reported in t.he literature. This dificult)~ as encountered particulai,l?- in t,he cases of succinic and lactic acids, fumaric and pyruvic acids, malic and osalic acids, and glycolic and oxalic acid-+. P~eliminary studies of ion eschallge techiiiques ( I ) indicated that such procedurrs were not entirrl). satisfactory for t,he isolation of labeled conipounds oii a 0.1- to 1.0-millimole scale, mainly because of two considerat,ions: the introduction of organic matter into column eluates by partial decoinpoeition of the resin e m p l o ~ ~ e d , resulting in lonered specific radioactivity values upon \vet combustion, and the difficulty of devising accurate analytical n ~ ~ t h o c l s for the determination (by titration) of organic acids in ;toidic or buffered column effluent^.

Conwquently, a method was devt~lopcd, using part,ition chro- iixxtography esclusively, which allov-eLl the separation from bin- logica.I materials of a numlwr of organic acid*. The following acids irere examined: acetic, a-ketoglutaric, citric, formic, fumitric, glycolie, kojic, lactic, malic, malonic, oxalic, perchloric. p>-ruvic, phosphoric, succinic, sulfuric$ ant1 tartaric. Satisfacto.ry

isol;ttions :mJ selxwatiorir W ( , I Y ~ ohtaiiicvl tiy the. use of tiyo solvent syst(aiii*: 1)utanol in chlorot'oin-0,5 .V sulfuric acid, and ethyl ethcr-0.5 .Y sulfuric acid. Ci,littx 545 \vas u ~ ( v 1 ininioldc ~ ) h ~ . ; e . In general. it \vas found that, of the arids Ftudied, those riot resolvctl ti?. c.hloroform-t~utanol could be separated Iiy i,cc.hromatogritphiiig with ether. Recover?- data ob- taiii<ltl iriclicatcxl that the mc,thod also afforded a fair quantitative wtiinatiori of these acidp i n :mima1 ti,+suw and fermentation liquids.

EXPERI3lEKTAL

The standard partition coluinns employed were borosilicate glass tubes, 45 x 1.0 em. in inside diameter, fitt,ed with a coarse fritted disk to support the body of the column and a stopcock to regulate the flow rate. For preliminary studies and small amounts of material (less than 0.1 millimole), 10 X 1.0 em. columns were often found satisfactory. For separating succinic, a-ketoglutaric and nialonic acids, slightly larger columns, 65 X 1.5 em., were found to give better separations. The fluid retention volumes of these three columns were 5 , 20, and 40 ml.

Both chloroform-butanol (CB j columns and ether columns were prepared in an identical manner: The stationary phase, 0.5 .Y sulfuric acid, was mixed very thoroughly with the inert support, Celite 545 (100- to 200-mesh), using 12 grams of Celite for a standard column. For most applications a ratio of 8 ml. of 0.5 -Y sulfuric acid to 10 grams of Celite was found satisfactory. The moist Celite \\-as then slurried with the organic phase and packed into the column by tamping ( 1 1 ) . The organic eluting agents were equilibrated with 0.5 S sulfuric acid prior to use; chloro- forin-butanol mistures were prepared as described by Isherivood

V O L U M E 2 4 , NO. 4, A P R I L 1 9 5 2

COMPARISON OF ELUTION BANDS OF ACIDS WITH CHLOROFORM-BUTANOL AND WITHETHYL ETHER AS THE MOBILE PHASES ON 45 x 1Ocm.CELITE PARTITION COLUMNS ( 2 0 ml.FREE VOLUME)

a THESE ACIDS NOTELUTED A T 2 0 0 0 m l

(A ) MOBILE PHASE CHLOROFORM-BUTPNOL - CB-IO +CB-35

Isotopic tracer studies of oxidative cycles in micro- organisms and animal tissues required methods for the separation and isolation of carboxylic acids on a milliniolar scale and w-ith a high degree of chemical and isotopic purity. The existing methods employ- ing chloroform-butanol partition chromatograms w-ere not entirely satisfactory because several pairs of acids could not be resolved with this solvent sys- tem. A number of organic acids w-ere satisfactorily separated, however, hy the successive use of two solvent systems-butanol in chloroforni followed by ethyl ether, with 0.5 4 sulfuric acid on Celite as the stationary phase in both cases. icids not resolved with chloroforni-biitanol could be separated by re- chromatographing on ethyl ether columns. The method results in satisfactor>- separations of chemi- call>- and isotopicall?- pure carboxylic acids and al- l o w - ~ a fair quantitative estimation of these sub- stances in animal tissues ;yld fermentation liquids.

(5 I .

:mw, l ~ u t this does not W P I ~ to :ifYc:ct the operation of the columi Thc. samples to be aiial\-zed \\-ere pfaced on the colunin

f'ollo\~.:: The aqueous solution or the sodium salts of the orgai a&ii n-as c:vaporated in vacuo to a volume of about 0.25 1111. or 11,~- in :I small, ~vidr-niouthrd conical flask. For some of the less staiilv nc%is-c.g., cr-ketoglutaric arid-the evaporation \vas cwricd out a t neutral pH and r o o ~ n temperature. Enough Celite (0.5 gi':iin or less) \\-:I$ addetl to the c-oncentrated aqueous phase (in thtl conical flask) to ni:1kc a fairly d r y misture of the same ap- pca:ir:inc'e as the original mi s tu~ ,e of CPlite and 0.5 ;\r sulfuric acid usc~ci in packing the rolumn. -1 fen- drops of aqueous phenol red intlicxtor solution \verc~ :~lways present in the sample: visual in-

colored \>anti gave an excellent indication in packing the column. This misture i v a . ~

iificvl with 4 to 8 drops (0.1 to 0.2 nil .) ot 23y0 sulfuric- acid, trani;irri,ccI quantitatively to about 1 to 2 1111. ofhlvcii t on top of the Celitcx, and tamped down. The liquid \vas (li:i\vn down exactly to the level of the Celite by opening thc stoi)cocli, and this rirlsing proc~ss was repeated by the additioii

('olunins r u n n-ith ctlltir usually have a "dried-out" appeal.-

%

66 1

of 1 to 2 ml. of the organic phase. The organic phase was then added in a reservoir attached to the top of the column.

Care 1%-as taken not to let the colunin become dry at any time after packing. Flow rates of approximately 0.5 to 1 ml. per min- u t e were found satisfactory for all types of columns, the slower rat'e being required for complete separation in some cases. The effluent was collected with an automatic fraction collector, and the organic acids were det,ermined by tit,ration with standard (0,100 S) alkali, making suit'able corrections for a blank (0.001 t o 0.004 nil. of 0.100 S sodium hydroxide per ml. of effluent,, depending upon the organic solvent used). Individual acids were identi- fied by comparison with knoivns run on standard columns, and also by the application of specific reactiona ( 1 2 ) , colorimetric procedures ( 4 , 6), and paper chromatography ( 7 ) . The known acids were used as obtained from the manufacturer without further liurification.

a

Table I . I<ecoieries of Organic .icids from Known Jlixtures

CB Columns", Ether Coluirins, i c id 7c c

93.0 77 2* 90 8 9 3 . 0 81.0 40 0 8 7 . 3

8 4 . 3 89 R * 9 2 . 0 85 .7 9 6 . 0 $45 0

. . . .

. . 9 3 . 9

9 2 . .i 9 2 6 94 0

88 0

. . . .

81 0 90 0 S i 3

, .

RESULTS 4x1) DISCUSSIO.\

Figule 1, -1, shons a compoPitcJ di:tgrain of the position of the vai.ious acids on a stand:trd chloroform-butanol chromatogram. ivith a 45 X 1.0 cm. column. Elution was started with 10% I~utanol in chloroform (CB-10) and con t inud with :3sG? butanol in chloroform, as indicatrd.

0

The inorganic acids perchloric, phosphoric, and sulfuric have heen inrludecl because they are often introduced in deprotein- ization procedures. The figure shows tha t a number of acids are resolved either partially or not a t all with this solvent mixture. It should k ) ~ borne in mind, how- ever, that many of these acids are further resolved by the use of sol- vent mixtures containing less hutanol, or hy the use of longer columns, as in the case of suc- cinic, m a l o n i c , a n d cu-keto- glutaric acids mentioned above.

Figure 1, B , s h o w a composite diagram for the standard ether chromatogram using a 45 X 1.0 cm. column. It is evident tha t most, of the pairs not resolved on chloroform-butanol separated satisfact,orily on ether chromato- grams. The differences in parti- tion coefficients from chloroform- butanol to ether are particularly striking for succinic and lactic acids, fumaric and pyruvic acids, and gl!-colic and oxalic acidg. For the separation of the two l a t t e r p a i r s s h o r t " e t h e r columns," 10 X 1.0 cni., n'ei'e

662 A N A L Y T I C A L C H E M I S T R Y

found to be convenient. For some applications ( 3 ) elution with ether followed by chloroform-butanol mixtures was sometimes advantageous.

Table I summarizes recoveries obtained for various known acids from both ether and chloroform-butanol chromatograms. The data indicate that t,he recoveries from Celite columns ma>- be somewhat smaller than those obtained rvith silica gel (6) . More- over, because acids appearing late on the chromatogram, such as citric (Figure 1, A ) , are released in increasingly larger effluent volumes, the maguitude of the blank can approach that of the measurement-for example, when 0.1 millimole of citric acid is chromatographed the volume of blank (in ml. of 0.1 S sodium hydroxide) is 12% of the total titration.

These methods have been applied in this laboratory in nunier- ous carbon isotope studies of intermediary metabolism (3, 9, 10). Cnder the experimental conditions employed, chemically and iso- topically pure acids were isolated from a number of biological systems.

LITERATURE CITED

# \ I ) .Inthony, D. S., Mosbach, E. H., arid Long, 11. Y., unpublished results.

Carson, S. F., Nosbach, E. H., and Phares, E. F., J . Bnct . , 62 ,285

Denison, F. JT., Jr., Ph.D. dissertation, University of Texas,

Dische, Z., Biochem. Z., 189, 77 (1927). Isherwood, F. -4., Biochem. J . , 40, 688 (1946). Long, C., Ibid. , 36, 807 11942). Lugg, J . W. H., and Overell, B. T . , .4ustraZian J . Sci. Resenwh,

Xarvel, C. S., and Rands, R. D., Jr., J . Am. Chem. .SIC,, 72, 2642

llosbach, E. H., Phares, E. F., and Carson, S. F.. Arch. Rio- chem. Biophys . , 33, 179 (1951).

llosbach, E. H., Phares, E. F., and Long, Vi, V., Federation Proc., 10, 226 (1950); Arch. B i o c h e m Biophys . (1961).

Peterson, RI. H., and Johnson, &I. J . , J . BioZ. Chem. , 174, 7 7 5 (1948).

Umbreit, W. W., Burris, R. H., and Stauffer, J. F , “.\fanometric Techniques and Related Methods for the Study of Tissue Metabolism,” Minneapolis, Burgess Publishing Co., 1946.

(1951).

1951.

1, 98 (1948).

(1950).

RECEIVED for review June 30, 1951. Work per- formed a t the Oak Ridge National L a b r a t o r y under Contract IV-i iOS-Eng- 26 for the .4tomic Energy Cornmiision.

Accepted October 2 , 1951.

lodometric Determination of Ozone C. AI. BIKDSALL, .A. C. JENKINS, a \ ~ EDWARD SPA1)INGEK

Linde Air Products Co., Diuision of Union Carbide and Carbon Corp., Tonuwanda, .V. 1’.

‘4 survey of the literature on ozone for the past 50 years discloses manj contra- dictory and confusing statements concerning methods of ozone determination. This work was undertaken to clarify the situation and establish a reliable method. A chemical method is described, \+hich proved suitable for ozone- oxygen mixtures containing up to 25Yo ozone. A physical method has been devised for use as a standard of comparison in testing this chemical method. Comparisons have been made with other chemical analytical methods for ozone. This work should clarifj the present unsatisfactory situation regarding the chemical analysis of ozone-oxygen mixtures. As the uses for ozone increase, these methods should be of increasing importance.

SVESTIGATIOSS on ozone hsve long been hampchred by I t,he lack of a standardized method of analysis. ;i survey of the literature reveals a host of claims and counterclaims for various methods, mainly modifications of the iodometric method; many of these contentions have little experimental basis. The neut,ral potassium iodide method has been shown to give correct results by Ladenburg and Quasig ( 4 ) and Treadwell and hnneler (IO). Riesenfeld and Bencker (6) also concluded that neutral potassium iodide gave correct results up to 2 0 5 ozone, although they present data only up to 8’3. Much of the controversy has been brought about by Itiesenfeld’s (5, 7 ) proposal tha t the potassium iodide solution be buffered with boric acid. The apparent check (wit,hin 2%) of this method with a gas density method is probably fortuitous, for the molecular weight of ozone as determined b>- the two methods was not run on the same sample of ozone and one analytical result out of three recorded gave a n ozone percentage of 107.7 ( 7 ) . Ruvssen (8, 9 ) found tha t boric acid-buffered potassium iodide gave high results, and Iirais and LIarkert (3) questioned Riesenfeld’s contention ( 6 ) that the use of a boric acid buffer was necessary.

To help clarify the situation, it was decided to study various methods for the determination of ozone in ozone-osvgen mixtures. The methods tested were the neutral and boric acid-huffered potassium iodide and the arsenious acid procedures. Acidified potassium iodide was not considered, because results nearly 50% too high have been repeatedly obtained by this method. .I pre- liminary experimental survey of these procedures using ozone concentrations up to 5 mole % showed tha t the accuracy of the

neutral potassium iodide method surpassed that of the boric acid-buffered method, This collclusion was based on a conipari- son of the chemical analyses with analyses by a physical method similar to tha t described below. The results obtained in this preliminary survey are given in Table I.

I n addition, a series of tests wa.i made to determine the effect of the amount of potassium iodide react,ed upon the accuracyof t h e method. An ozonizer with an output of 1.3 mole C;. ozone \vas used in these tests and six series of three analyses xere made, in which the first and last were on low volumes of the ozone-oxygen mist,ure and the middle one on increasingly larger volumes. It. was found tha t u p to SiV, of potassium iodide consumed, the amount used up had no effect upon the result. The p H of sevc.raE

Table I. Results of Preliminary Surve: Mole Per Cent

for Neutrala Buffered6 Absolute Corrected

Pressure conipressi- iodometric iodometric difference, Relative change bility method method mole ’% Oa Error, %

3 . 3 7 4 29 4 . 2 8 5 . 1 3 4 . 1 3 4 . 2 1 4 48 4 58

3 . 3 6 4 . 2 7 4 . 2 6 5 . 1 1 4 . 1 1 4 . 1 9 4 . 4 6 4 . 5 6

3 . 4 0 4 . 3 9 4 . 3 3 5 . 2 3

. . . . . . . . . , . . 1.86 4 . 8 3 5 , 16 .5 ,33

+O 04 +o. 12 t O . 0 7 +o. 12 +o 75 + O . 64 +O 70 T O 77

+ 1 . 2 4 - 2 . 8 - 1 . 6 - 2 . 4

+ 1 8 . 2 - 1 5 . 3 t l 5 . 7 t16.4

2 7 unbuffered aqueous KI solution 5 7c7c aqueous KI solution, saturated w i t h boric acid.

_________ -