660
ANALYTICAL CHEMISTRY
volvw only the cell t,hicknc chromatic light employed.
ind t h e ~v:ive length of rhe mono-
ACKSOWLEDGMENT The authors are indebted t o L. R. C Dr J3roske of the Instrunient Design and Research Section, n'atioixil 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 t o espress t,heir great appreciation to l.:mil Maier, Pyrocell Nfg. Co., Xew Tork, X . Y.,for constructing t h r 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, 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. . (~oitcioii), .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., Res. Sei.Instrunicrzts, 20, 209 (1949). ( 7 , Gosting, L. J., and ?Jori.i-. &I. S., .J. A m . C'hcm. Soc.. 71, 1998
s.,
~
(1949).
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. Longsfforth. L. G., IND.EX(?. C H E M LI,. E D . ,18, 219 (1946). Longsworth, L. G.. J . Am. ('hem. Sor., 61, 529 (1939). Longsworth, L. G. Ibid., 69, 2510 (1947). Loitgsworth, L. G.. Rec. Sei. Instruments, 21, 524 (1950); . ~ s . \ L . CHEM., 23, 346 (1981). Perlmann. G. E., and Longs~vorth,L. G., .J. d m . ( ' h e m . Soc., 72,
2719 (1948). 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 NCHmite.Li. SOCIETY, April 1950; .I. A m . ('hem. .%IC.. in p r e s . Solier, H. A , , Kegeles, G . , a n d Glitter. 1;. .J., (1 949).
teiit. I\-. H.. and Moore. tcim. K. G . , L-. S . Patent ~ ~ I I ~ WH., I I . d r t u C'hem. 5, 7 2 (1951).
, .J. Bioi. Chem., 176, 337 (1948). 495,297 (.Jan. 24, 1950). m d . . 3, 1170 (1949,; 4, 399 (1950):
S v e n ~ w i iHarry. . Kolioirl-Z.., 87., 181 (19.'39) : 90._111 (1940). . , , ~veii>-oi~, Harry, personal eommunicatioti anti Swedkh Patetit .11>~>lic.atioii 1621 150. 'Tltoma~.G. R.. O'Kon&i, C . T.. aiid Hiird. C. D.. .Is\I,. CHEII..22, 1221 (1950).
(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). Hm . f . , 23, 794 (1951). (11) Holman, R. T., and Hagdahl. L..a l s . \C ~ (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, ~. 21, 1470 (19491.
Separation of Biosynthetic Organic Acids by Partition Chromatography E. t.'. I'lIiRES, E. H . .1IOSB4CII, 1;.J '
. DEUISOK, J R . ,
S. F.CARSON, with the technical assistance of AI. \ 4. GWIX Biology Division. Ouk Ridge .Vational Laboratory, Oak Ridge. T e n n .
A
4YD AhD B.
S I l l P O R T . ~ S Tprerequihite in radioactive tracer studies of
organic acids in various biological s>-stemsis t h e separation of chcmically a n d 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) 1)- partition chromatograph>-, several pairs of acids ~ v e r eohtsined which could not be separated t o a satisfactory degree of purit>- on t h e chloroformbutanol 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~eliminarystudies 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 ~ ~ eresulting d, in lonered specific radioactivity values upon \vet combustion, and the difficulty of devising accurate analytical n ~ ~ t h o cfor l s the determination ( b y titration) of organic acids in ;toidic or buffered column effluent^. Conwquently, a method was devt~lopcd,using part,ition chroiixxtography esclusively, which allov-eLlthe separation from binlogica.I materials of a numlwr of organic acid*. T h e following acids irere examined: acetic, a-ketoglutaric, citric, formic, fumitric, glycolie, kojic, lactic, malic, malonic, oxalic, perchloric. p>-ruvic,phosphoric, succinic, sulfuric$ant1 tartaric. Satisfacto.ry
. LONG
isol;ttions :mJ selxwatiorir W ( , I Y ~ohtaiiicvl tiy the. use of tiyo solvent syst(aiii*: 1)utanol in chlorot'oin-0,5 .V sulfuric acid, and ethyl ethcr-0.5 .Y sulfuric acid. Ci,littx 545 \vas u ~ ( v 1 ininioldc ~ ) h ~ . ; e In . general. it \vas found that, of the arids Ftudied, those riot resolvctl ti?. c.hloroform-t~utanol could be separated Iiy i,cc.hromatogritphiiig with ether. Recover?- data obtaiii-separations of chemicall>-and isotopicall?- pure carboxylic acids and all o w - ~a fair quantitative estimation of these substances in animal tissues ;yld fermentation liquids.
('olunins r u n n-ith ctlltir usually have a "dried-out" appeal.:mw, l ~ u this t does not W P I ~t o :ifYc:ct t h e operation of t h e columi Thc. samples t o be aiial\-zed \\-ere pfaced on t h e colunin f'ollo\~.:: T h e aqueous solution or the sodium salts of the orgai a&ii n-as c:vaporated in vacuo t o a volume of about 0.25 1111. or 11,~- in :I small, ~vidr-niouthrdconical flask. For some of the less (5
I .
staiilv nc%is-c.g., cr-ketoglutaric arid-the evaporation \vas cwricd out a t neutral pH and r o o ~ ntemperature. Enough Celite (0.5 gi':iin or less) \\-:I$ addetl t o the c-oncentrated aqueous phase (in thtl conical flask) t o ni:1kc a fairly d r y misture of t h e same appca:ir:inc'e as the original m i s t u ~ , eof CPlite and 0.5 ;r\ sulfuric acid usc~ciin packing t h e rolumn. -1fen- drops of aqueous phenol red intlicxtor solution \verc~:~lwayspresent in the sample: visual incolored \>anti gave an excellent indication in packing the column. This misture i v a . ~ iificvl with 4 t o 8 drops (0.1 t o 0.2 nil.) ot 23y0 sulfuric- acid, trani;irri,ccI quantitatively t o about 1 to 2 1111. ofhlvciit on top of the Celitcx, and tamped down. T h e liquid \vas (li:i\vn down exactly t o the level of the Celite b y opening thc stoi)cocli, and this rirlsing p r o c ~ s swas repeated by the additioii
of 1 to 2 ml. of the organic phase. T h e organic phase was then added in a reservoir attached to the top of the column. Care 1%-astaken not t o let the colunin become dry at any time after packing. Flow rates of approximately 0.5 t o 1 ml. per minu t e were found satisfactory for all types of columns, t h e slower rat'e being required for complete separation in some cases. The effluent was collected with a n 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 identified by comparison with knoivns run on standard columns, and also b y 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.
Table I.
I
