Automatic Recorder for Interferometry and Refractometry of Streaming Solutions Applicable to Chromatographic -4 nalysis GERSON KEGELES' AND HERBERT .4. SOBER National Cancer Institute, National Institutes of Health, Public Health Service, Bethesdu 1 4 , M d .
To permit continuous analysis of the liquid a t the point of emergence from the adsorption column during chromatographic experiments, two instruments were developed by Tiselius and Claesson: a manually operated interferometer, using white light, and an automatic recording prism cell arrangement. Using a cell-focusing cylindrical lens optical system, essentially the arrangement of schlieren optics, with the addition of a Fertically driven photographic plate scanning the liquid a t efflux from t h e adsorption column as a function of time, the advantages of t h e two instruments of Tiselius and Claesson have been combined in a single instrument. By minor rearrangements, the instrument is employed as an automatic recording prism cell device, as a continuous recording interferometer, using monochromatic light, or as a continuous concentration gradi-
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ITH the developnic~nt by Tiselius (34) of refractometric methods for analyzing the colorless effluent from adsorption columns, t'he need arose for equipment that would produce a continuous refractive indes record. This need was met by the two instruments developed by Tiselius and Claesson (56) and by Claesson (3). One of t'hese ( 3 ) was a completely automatic instrument utilizing a 45' angle double-prism cell with a split light beam and two photocells, arranged to plot continuously on a sensitized emulsion a direct record of refractive index increment versus volume of effluent. The other instrument, ( 3 ,56),based on the Rayleigh interferometer ( 2 4 ) ,at>tainedhigher precision. This, however, rrquired manual operation by the invest#igator,limiting the duration of the experiments to a very few hours. Further improvements in this instrument have been described by Holninn and Hagdahl(11). These interferometric arrangements possess nearly the same inherent precision as the one described here, but bemuse white light is used for the analysis they suffer somewhat f1~omdifferences in dispersion between the glass compensators and the solutions analyzed (1). TThen layering occurs during the p of a sharp boundary through the cell (IO),these arrangements also suffer considerably in the loss of detail. Sevei.al recent publications from this country ( 2 , 6 , 13, 33, 51')have described instruments with various prismatic arrangements, some of tvhich are rugged and compact and are designed to producc records on paper. From the standpoint of sensitivity, these are generally suitable when the high precision of an interferometer is not required. However, a general research instrument for chromatogi,aphic analysis should produce a concomitant record of effluent volume, as the flow rate cannot always be maintained constant. Such an instrument, if based on a refracting prism arrangement, should, if possible, use monochromatic light (2, 6 ) and not white light. For, in addition to the absence of suitable refractive indes standards for white light calibration, the dispersion of the liquids being examined may be of such magnitude as to impair seriously 1
e n t recorder. Through the use of the new grating interferometer principle of Svensson, the interfemmeter provides for automatic plotting as well as automatic recording of refractive index changes in streaming solutions. The volume of effluent liquid is also automatically recorded with each arrangement. Operating accuracy and reproducibility of one to two units in the sixth decimal place of refractive index have been achieyed with the interferometer, using a 25-111ni. cell and the green mercury line, thereby providing an independent precision instrument fo; the measurement of refractive index increments. Because of the automatic recording principle, the high accuracy of the interferometer is made available for long experiments, using slow flow rates, and assuring the approach to equilibrium in chromatographic anal) sis.
the accuracy attainable with :111 otherwise sensitive in.;t,runient. Moreover, refracting prism arrangcment's designed to plot the refractive indes increment directly should take account of nonlinearity terms in the piism cell deflections ( 1 4 , 1 ; . '71) which become increasingly important a t high sensitivity. Chromatographic esperiments in this laboratory: resulting in the resolution and analysis of rertain protein niistuws (25, 26, ZT), were originally followed with the schlierrn scanning optical system of Longsworth (18). To obtain intermittent records of such esperinients, 12 to 24 hours' observation hy the investigators was required. Even with such attention poor photographs were obtained, Ilecause the boundaries whirh had entered the cell first became blurred owing to diffu~ion. This sit,uation again presented the need for an automatic recording refractometric instrument to analyze the liquid a.5 it leaves the adsorption column. Because of the high inti,insic accuracy and versatility of the basic lens system of scphlieren optics, the authors have chosen to develop this sj-stoni as R photographic recording instrument. This instrument utilizes the same lens arrangement to record as a function of time eit'her interferometric d i a g r a m or piism cell refractive index deflections, or the more conventional refraistive index gradient patterns. Xonochromatic light is ordinarilj- used to analyze the effluent liquid from the adsorption column. Liquid flow is recorded simultaneously on the photographic pl:tte hy a tungsten lamp. The effect of any vertical layering in t h e interferometer cell is overcome by the combination of a cylindrical lens with vertical asis arranged to permit simultaneous focusing of the cell and the interference fringes, and a horizontal scanning slit conjugate to only one level in the cell hole. By calibration of the prismat,ic system with a series of interferometrically standardized liquids, the nonlinearity in the dependence of the light deflection on the refractive indes incrrment is taken into account,. MECH4NICAL CO5STRUCTIOY 4WD OPTICAL A R R 4 5 G E M E Y T
Present, address, Department of Chemistry, Clark Unix-ersity, Worces-
The optical tract
ter, XIaEs.
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41on11 in a scale diagram in Figure 1.
V O L U M E 24, NO. 4, A P R I L 1 9 5 2
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The optical bench and the brackets supporting the optical elements are similar t o those in the electrophoresis equipment described by Longsworth (17). As 8 support for this bench, pyramidal concrete blacks have been cast on steel plates and isolated from the floor hv uads of Keldur (Hobsite Products Co.. 550 Cortlandt St., Beil-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.,A c t a 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. Tenn.
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S I l l P O R T . ~ S Tprerequihite in radioactive tracer studies of
organic acids in various biological s>-stemsis 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) 1)- partition chromatograph>-, several pairs of acids were ohtsined which could not be separated t o a satisfactory degree of purit>- on the 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 (by 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*. 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
. 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
