glected without departure from Equation l . I'essels made from tubing of the same inside diameter need not be individuall\calibrated for membrane adsorptioii. because the area of membrane exponrti to the solution is the same in each vesse!. For vessels of different inside diameter the amount of drug bound by
the membrane a t any equilibrium free drug concentration can be calculated by the ratio of membrane areas. The accessibility of dialyzing solutions allows ana1j;sis at any time during the dialysis. Other applications of the vessel include removal of the dialyzable impurities from a small volume of solution of macromolecules, effected
rapidly and without the use of large rolunies of solution by passing fresh solution through the opposite side of the vessei either continuously or in aliqiio: volumes. Further, a dialyzable coniponent may be separated quantitatively by the same procedure, and n-ili be more easily recovered from the smaller volumes invdlved.
h n improved Phenylhydrazine Reagent W. Knowlton Hall and Teckla
S. Decker,
Department of Biochemistry, Medical College of Georgia, Augusta, Ga.
phenylhydrazine rewhich obi-iates the usual dficult!. of tar formation has been in use in this laboraton for 15 years. Sodium bisulfite stabilizes the phenylhydrazine and prevents tar formation in the reagent on standing and in use. Hamilton [ J . .4m.Chem. SOC.56, 487 (1934) ] had reported using sodium bisulfite in a similar manner in a quantitative study of the osazone reaction. ISfPRoi-Eri
A'sagi.nt
The improved osazone reagent as used for the identification of sugars is prepared by dissolving 20 granis of phenylhydrazine hydrochloride and 20 grams of sodium acetate in 200 nil. of 3ci; sodium bisulfite solution a t room temperature with stirring. In use, this reagent is added to an equal volume of the sugar solution (0.2.V' to he tested, the mixture is heated, and tlie test is carried on in the usual manner.
To determine the most suit.able concentration of sodium bisulfite,' three series of phenylhydrazine reagents were
prepared a t different times. with a range of concentrations of sodium bisulfite in each series varying from 1 to 5%. Each preparation was tested with the more common sugars.at intervals of 2 weeks to a month for a 2-year period or as long as that reagent preparation was usable. The reagent made with 370 of sodium bisulfite Kas the most satisfactory, as it gave maximum stability with niininiuni interference in the forniation of typical crystals. This confirmed impressions gained from less systematic trial$ in student laboratory usage. The improved phenylhydrazine reagent with 3% bisulfite was stable for at least 6 months. After 6 months the time necessary for the formation of the osazone crystals increased, although the reagent could still be used and produced typical crystals. After 12 or more months, tar formed in the reagent and it became unusable. T h e n it was approximately 2 months old, long, needlelike, white crystals began to form
in the reagent and increased in aniouri~. Qualitative analysis showed that thew crystals contained carbon. nitrogen^ sulfur. and sodium. That the crJ-stai. probably xere compounds similar t o one such compound (C&N,SO2Sa. H C prepared hi- Bucherer and Schmidt [ J . p r a t t . Chem. 79, 369 (1909)l x a i indicated by the formation of phen!-lhydrazine on heating. When attempts were made to anal>-ze the crystal. quantitatively for carbon, hvdrogcr , nitrogen, and sulfur. variable resulr-: were obtained even niter repeated rf'crystallization. This indicated tllat ail tinstable misturr of reaction products of phenylhydrazine and bisulfite hac1 formed in the reagent. The formation of the cr>-stals in the reagent did not impair its utility within the limits described. It is advisable to avoid heat in the prepamtion of the phenylhydrazine reagent, to minimize the formation of bisulfite derivatives of the phenylhydrazine,
Radioasray of Aqueous Sampler Alicia Marc6, Jean C. Scott,
J. C. Elwood,
and J. T. Van Bruggen,
Department of Biochemistry, University of Oregon Medical School, Portland, Ore. ~ ~ O A S S A of Y
aqueous samples concarbon-14, by conventional Geiger assemblies, usually involves precipitation or evaporation of the solution to obtain solid residues and/' or combustion of organic material in soiution to carbon dioxide, which can be plated as barium carbonate. The evaporation of solutions containing volatile tracers is not practical. Direct countine: of aqueous samples under end xindow counters is hazardous because of t : i t problem c,f contamination, and r t q ~ s'ous samples cannot be count.ed in ya:. flox counters. liquid sample .*ountingtechnique has been developed hew that pro!-ides a high degree of acwracy and ease of manipulation and ssiving of both time and materials.
R-taining
The liquid simple, not less than 174 *
ANALYTICAL CHEMISTRY
0.5 ml., is added to stainless steel cupped planchets (Suclear-Chicago SS-10j. -\ddition of a small drop of a 1% aerosol
solution assures the even spreading of the 0.5 ml. over the surface of the cup. Water samples of less than 0.Snil. volume do not cover the surface of the planchet. The sample is now covered by a thin film of Nylar to prevent evaporation and/or exchange of the sample with the atmosphere; the Mylar film is stretched across the face of the cup and held in place with a rubber band around the side of the cup. To attach the film to the cup, disks of Mylar film 33-nun. in diameter (Du Pont O.2j.mil Mylar, 0.85 mg. per sq. cm.) are cut with a cork borer assembly. A S o . 8 rubber band is doubly looped around the middle of the tapered sides of a S o . stopper. The disk of Mylar is centered on the bottom of the stopper, where it is held electrostatically. The stopper
is no" used as a handle and the film centered on the cup, the stopper pressed firmly on the cup edge. and the rubbeband rolled off the taper of the stopper onto the cup. In so doing, the edges of the film are crimped against the side of tlie cupped planchet and held in place by the rubber band. The sample can now be counted under the desired counter without fear of loss or contamination. It should, honever, be counted n-ithin 1 hour, because of a tendency for the liquid to condense OK the under surface of the SIylar film: the condensate then acts as a filter and further reduces the sensitivity of th? assay. This method of sample preparation is less sensitive than some others and is used only where adequate activity is available. When samples of carbon-14-labeletl
