F i d by trial the amount of sample fluid that will provide the optimum number of particles on the grid. Measwe the height of the meniscus above the grid. For very dilute suspensions fill the tube. For very concentrated suspensions centrifuge the liquid in another tube and dilute the sample with some of the clear'liquid. Stopper the tube and centrifuge the contents for 5 to 30 minutes, or until the liquid is CAW. The glaas vial has withstood full speed (2200 r.p.m., 700 relative centrifugal force) without breaking. After centrifuging, remove most of the liquid with a suction pipet down to 1 cm. above the grids. Tilt the tube shghtly, pull the fixture slowly out of the tube by means of the tweezers, and place it on fiiter paper to drain. Blot the sides of the grids with a piece of filter paper to remove most of the liquid. Grasp and lift a specimen grid with the fine tweezers and with the other hand slide a half piece of filter paper up into the closed tip of the tweezers as f s as possible. This removes the liquid trapped between the tweezer tips and prevents a wash of particles from the grid as it is released. Set the grid on
filter paper for final draining. After a few minutes, i t is ready for examination.
to obtain a deposit of particles without having to dry down the volume of the
Although only a limited amount of work has been done with this fixture, it has been found useful in filtration efficiency studies and in fuel oil sediment were as studies where particle sismall as 0.1 micron. This technique is adaptable to particle counts, assuming that no particles are lost from the grids as they are removed from the liquid. In the case of fuel oil, the particles were held firmly on the grids; no cloudiness \vas observed in the "centrifugate" and no solid material was observe-! after rccentrifuging the liquid remainder. Possibly, the type of vpecimen grid coating could be varied to provide a stronger attachment for the particle to be separated. No comparison was made with the microdrop or spray technique. However, in the present method very dilute suspensions are concentrated, thus saving time in finding the particles under the microscope. It is possihle
sample fluid, chus avoiding precipitation of dissolved material. And it should be possible to obtain differential d e p osition of particles depending 011 size, shape, or density by varying the centrifugal force and the time. The number of particles to be counted and the number of areas to be viewed are governed by statistical methods. The volume of the sample represented by one area of view is: Height of liquid X 0.7854 X diameter of viewing screeel) magnification /
(
or. if a picture is used:
Height of liquid X
picture width
X
picture length magnification Particle. sizes may be estimated visually by comparison with measured particles in electron micrographs or by comparison wibh a inillimeter scale.
Equilibrium Dialysis Vessel for Analysis of Drug-Macromolecule Interactions Ronald G. Wiegand, Department of Pharmacology, Abbott Laboratories, North Chicago, Ill.
HE error involved in equilibrium diT alysis measurements of drug-macmmolecule interactions at low drug con-
centrations, using the conventional dialysis tubing bag, is sometimes so great as to make the procedure of doubtful value. This error a r k from the variation in the amount of drug adsorbed by the membrane and is proportional to the drug-membrane affinity. The equilibrium dialysis vessel here described reduces the error caused by membrane binding by two means: The membrane area is a fraction of that required with the conventional bag, and the area of membrane available for binding is constant. The average e m r in amount of drug adsorbed by the membrane in this vessel is =t2% of total drug in the concentration range 10-5 to 1 0 - 7 ~ . Each half of thc vase1 has a volume of 5 ml. and is made individually from a length of b o d c a t e glass tubing 25 m. in outside diameter by flattening the end, attaching side arms and hooks, and finally cutting off the tube and grinding the cut surface on a lap. Halves of B pair are numbered, and hand-ground to a water-tight seal with No. 240 Carborundum powder. To aesembie, the ground surfacea are given a light coating of gnkse to prevent leakage while shaking, a single dialyeis membrane is placed between the two halves, and the unit is secured by rub-
A i
lxww SURFACE
SECTION A-A
A '
ber bands between the hooks. After filling, the side arms of each side are connected with rubber tubing to prevent evaporation. During equilibration, the vessel is continiiously shaken on a rotary shaker at 185 r.p.m. For measurement at constant temperature a Dubnoff metabolic shaker is used at 120 cycles per minute, with a rack with rubber tubing-overed bolts appropriately spaced to support the vessels.
In equilibrium dialysis experiments the concentration difference of the freely dialyzable solute on the two sides of the membrane decreases from its initial value to zero. If this solute is placed in side a of the vessel and pure solvent in side b, the concentration dzerence decreases according to the equation
where
C.
and c) are the solute con-
centrations on sides a and b, e, is the initial concentration of solute on side u, k is the rate constant in units of reciprocal time, and 1 is time. With a cellulose membrane (Visking, 44 mm.) and a drug of niolecular weight 3:?, k was experimentally determined to 0 1 0.87 hour-'. Calculation based on tlrx value of k indicates that the concentrations on the two sides differ by no mort %an 0.1% within 8 hours. Equation 1 describes the approach of the system to equilibrium if the drug is adsorbed by the membrane or is not. For the case of no adsorption by tne membrane, at equilibrium c. = cb = c.V./(V, VJ, where V. and Vb aw the volumes of sides a and b. If the membrane does adsorb drug, at equilibrium the amount of drug adsorbed is coVa - c.(Vo 4- VJ. If a macru molecule to which the membrane IS semipermeable is placed on side b at a concentration low enough to makc Donnan equilibria effects negligible, the amount of drug bound by the macromolecule is found by subtracting iron c.V. the amount of free drug a t equiT'b), and the amount librium, c.(V. of drug adsorbed by the membrane .k the equilibrium concentration of drug on side G . A t concentrations of macromolecule nv enough to neglect Donnan equilibria effects, the volume flow due to the osmotic pressure difference b o tween sides c and b can also be ne-
+
+
VOL. 39, NO. 10. OCTOBER 1959
1745
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.
of 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. OIOASSAY
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. cni.) 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
