Procedure for Determination of Diffusion ... - ACS Publications

longer time intervals. ... served in Conway's and Kirk's titrimetric procedures. The ease .... An elapsed time of 20 to 30 minutes is allowed to estab...
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ANALYTICAL CHEMISTRY

838 processes involved in thia method. When such heating would lead to significant hydrolysis of amides present, t h e diffusion should be carried out a t lower temperatures for correspondingly longer time intervals. The accuracy of the Sessler spectrophotometric method appears to be somewhat inferior to t h e within 1% accuracy observed in Conway’s and Kirk’s titrimetric procedures. T h e ease of manipulation in t h e present procedure, in which the color is developed directly in t h e absorption cell, is advantageous, however, when large numbers of microanalyses are to be run in a relatively routine way: The analyses of t h e large numbers of nitrogenoufi materials separated, for example, from chromatographic columns or from the products of tissue slice or homogenate metabolism could be facilitated b y this simple procedure. The ammonia can be stoichiometrically produced (3) from urea, amides, amines, total nitrogen, and nonprotein nitrogen. The microdiffusion technique has proved to be highly satisfac-

tory and convenient for preparing nitrogen samples for isotope ratio determination with t h e mass spectrometer. The ammonia sample is obtained directly in an interference-free form for t h e usual treatment with hypobromite (6) t o produce nitrogen gas. LITERATURE CITED

Conway, E. J., and Byrne, A., Biochem. J . , 27, 419 (1933). Hawk. P. B.. Oser. B. L.. and Summerson. mi. H.. “Practical Physiological Chemistry,” 12th ed., p. 1230, Blakiston, Philadelphia, 1947. Kirk, P. L., “Quantitative Ultramicrcanalysis,” Chap. 7 , Wiley, Kew York, 1950. Linderstrom-Lang, L., and Holter, H., 2. physiol. Chem., 220, 5 (1933).

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Seligson, D., and Seligson, H., J . Lab. Clin. M e d . , 38, 324 (1951). Sprinson, D. B., and Rittenberg, D., ,Vaval Medical Bull., Supplement on Preparation and LIeasurement of Isotopes, p. 82, 1948. RECEIYED for review July 15, 1954.

Accepted November 12, 1Q.54.

Procedure for Determination of Diffusion Coefficients of Gases and Nongaseous Solutes for Membranes SVEND G. JOHNSEN and JOHN ESBEN KIRK Division o f Gerontology, Washington University School o f Medicine, St. Louis, M o .

A procedure was developed for determination of the diffusion rates of gaseous and nongaseous solutes from one fluid phase to another through biological or inanimate membranes. The procedure permits determination under a constant total pressure of the diffusion coefficients for several solutes within a single experimen t

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ROCEDURES for determination of diffusion rates of gases through membranes have been described by Krogh (5) and

Wright ( 5 ) . The apparatus devised by Krogh permits measurement of diffusion rates of oxygen and carbon dioxide in separate experiments, whereas the apparatus used by Wright is applicable only to the study of the diffusion of carbon dioxide. T h e procedure desiribed in the present publication represents a further development of Krogh’s diffusion method. It permits measurement of the diffusion rates of gases and nongaseous solutes from one liquid phase to another through biological or inanimate membranes, utilizing the principle for sample transfer and gas analysis employed b y Kirk and Hansen (2). PROCEDURE

Apparatus. DIFFUSIONAPPARATUS.T h e details of the assembled diffusion apparatus are shown in Figure 1. The apparatus consists of two 50-ml. glass syringes (A. S. Aloe precision syringes): the bottoms of the barrels have been removed and the barrel tops have been ground plane. The membrane is interposed between the ground barrel tops of the syringes and is held in place by two metal diaphragms with circular openings and two hard rubber 1.25-inch slip joint washers. A cannula of stainless steel about 1 mm. in outside diameter (hypodermic needle, gage 19) is inserted through each rubber ring. The tips of the needles are cut off a t a right angle and do not extend beyond the inner surface of the rings. Airtightness of the perforation canal is ensured by sealing the place of entrance of the needle with cement (Sealit, Fisher Scientific Co.). The outer ends of the needles are closed by insertion of a oneway metal stopcock with two male outlets. Rotation of the diffusion apparatus is provided by an electric motor, as shown in the insert of Figure 1. A screw-shaped glass piece about 1 cm. in length, placed in each compartment, effects stirring of the solution during the rotation of the apparatus. The metal diaphragms used in the diffusion apparatus are

made of chromium-plated steel. Sets Kith beveled rircular openings 25, 20, 15, and 10 mm. in diameter have been found suitable. The rubber washers should be tested for gas permeability. With the hard-rubber washers used by the authors no measurable loss of gas was observed over a 2-hour period in experiments in which the compartments were filled with carbon dioxideaerated water and a steel plate interposed between the rubber rings. EQUIPMEXT FOR GASANALYSIS.The equipment for gas analysis includes one, or preferably two, Van Slyke manometric apparatus with extraction chambers provided with calibration marks a t 0.5-, 2.0-, 10.0-, and 50.0-ml. volumes. A stainless steel cannula 7 5 mm. in length and about 2 mm. in outside diameter (hypodermic needle, gage 15) with attached rubber tip is used for transfer of samples from the diffusion apparatus to the extraction chamber of the Van Slyke apparatus (2). Reagents. The reagents for gasometric determination of carbon dioxide, oxygen, and nitrogen are described by Kirk and Hansen (2). Technique for Use of Diffusion Apparatus. I n diffusion studies on animal membranes the experiments are preferably carried out under sterile conditions. After the membrane, the metal diaphragms, and the rubber washers have been inserted between the syringe barrels, the apparatus is screwed firmly together. The metal stopcocks and the outside parts of the needles are secured in a fixed position by means of thin copper wire. For evperiments on gas diffusion about 50 ml. of buffer medium are heated in a beaker t o 43” C., and the solution is aerated with the appropriate gas as described by Kirk and Hansen ( 2 ) . Thirty to 40 ml. of the solution are then poured into one of the compartments of the apparatus, the screw-shaped glass piece is placed in position, and the plunger is inserted. Anv free gas present is ejected, and the volume of solution introduced is determined by weighing. Buffer medium for the other compartment is then prepared and similarly introduced. The plungers require no special fastening, but n-ill stay in place during the rotation of the apparatus. The diffusion apparatus is finally placed horizontally in the belts of the rotator (see insert, Figure 1) in a thermostat a t 37” C., and rotation is started. It is convenient to place a shield of paper board or a rubber ring around each syringe barrel to keep the belts in place during the rotation. An elapsed time of 20 to 30 minutes is allowed to establish temperature equilibrium and initial penetration of the gas through the memhrane. The apparatus permits determination of the diffusion coefficients of several gases in the same experiment. Buffer medium aerated with oxygen may be used in one of the compartments,

V O L U M E 27, NO. 5, M A Y 1 9 5 5 and medium aerated with nitrogen or a nitrogen-carbon dioxide mixture in the other compartment. If the diffusion rate of carbon dioxide alone is studied, buffer medium aerated with this gas is employed on one side and unaerated medium on the other Bide. For experiments with nongaseous solutes a mixture of equal volumes of buffer medium and an isotonic solution of the compound t,o be studied is introduced in one of the compartments, and buffer medium is employed on the other side. At the end of the diffusion experiment the volume of solution remaining in each compartment is measured; this control ensures that holes in the membrane will not be overlooked. After the apparatus is disassembled, the membrane is removed. The part inside the openings of the diaphragms is carefully cut out and its area and weight are determined. Analytical Technique for Gas Determinations. The transfer of samples from t,he diffusion apparatus to the extraction chamber of the Van Slyke apparatus is carried out as described for Procedure B by Kirk and Hansen ( 2 ) . About 1 ml. of solution is ejected from the compartment of the diffusion apparatus Ilefore the introduction of the sample into the chamber. The exact measurement of the saniple and the gasometric analysis are performed as described previously ( 2 ) . In experiments in which the diffusion of oxygen and nitrogen are studied a 9-ml. sample is used for maximum accuracy. When carbon dioxide alone is studied samples of 1 t o 2 ml. may be employed without loss of arruracy. Interval between Withdrawal of Samples; If conditions of analysis permit, samples should be withdrawn in immediate succession from t8hetwo compartments of the apparatus a t the beginning and end of a diffusion period. This will be possible in t,he rase of diffusion studies on most nongaseous solutes and in investigations on gas diffusion when two Van Slyke apparatus are avai1:ible. If only one Van Slyke apparatus is available, an interval vorresponding to the time required for performing a gas analysis must elapse between the withdrawal of samples from the two compartments of the diffusion apparatus. This period is about 15 to 18 minutes in the case of oxygen and nitrogen determinations. and 5 to 6 minutes for carbon dioxide and bicarbonate deterndnations. The :ipproprinte length of each diffusion period depends on the

Figure 1. 1.

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: plullyc'

ss steel cannula (hypodermic needle, gage 19) a i t h tip cut off a t

riplit angle

area and thickness of the membrane, the diffusion coefficient of the compound, and the sensitivity of the analytical method. I n gas diffusion studies on animal membranes 3 to 4 sq. cm. in area and 0.04 to 0.08 cm. in thickness, a 1- to 2-hour interval has been found suitable. CALCULATION OF DIFFUSION COEFFICIENT

The diffusion coefficient is defined as the number of units of the substance diffusing through 1 sq. cm. of the membrane in 1 minute at a concentration gradient of 1 unit per nil. per cm. (1). For experiments in which samples are withdrawn in immediate succession from the two compartments at the beginning and end of a diffusion period the coefficient may be calculated from the equation given by Pletscher and coworkers ( 4 ) :

where k = diffusion coefficient cI = concentration of solute on donor side at beginning of period cz = concentration of solute on recipient side a t beginning of period cg = concentration of solute on donor side at end of period c4 = concentration of solute on recipient side at end of period A = area of membrane in square centimeters L = thickness of membrane in centimeters VI and TiZ = volumes of solution in the two compartments expressed in milliliters t = time in minutes In diffusion studies in which an interval elapses between the withdrawal of samples from the two compartments of the apparatus another formula for calculation of the diffusion coefficient must be employed:

whwr q represents the decrease in quantity of the compound

Schematic illustration of assembled diffusion apparatus

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4. 5. 6.

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7. One-way metal stopcock with two male outlets 8. Screwshaped glass piece t o provide stirring of solution 9. Wooden clamp with brass screws 10. Motor 11. Metal axis 12. Belt 13. Paper board shield

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ANALYTICAL CHEMISTRY

(expressed in units) on the donor side or the increase in quantity on the recipient side, and d is the mean concentration difference (expressed in units per milliliter) during the diffusion period. The value of d is obtained by plotting the observed concentrations on the donor and recipient sides on graph paper in relation to time. Smooth curves are then drawn to connect the values on each side, and the mean concentration difference is calculated from the area limited by the curves. Correction for Tissue Respiration. I n diffusion studies on living membranes some oxygen will be used by the respiration of the tissue. If the experiment is carried out by withdrawal of samples in immediate succession from the two compartments, no correction for the tissue respiration is necessary if the volumes of fluid on the two sides are the same, the supposition being made that an equal amount of oxygen is consumed on both sides of the membrane. I n experiments in which a time interval elapses between the withdrawal of samples from the two compartments, the value of p in Formula 2 must be corrected for the amount of oxygen consumed by the tissue. If it is aPsumed that the oxygen used for the tissue respiration on the average has diffused through half of the membrane, the correction to be applied is equal to one half of the difference between the decrease in quantity of oxygen on the donor side and the increase in oxygen on the recipient side. If a solution aerated with pure carbon dioxide is used in the donor compartment of the apparatus, the respiratory carbon dioxide can be disregarded.

DISCUSSION

The main advantages of the present procedure as compared with previous methods are the following: It permits determination of the diffusion coefficients of several solutes in the same experiment; it provides for the withdrawal of samples for analysis a t any time during an experiment,, thus making it possible to carry out successive diffusion periods within a single experiment; it permits an accounting for the diffusing quantities of a compound by comparison of the amounts disappearing on one side of the membrane with those appearing on the other side; and it involves the use of easily movable syringe plungers, thus ensuring against differences in total pressure on the two sides of the membrane. LITERATURE CITED

(1) Hill, A. V., Proc. Roy. Soc. London, 104B,39 (1928-29). Kirk, J. E., and Hansen, P. F., J . Bid. Chena., 199, 675 (1952). (3) Krogh, A,, J . Physiol., 52, 391 (1918-19). (4) Pletscher, h.,Staub, H., Huneinger, W., and Hess, W., H e h . Physiol. et Pharmacal. Acta, 8 , 306 (1950). (5) Wright, C. I., J. Gen. Physiol., 17, 657 (1933-34). (2)

RECEIVED for review September 7, 1954. Accepted December 13, 1954, Investigation supported in part b y a research grant (PHS-891) froin the National Heart Institute of the National Institutes of Health, Public Health Service. A demonstration of the technique was given a t the 43rd meeting of the American Society of Biological Chemists, April 14 t o 18, 1952 S e w York. N. Y.

Effect of Particle Size on the Characteristics of Silicic Acid Chromatographic Adsorbent EARL W. MALMBERG McPherson Chemical Laboratory, The O h i o State University, Columbus, O h i o

The effect of particle size on the efficacy of silicic acid in affording chromatographic separations has been studied with the purpose of obtaining the best adsorbent from commercially available materials. Procedures of sieving and ball-milling have been used to provide samples whose effectiveness was tested by their ability to separate the 2,4-dinitrophenylhydrazonesof acetaldehyde and formaldehyde and the dinitrophenylosazones of glyoxal and methyl glyoxal. A procedure for obtaining a satisfactory adsorbent from a commercially available silicic acid is described.

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HE influence of physical characteristics on the behavior of

chromatographic adsorbents is geiierally ignored until difficulties arise which necessitate bringing certain variables under control. T h e influence of particle size on the chromatographic behavior of silicic acid is t h e subject of t h e present investigation. For some years commercially available silicic acid has been used as a chromatographic adsorbent and has given uniform characteristics except for certain variations in adsorptive strength which are to be expected. Recently t h e samples of silicic acid from t h e usual source have been completely different in particle size and apparently in their adsorption characteristics in general. Studies of particle size distribution, grinding, and chromatographic separations were made on various samples of silicic acid. A procedure for preparation of a very satisfactory adsorbent from a commercially available Mallinckrodt silicic acid is described as devised from these investigations.

GENERAL EXPERIMENTAL PROCEDURES

Throughout this work two rather sensitive chromatographic problems, the separation of the 2,4-dinitrophenylhydrazones of formaldehyde and acetaldehyde and of the 2,+dinitrophenylosazones of glyoxal and methylglyosal, were used as criteria of the chromatographic power of a n adsorbent. These tests are referred to below simply as the formaldehyde-acetaldehyde and glyoxal-methylglyoxal separations, respectively. T h e procedure (1) for the formaldehyde-acetaldehyde separation requires a prewash of Vlj0ml. ( 2 ) of 10% of acetone in ligroin (Skellysolve B), 0.5 mg. each of hydrazones in 5 ml. of 1 : 4 chloroform-ligroin (on a column of 14-mm. inside diameter), and development with 10% of ether in ligroin. Some of the best adsorbents did not, require the prewash, but even with the best the separations were improved. The osazones, 0.1 mg. of each in 1 to 14 nitrobenzene-benzene, were placed on a column wet with benzene and developed viith a solution of 5% of ether in 1 to 1 benzene-ligroin. This system has a complicating feature: Some adsorbent,s n 3 l not afford a satisfactory separation under any conditions, while other samples ail1 give a good separation a t slow flow rates but not a t fast. From the profiles of the zones, this rat,e effect seems to be a result of a slow rate of desorption for some of the adsorption sites. The ball-milling of the adsorbent was done in a Paul 0. Abbe mill of 6-gallon size, 55 r.p.m. The mill xvas charged wit8h 0.5 kg. of silicic acid and 1.5 kg. of 1-inch porcelain balls. Combinations of larger amounts of material and balls were found to be ineffective. I n all cases the adsorbent was heated 4 hours a t 200" C. before testing. Each column was packed with the tube in position on a suction flask, the adsorbent being poured in with a stopcock on a safety bott.le open to the atmosphere; after initial set'tling was completed, the stopcock was closed, and as the vacuum was established, the column further contracted. T o prevent surface spreading, the upper surface of the adsorbent must be pressed