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INDUSTRIAL AND ENGINEERING CHEMISTRY
proper size of tube required for most accurate results. I t has been the author’s experience that, when capillaries of less than 0.3-mm. internal diameter are used, the boiling point is too low; with larger capillaries, the boiling point is t,oo high (Table III). LITERATURE CITED
(1) Benedict, H.C . , IND. ENO.CHEM.,ANAL.ED., 2,91 (1930). (2) Dowzard, E., and Rusao. M.. Ibid., 15,219 (1943).
Vol. 16, No. 2
(3) Emich-Schneider, “Microchemical Laboratory Manual”, pp. 118-35, New York, John Wiley & Sona, 1932. (4) Gettler, A. C.,and Fine, J., IND.ENG Cmm., ANAL.ED., 11, 469 (1939). (6) Hubacher, M. H., Ibid.. 15,448 (1943). (6) Morton, A. A., and Mahoney, J. F., Zbid., 13,494 (1941). (7) Morton, A . A., Mahoney, J. F., and Richardson, Graham, Ibid., 11, 460 (1939). (8) Shriner and Fuson, “Identification of Organic Compoundd’, 1st ed., p. 51, New York, John Wiley & Sone,’1935.
A DIALYSIS CELL For Rapid Quantitative Analytical Determination of Diffusible Components in Blood Plasma PAUL B. HAMILTON AND REGINALD M. ARCHIBALD P i , N. Y.
Hospital of Rockefeller Institute for Medical Research, N e w York
A simple dialysis apparatus is described which in P to 3 hours provides quantitative equilibrium of diffusible constituents in the system and a dialyzate convenient for analyses.
Tit HE
dialysis cell here described has been of use in a variety of procedures. The technique is especially applicable when is desired to prepare a protein-free solution of dialyzable components with any one or a combination of the following conditions: minimal dilution, absence of foreign ions, or exposure to acid or alkaline reagents or to reagents capable of denaturing protein. The apparatus has proved so simple and effective that it can be recommended for general use in analytical dialysis.
cates that the diaphragm is intact and secure. Sausage casing (27/32) has proved satisfactory (supplied by the Visking Corp., 6722 West 65th St., Chicago, Ill.). Some cellophane products are, however, impermeable to water and of course are useless for this purpose. A simple test of permeability is to introduce a measured volume of 0.1 N hydrochloric acid into the central tube and dialyze against 2 volumes of distilled water. If the intact membrane is permeable, at equilibrium the concentration of acid throughout will be 0.0333 N .
APPARATUS
The dialysis cell consists of a small wide-mouthed bottle of about 120-cc. capacity, closed by a So. 8 stop er, through which passes a straight glass tube of 28-mm. out& diameter and 11 cm. long. The central tube is closed a t its lower end by a cellophane diaphragm held securely in place by many turns of an elastic band, and a t the top by a S o . 5 rubber stopper. Both rubber stoppers have intravenous needles (No. 18) passing throu h to allow equilibration of pressure with the atmosphere wikout loss of liquid by evaporation. The apparatus is assembled as shown in Figure 1. The technique is like that employed by Hamilton and Van Slyke (1). Two cubic centimeters of plasma were pipetted into the central tube, a glass marble was introduced to give mechanical stirring as described by Northrup and Kunitz (S), and the central stopper was set in place. Eleven cubic centimeters of distilled water were pipetted into the bottle, and the central tube was pushed into place with glycerol as lubricant, and pushed down till the diaphragm was within 1 or 2 mm. of the bottom of t,he bottle. The bottle was then gently rocked for 2.5 hours, by which time all diffusible amino acids had become u n i f o r m l y d i s t r i b u t e d throughout the total 13 cc. of liquid in the system. A convenient rocking device is depicted in Figure 2. Each assembled cell is secured to a narrow board by a stout elastic band. The board is rocked back and forth by B windshield wiper motor, as described by Kunitz and Simms ( 2 ) . There are several points to note in the operation of the cell. The diaphragm is tested for leaks by immersing it in water and blowing down the open end of the tube; Figurel. DialysisCell the absence of any bubbles indi-
Figure
2. Rocking Device for Dialysis Cell
If the cell is used to prepare dialyzates suitable for ultraviolet spectrographic analysis it is essential to wash the membrane free of substances that absorb light in the ultraviolet. The blanks are easily reduced to low constant amounts by cleansing the membranes in four washes of 100 cc. each of distilled water: each wash is continued for 3 hours with gentle rocking, For exact quantitative York it is necessary to avoid isolation of droplets on the glass walls. This may be accomplished by having the glass parts, especially the central tube, scrupulously clean and free from grease, or the central tube may be coated within and without by a layer of paraffin. In this latter case the glass marble provides the wettable Furface on which to drain the pipet. In this apparatus a shift of fluid across the membrane in either direction does not influence the final concentration of a freely diffusible constituent (one independent of a Donnan equilibrium), since its concentration will be equal on both sides of the membrane. The length of time a cell has to be rocked, in order to have equilibrium of diffusible amino acids throughout the total fluid volume, was established by dialyzing 2 cc. of plasma against 11 cc. of water. Cells were rocked for 15, 30, 45, 60, 90, 120, 150, 180, and 310 minutes and the dialyzate mis analyzed for free alpha-amino acids by the ninhydrin-carbon dioxide method (1). All analyses were in duplicate. Figure 3 shows that equilibrium across the membrane with respect to diffusible free alpha-amino acids was achieved within 2 hours of rocking,
ANALYTICAL EDITION
February, 1944
137
2.5 hours against 11 cc. of distilled water. Free amino acids were determined on aliquot portions of the dialyzate by the ninhydrin reaction ( 1 ) and were compared with amino acid concentrations found by analyses of plasma filtrates obtained by protein precipitation with picric acid (1) (see Table I). With a plasma-water ratio of 2 to 11 a 5 4 0 . aliquQt of the dialyzate contains the amino acids from 0.77cc. of plasma. If, however, a more concentrated dialyzate is required, the ratio of plasma to water could be made 12 to 6, so that a 5-cc. aliquot of the dialyzate would contain amino acids from 3.33 cc. of plasma. Another experiment exemplifies separation of free amino acids from egg albumin.
.$0 . 5 r 0
I
l
l
l
30
60
l
l
I
I
I
I
90 120 150 Time in minute3
Figure 3.
I
l
180
i 210
Time of Rocking
Maximum constant value obtained a k a P boun' dirlysir indicates equilibrium d dlffurlblc amino rcidr
Table
1.
Dialyzate 1
2 3
Av. Picrio said filtrate
Free Amino Acids Alpha-Amino Acid Nitrogen Mg./iOO CC. plaama 4.64 4.71 4.66 4.67 4.67
Table
II. Amino Acids in Egg Albumin Alpha-Amino Acid Nitrogen M g . / 1 0 0 cc. plasma
From analysis of dialyzate of egg albumin plus added amino acids From analysis of dialyzate egg albumin Added amino acid N by difference Amount of added amino acid N
2.88 0.61 2.37 2.44
To a 7% e g albumin solution was added an amino acid mixture o b t a i n e j by the hydrolysis of edestin with strong hydrochloric acid. The amount of amino acid alpha-nitro en added, calculated from results of a ninhydrin-carbon dioxije analysis on the hydrolyzate (4),was 2.44 m per 100 cc. After 2.5 hours of dialysis aliquot portions of t%e dialyzate were analyzed (Table 11). LITERATURE CITED
The use of the dialysis cell in quantitative analytical analysis is demonstrated by the following experiments: To illustrate the precise quantitative analytical data that can be obtained by means of the apparatus, three separate dialysis units were set up with 2-cc. portions of human plasma dialyzed
(1) Hamilton, P. B., and Van Slyke, D. D., J . B i d . Chem.. 150, 231 (1943). (2) Kunitz, M., and Simms, H. S., J . Gen. Physiot., 11, 641 (1928). (3) Northrup, J. H., and Kunitz, M., Ibid., 9, 351 (1926). (4) Van Slyke, D. D., Dillon, R. T., MacFadyen, D. A., and Hamilton, P. B., J. Bid. Chem., 141, 627 (1941).
Determination of Small Amounts of Molybdenum in Plants and Soils M. L. NICHOLS AND LEWIS H. ROGERS, Cornell University, Ithaca, N. Y. Spectrographic, colorimetric, and polarographic procedures For the determination of small amounts of molybdenum in plants and soils have been studied. I t i s concluded that, for the ordinary laboratory, the colorimetric procedure i s superior if reasonable amounts of sample (1 gram or more of soils, 10 grams or more OF air-dried plant material) are available. However, iF only small amounts of sample are available (100 mg. of soil, 1 gram of air-dried plant material), the spectrographic procedure is recommended. The polarographic procedure has no particular advantages over the other two.
.
EVERAL recent papers (1, 4, 20) have indicated that molybdenum will need t o be considered in future studies on the role of various elements in plant and animal nutrition. In one case (1) it was thought to be essential for plant growth; in another case (4) excessive quantities had a deleterious effect on cattle. The work reported here was undertaken as a result of two observations on the occurrence of this element in plants and soils. In one study (16) it was found that many mineral soils in Florida contained no spectrographically detectable molybdenum. In another study, certain Florida muck soils and some plants grown thereon showed readily detectable quantities of this element (unpublished data). These data made it desirable t o study the methods of analysis for very small amounts
S
of molybdenum with respect to their precision, sensitivity, and other factors. The methods for the quantitative determination of molybdenum include gravimetric, volumetric, spectrographic, colorimetric, and polarographic procedures. The three latter methods should be more suitable for the determination of very small amounts. Spectrographically, molybdenum may be determined by a variety of procedures, but there is a growing tendency among workers in this country to use microphotometric methods, with either an internal standard line of the matrix material or an added line standard. The essential feature of this latter method is the introduction into the sample in constant known amounts of an element not originally present. This added element furnishes spectrum lines of constant intensity which, measured with a microphotometer in comparison to the line intensities of the unknown element, give a method of determination. Colorimetrically, molybdenum is most often determined by the yellow-amber color of its thiocyanate, either directly in the original solution or by first extracting with an organic solvent, immiscible with vvater (6, 16). Several reducing agents have been used, but stannous chloride has been employed more often than the others. The reaction is affected by several compljcat-
