results on four compounds, including acetanilide, so treated. Consequently, the amount of sulfuric acid was increased t o 1.5 ml. which gave a mixture that did not become dry, and which when boiled vigorously, attained a temperature of only about 340’ C. (section A, Table I). The main disadvantage of the zinciron reduction method is the length of time required. However, because a large number of reductions and subsequent digestions can be done simultaneously, the time element becomes important. The method has the advantages of requiring only the regular Kjeldahl equipment and also, that certain types of compounds-for example, hydrazines, nitro compounds, and others-mhich are apt to be troublesome when analyzed by the Dumas method are easily handled by the zinciron reduction method with accurate results. EXPERIMENTAL
Apparatus. The apparatus has been described (16), the one-piece distillation unit being preferred for distillation. Procedure for Determination of Nitrogen in Azo and Nitro Compounds, Oximes, Isoxazoles, Hydrazines, and Hydrazones. From 5 t o 8 mg. of sample, 0.2 ml. of 98 to 100% formic acid, and 0.1 ml. of concentrated hydrochloric acid, specific gravity 1.18, in a 30-ml. micro-Kjeldahl flask (preferably, Soltys type) (12), are heated in a water bath (80’ to 85’ C.) until the sample is dissolved. To this are added 80 mg. of zinc dust (nitrogenfree), and the contents mixed by swirling for about 2 minutes, and heated in the mater bath for 5 additional minutes. Then 40 mg. of iron powder (prepared by hydrogenation) are added and the
flask is swirled for 2 minutes to effect mixing. To this are added 0.1 ml. of concentrated hydrochloric acid, specific gravity 1.18, and 0.15 ml. of ethanol, and the mixture is again heated for 5 minutes (water bath). Every 5 minutes a 0.1-ml. portion of concentrated hydrochloric acid is added, with swirling and heating in the water bath, until the iron is dissolved. While the flask is swirled under a hood, 1.0 ml. of concentrated sulfuric acid, specific gravity 1.84, is added (cautiously), and the contents are warmed until the evolution of gas (hydrogen chloride) has subsided. Then 0.65 gram of potassium sulfate, 0.016 gram of mercuric oxide, and, finally, 0.5 ml. of concentrated sulfuric acid, specific gravity 1.84, are added. (It is preferable to add the sulfuric acid in this manner rather than all a t once, because the final 0.5 ml. can be used to wash down any materials adhering to the walls.) The mixture is boiled on a microKjeldahl digestion rack for 4 hours, making certain that boiling is vigorous enough so that refluxing takes place almost half-may up in the neck of the flask. The digest is transferred to the micro-Kjeldahl distillation apparatus (preferably the one-piece model) ( l a ) , 7.5 ml. of sodium hydroxide-sodium thiosulfate mixture (12) are added, and the ammonia is distilled out into boric acid solution (12) (8 minutes with the delivery tube below the boric acid solution, 2 minutes with it above). The distillate is then titrated with 0.OliV hydrochloric acid, using bromocresol green-methyl red mixture (16) as the indicator. A blank determination should be run using all of the materials, minus the sample, and going through the entire procedure. Any blank value so obtained should be subtracted. Procedure for Determination of Nitrogen in Nitrates. I n a 30-ml. micro-Kjeldahl flask (12) are placed 5 to
8 mg. of sample, 35 mg. of salicylic acid, and 1 ml. of concentrated sulfuric acid, specific gravity 1.84. The mixture is allowed to stand a t room temperature for 30 minutes, during which time nitration of the salicylic acid takes place. Then 100 mg. of crystalline sodium thiosulfate are added and the mixture is allowed to stand a t room temperature for an additional 10 to 15 minutes. Finally 0.65 gram of potassium sulfate and 0.016 gram of mercuric oxide are added, and the mixture is boiled on the digestion rack in the usual manner for 4 hours. Distillation, titration, and blank determination are done as described. LITERATURE CITED
(1) Baker, P. R.
W.,Analyst
80, 481
(1955). (2) Bradstreet, R. B., ANAL. CHE~U. 26, 185 (1954). (3) Ibid., p. 235. (4)Clark, E. P., “Semimicro Quantitative Organic Analysis,” pp. 37-43, dcademic Press, New York, 1943. (5) Dickinson, W. E., ANAL. CHEM.26, 777 (1954). (6) Ibid., 30, 992 (1958). (7) Friedrich. A.. 2. .phwsiol. Chem. 216, ” 68 (1933). ’ (8) Furman, N. H., ed., “Standard Methods of Chemical Analysis,” bv TV. W. Scott, 5th ed., Vol. 11, p. 633. vnn Nostrand, New York, 1939. (9) hla, T. S., Lang, R. E., hlcKinley, J. D., Jr., Mikrochim. Acta 1957, 368. (10) Ogg, C. L., Willits, C. O., J . Assoc.
0 , f i c . Agr. Chemists 35, 288 (1952). (11) Steyermark, A., Zbid., 38,367 (1955); 39, 401 (1956); 40,381 (1957); 41,297,
299 (1958).
(12) Steyermark, .4.,“Quantitative Organic Microanalvsis.” m. 134-55. Blak&ton, Philadelpfiia,’19’ji. (13) Willits, C. O., Ogg, C. L., U.S. De-
partment of Agriculture, private communication. RECEIVED for review February 18, 1958. Accepted May 21, 1958.
Determination of Total Protein in Cerebrospinal Fluid by an Ultramicro-Kieldahl Nitrogen Procedure WALLACE W. TOURTELLOTTE, JULIUS A. PARKER, R. ERIC ALVING, and RUSSELL N. DeJONG Deparfmenf of Neurology, Universify o f Michigan, Ann Arbor, Mich.
b An ultramicro-Kjeldahl procedure is presented which is capable of determining 28 y of protein (approximately 4 y of nitrogen) with a coefficient of variation of loyo; in the working range, 200 y of protein, the coefficient is less than 1%. The procedure is unique in that the collecting flask for potentiometric titration is attached to the still and that the anhydrous ashing mixture is useful for carrying out digestion in the same tube in which the pro-
tein is precipitated and freed of nonprotein nitrogen. Several tests of the validity of the procedure are presented.
T
HE Kjeldahl method has been the standard by which all protein determinations on the cerebrospinal fluid are evaluated; previously adaptations have required from 2 to 15 ml. of fluid. The micromethod of Peters and Van
Slyke (9) can be carried out with only 2 ml., but Samson (10) showed that 10 ml. of fluid were necessary for reproducible results. The method described permits the determination of 28 y of protein, the amount present in approximately 0.2 ml. of normal cerebrospinal fluid. The total protein is separated from other nitrogenous materials by precipitation with trichloroacetic acid. Results are most consistent if the precipitation is VOL 30,
NO. 9,
SEPTEMBER 1958
1563
carried out before the nitrogen is determined (3, 4). The difficulties in the Kjeldahl determination are caused by digestion. Kirk ( 7 ) stated that a combination of mercury with selenium catalysis, properly used, is superior to either alone for quantitative digestion of proteins. An anhydrous digesting solution which contains both mercury and selenium makes it possible to digest without bumping in the same small tubes in which the protein is precipitated REAGENTS
Water, distilled from alkaline permanganate in an all-glass conductivity still, is used throughout. Trichloroacetic acid, 5 and 55%. Reagent grade acid is vacuum distilled at 148' to 153' C., and the purified crystals are dissolved in a minimum amount of water. About 1500 grams will dissolve in 120 ml. of mater in about 1 week. The concentrated solution is diluted 1 to 10 and is titrated against 0.977N sodium hydroxide t o the phenol red end point. From the normality of the concentrated solution, the grams of trichloroacetic acid per 100 ml. are calculated. Appropriate dilutions are made to give 5 and 5574 trichloroacetic acid. Digesting mix. After 6 grams of anhydrous potassium sulfate, reagent grade, are made t o 25 ml. with reagent grade sulfuric acid, 25 mg. of crystalline Mercurochrome and 5 pl. of selenium oxychloride are added. Sodium hydroxide, 40%. Reagent grade sodium hydroxide pellets, 40 grams, are made to 100 ml. with water. Sulfuric acid, 0.0200N. The normality should be determined precisely. Sodium hydroxide, O.OlOON. This reagent should be carbonate-free and the normality should be determined precisely prior to every refilling of the gas-free microburet reservoir. Ammoniuni sulfate standard. Reagent grade ammonium sulfate is dried t o constant weight and dissolved in 0.l.h' sulfuric acid to make 400 y of nitrogen per ml. Lang-Levy pipets (Microchemical Specialties Co., 1834 University Ave., Berkeley 3, Calif.) were used throughout ( 8 ) . Reaction tubes, 12 x 7 5 mm. rimless culture tubes of borosilicate glass. Mixing is accomplished with the equiDment described bv Lowrv and his associates (8). An electric ashing. furnace (Microchemical Specialties "Co.), A sheet of Transite (Johns-hIanville Co., S e w York, S , Y , ) , 3/8 inch thick with 12mm. holes countersunk on the underneath side, was substituted for the face of the oven to adapt this oven to the heating of the small reaction tubes. The small reaction tubes are held in place with a bent metal pencil clip. An exhaust manifold is not required. Transferring device. The easily constructed transferring device described by Sperry ( 1 2 ) was modified by substituting a 19/22T ground-glass joint for the rubber stopper (Figure I). 1564
ANALYTICAL CHEMISTRY
Figure 1. Gas-free reagent vessel added to Gilmont ultramicroburet (left), schematic drawing of ultramicro Kieldahl still and collecting vessel (center), and transferring device (right)
Micro-Kjeldahl vessels, 10-nil. capacity with 19/22 T (Microchemical Specialties Go.). Ultramicro-Kjeldahl apparatus. A Kjeldahl apparatus designed by Jenden and Taylor (5) (Microchemical Specialties Co.) was modified as shown in Figure 1. It was convenient to add a steam generator, A . Trap B functions as a steam splash trap and C regulates the steam pressure. A buret, D , to deliver sodium hydroxide through stopcock E was attached to neutralize the diluted acid digest prior to distillation in a closed chamber; this prevented the escape of ammonia. The valve, F , is similar to that used by Jenden and Taylor. Trap J functions as a distillate splash trap. The most prominent improvement is in the method of trapping the distillate. The first few drops of distillate form an effective liquid seal in the condenser, K , because of the introduction of an eccentric glass bead, L. Furthermore, the distillate is immediately neutralized by collection in flask, M, approximately 15 ml. containing acid which is rapidly stirred. The stirring is accomplished by driving a flea, S,with an Alnico button magnet, 0 (Grneral Magnetic Corp., Detroit, Xich.), mounted on a phonograph motor. The orifice, R , of collecting flask ,If permits the tip of a microburet to be placed under the distillate for back-titration of excess acid. A Beckman p H meter Model G was used for potentiometric titration. The glass electrode and saturated potassium chloride reference cell are indicated by P . The electrodes are secured t o collecting flask M by rubber gaskets, Q. It was necessary to shield and
ground the leads from the electrodes to the meter. Gilmont ultramicroburet (The Emil Greiner Co., 2C-26 Korth Moore St., Xem York 13, S . Y . ) . T o the buret (Figure 1) was added a gas-free reservoir, W , betveen the mercury micrometer drive, T,and the delivery tip, S, to facilitate filling and to store sodium hydroxide. The delivery tip of the buret was drawn to a fine tip and bent t o adapt the delivery end for titration in the collecting vessel. To fill reservoir W , the ground-glass joint, Y , is connected to the stock sodium hydroxide bottle (not shown). The two-way stopcock, X , is turned to admit the stock sodium hydroxide through Y to W . h mercury reservoir (not shown) attached to is then lowered and the sodium hydroxide is drawn into reservoir W . Then stopcock X is closed and the mercury reservoir is raised. The leader from the stock sodium hydroxide bottle is disconnected a t Y . The sodium hydroxide remaining in Y is aspirated and mercury is replaced. The micrometer drive is reset t o zero, and stopcock X is turned to connect W with the buret which automatically fills, Stopcock X is turned to connect the buret with Y which forms a mercury seal in the stopcock. Stopcock X is then closed. A coarse sintered glass filter, Ti, is attached to a vertical manometer. PROCEDURE
I n a reaction tube, 1 ml. of cerebrospinal fluid is mixed without foaming with 222 pl. of 55% trichloroacetic acid. allowed to stand for a t least 10 minutes, and centrifuged for 15 minutes at 2600 r.p.m. The centrifuge is allowed to coast to a halt. The supernatant fluid is removed by aspiration with a fine tipped pipet. The residue is then extracted once with 1 ml. of 5% trichloroacetic acid. T o the extracted protein residue 100 pl. of digesting mix are added and miued. The tubes are heated on the oven a t a temperature (300" to 320" C.) sufficient to reflux the sulfuric acid to a height of about 1 inch but not hot enough to drive off sulfur trioxide fumes. Total digesting time is usually 2 hours or about four times the clearing time if the latter is prolonged. The cooled digested contents of the reaction tube are transferred to the microKjeldahl distilling vessel by the transferring device (Figure 1). TO use this device, several drops of water are added to the micro-Kjeldahl vessel, I . With the vacuum on, the micro-Kjeldahl vessel on G, and the tip of device in the reaction tube (not shown), the sample is transferred by placing a finger over orifice 0. The transfer is repeated three times with 1 ml. of water each time. The ground-glass joint, G, of the micro-Kjeldahl vessel is moistened with 40% sodium hydroxide before the vessel is attached to the still by spring H . Before distillation is begun, the trapping acid (0.5 ml. of 0.02N sulfuric acid) should be introduced into flask 41, stirring started, and outlet
R loosely covered. The distillation procedure is begun with stopcock C open. Sufficient 40% sodium hydroxide is added from buret D to alkalinize the digested sample in the micro-Kjeldahl vessel, I ; 0.5 ml. is sufficient when 100 pl. of digesting mix are used. Stopcock C is then closed, diverting the steam from flask -4 through the apparatus. Distillation should be a t the rate of 2 to 3 ml. per minute until the distillate covers the active surface of the p H electrodes, P . The same amount of distillate, 8 ml., should be collected each time, The excess acid is then titrated over 3 p H units to pH 6.0 by introducing the tip of the ultramicroburet through orifice R. The tip of the buret should be below the liquid surface. After titration of the excess acid in collecting flask M , the fluid is aspirated. The flask is filled with water and aspirated three more times. No traces of ammonia could be detected after three successive rinses; furthermore, it was not necessary to steam rinse the still between successive distillations. Blank determinations should be run with each series of distillations. This is accomplished by adding 100 p1. of ashing mix to clean reaction tubes, digesting, transferring t o micro-Kjeldahl vessels, neutralizing, distilling, and titrating as described. The normality of the sodium hydroxide is then recalculated on the basis that the more stable standard sulfuric acid solution has not changed. This normality of the sodium hydroxide is used for the usual Kjeldahl calculations. The exact determination of the normality of the sulfuric acid and sodium hydroxide can bP avoided by determining a nitrogen standard curve with each series of distillations. Three different concentrations of ammonium sulfate standard are dried in a centrifuge evaporator (1) and carried through the entire procedure. The equation of the line is calculated ( 8 ) . X is the micrograms of nitrogen and Y is the microliters of sodium hydroxide used for the blank titration minus the microliters of sodium hydroxide used for the back-titration of the saniplei.e., the amount of sulfuric acid, which is expressed in microliters of sodium hydroxide, neutralized by the ammonia. The micrograms of nitrogen in the unknown sample are calculated by substituting the experimentally determined value for X and solving for 1'. Xicrograms of protein nitrogen in the sample are converted to micrograms protein by multiplying by the arbitrary factor 6.25. VALIDATION
OF
METHOD
The precipitation of cerebrospinal protein, the removal of nonprotein nitrogen, and the conversion of the protein nitrogen to ammonia were carried out in 3-ml. tubes. Significant amounts of \yater in the small reaction tubes cause splattering during digestion; this is avoided when the nitrogen content of solutions is determined by a preliminary drying step (1). It was necessary to use an anhydrous ashing
mixture, too. Sulfuric acid has been used with or without added catalysts as a digesting agent, but more complete digestion Jvas obtained when selenium and mercury were used as catalysts. Selenium oxychloride (SeOC12) is readily soluble in sulfuric acid. Mercuric sulfate is almost insoluble in concentrated sulfuric acid,
