Apparatus for Continuous Delivery of liquid at Constant Rate A. R. RICHARDS', Trinidad Leaseholds, Ltd., Pointe-a-Pierre, Trinidad, B. W . I .
w
amount, provided that gas is escaping slowly from the bubbler. Tube L should be of the same diameter as K ; if the pressure in E can be maintained constant by any external device, the closed tube, L, and the connection to E are unnecessary. The gas from J is admitted to either of reservoirs A or B, or to both, through the common 3-way cock, M . Cocks N a n d 0 communicate with the capillary leaks, P. These are unnecessary if the pressure in E is constant. When delivery from A is almost complete, B is filled and cocks G and S are closed. Anv increase of Dressure in E raises the liauid level in K and the pressure in J is-increased an equal a m o h t . Gas passes through cock M and bubbles through the charge and so increases the pressure in A . Thus the li uid head across D is maintained. If the pressure in E is reduce1 the pressure in!J is reduced, but unless gas leaks through N the gas pressure above the charge in A will not be reduced. Therefore the leak should be adjusted so that the rate of pressure drop in the empty reservoir with M closed is a t least equal to the maximum rate of pressure drop in E. I n practice this is slow-rapid pressure fluctuations in E do not reach J,as they are damped by the capillary, &. To change reservoirs M is slowly turned, so that J is in communication with both A and B and 0 is opened. There is a slight surge of liquid from A if M is not turned slowly, and it is advisable to locate the cock well above the bottom of the reservoir as shown. Cis then slowly turned, so that both reservoirs are in communicstion with D. At this stage although the liquid levels in A and B are different and J and D are in communication with both reservoirs, there is no relative change in liquid levels. Delivery will take place from both reservoirs if required. Cocks C, M , and N are turned to isolate reservoir A and complete the change-over. The volume of liquid in B should be read before the chenge-over and the volume in A after. A may now be refilled, the gas being vented through R.
HEN a large quantity of liquid is to be delivered continuously it is common practice to provide two containers, refilling one while liquid is being delivered from the other. However, there is a possibility that the operation of the system will be disturbed while the delivery rate from the refilled reservoir is being adjusted. This is particularly irksome when the delivery rate is low or the setting of the control cock is difficult, because it requires some time t o detect and then'to correct the changed rate. The simple apparatus shown in Figure 1 overcomes this difficulty by maintaining a constant liquid head across the control cock. It has been applied successfully to continuous fractionating columns and to continuous reactors. "
F
Y
Y G
Figure 1
The advantages offered over two independent constant feed reservoirs are: maintenance of feed rate without resetting the control cock; independence of pressure changes on the delivery side of the device; reduction of the reservoir size with consequent reduction of fire hazard if inflammable liquids are being used. As a result, the reservoirs may be thermostated economically.
The two calibrated reservoirs, A and B, discharge through the common 3-way cock, C, and control cock D into the drop counter and sight glass, E. The reservoirs are filled through cocks F and G.( ,:Air or an inert gas is admitted through cock H into tube J, where it is maintained a t a pressure determined by the depth of immersion of t h e tube in the water or mercury bubbler, K . This is, in turn, dependent on the pressure in L and in the sight glass, E, so that the pressure in J exceeds that in E by a constant 1
ACKNOWLEDGMENT
Thanks are due to the management of Trinidad Leaseholds, Ltd., for permission to publish the details of this apparatus.
Present address, Caribbean Development Co., Port-of-Spain, Trinidad,
B. W. I.
Micro-Kjeldahl Determination of Nitrogen Use of Potassium Biiodate in Iodometric Titration of Ammonia ROBERT'BALLENTINE~ AND JOHN R. G R E G G ~ Department of Zoology, Columbia University, New York, N . Y . tion of the particular procedure employed. However, the Kjeldah1 method, like any other, is of real value only when performed under carefully standardized conditions which f i s t must be determined if dependable results are to be obtained. These conditions necessarily vary with the apparatus and reagents used, with the material to be analyzed, and with the various manipulstiona in the whole analytical process (5). The data reported here have been obtained in working out a modification of the Kjeldahl method for use in a study of the nitrogen metabolism of amphibian embryos (1). The chief fea-
H E Kjeldahl method for the determination of nitrogen has Tbeen widely used in biological chemistry, and a great many modifications have been described. I n spite of the fact that it is largely empirical, its almost universal acceptance has caused its accuracy tqo often to be taken for granted. Work whose final validity has depended on the accuracy of nitrogen determinations often hss been published without any apparent critical examina-
* Present addresa, California Institute of Technology, Pasadena, Calif.' Present address, Department of Biology, The Johns Hopkins University, Baltimore 18, Md. I
281
282
ANALYTICAL CHEMISTRY
ture of the method is the use of biiodatp in the iodometric titration of ammonia. Potaasium biiodate may be readily obtained in a very pure state and ita solutions are stable indefinitely. These factors, among others, have led to ita recognition as an excellent primary standard in acidimetry and iodometry. As a stwdard acid it has been successfully used as a receiver for the ammonia distilled from Kjeldahl digests (6). It seems never to have been used as an iodometric reagent for this purpose, although iodometric determination of excess acid after ammonia distillation is a common procedure (5). I n addition to the desirable characteristics of biiodate mentioned, the advantages offered by the sharp end point obtainable with starch indicator in iodometric titrations led to the use of biiodate in the Kjeldahl analyses reported here. Since biiodate simultaneously serves as an iodometric and an acidimetric reagent, the whole method requires only one standard solution, which is readily prepared by direct weighing and dilution.
Table I. Reproducibility of Blank Titrations No.
0.01022 M Biiodate MI.
tained from the Scientific Glass Apparatus Company Bloomfield, N. J., catalog No. M-3074. It may be connected with an ,adapter to the center outlet of a 500-ml. three-arm round-bottomed flask. Inside the flask is placed a heating coil of Nichrome resistance wire. Leads through the side arms connect the coil to a 110-volt outlet. An adjustable resistance in the circuit enables the rate of heating of water in the flask to be precisely controlled. With this arrangement a steady rate of steam evolution is assured during the distillations. A sand bath is easily made by nearly filling a saucepan with sand. This can be placed on a tripod and heated with a Fisher burner. A 22.5-cm. (9-inch) sand bath will accommodate about eighteen 10-ml. Kjeldahl flasks. An ordinary calibrated 10-ml. buret, graduated in 0.05-ml. intervals, was used in nearly all the titrations reported here. REAGENTS
Digestion Mixture. Mix 3 volumes of concentrated sulfuric acid and 1 volume of concentrated phosphoric acid (6). Catalyst. Potassium persulfate, solid. Sodium Hydroxide, 20%. Dissolve 100 grams of sodium hydroxide in 500 ml. of distilled water. Sodium Thiosulfate, approximately 0.011 molar. Dissolve 2.75 grams of sodium thiosulfate pentahydrate in 1 liter of distilled water, and add 3 drops of chloroform (2). Standard Potassium Biiodate, 0.05 molar, stock solution. Dissolve 19.5 grams of potassium biiodate in distilled water and make up to a final volume of 1liter. Dilute 5 times to obtain a 0.01 molar solution (0.01 N with respect to hydrogen ions). Starch Indicator, 1%. Make a paste of 0.5 gram of soluble starch and a little distilled water. Pour in, with constant stirring, 50 ml. of boiling 20% sodium chloride solution (4). Potassium Iodide, solid. PROCEDURE
Digestion. The sample to be analyzed (containing, 0.1 to 1.4 mg. of nitrogen) is weighed or pipetted into a 10-ml. Kjeldahl flask, and 1 ml. of the sulfuric-phosphoric acid digestion mixture is added along with a clean Pyrex bead. Excess water is driven off over the low flame of a microburner or in a drying oven a t approximately 125' C. The flask is then allowed to cool and about 100 mg. of solid potamium persulfate are added along with 5 to 8 drops of distilled water. The flask is again heated over the microburner until white fumes start coming off. The flask is allowed to cool again, and if the digest is not clear, the treatment with persulfate is repeated. Finally, about 50 mg. of potassium persulfate are added (along with 5 to 8 drops of water) and the flask is placed in the sand bath at approximately 375" C. After at least 5 hours in the sand bath, the flask is removed and allowed to cool to room temperature. All these operations should be carried out in a hood. Distillation. Ten milliliters of the 0.01 molar potassium biiodate are pi etted into a 50-ml. Erlenmeyer flask and placed under the defkery tip of the still. The delivery tip should be placed well beneath the surface of the receiver contents. The cooled digest is washed into the still with five !-ml. portions of dietilbd water containing a httle phenolphthalein inhcator, and is followed by 20% sodium, hydrovde in, ampunt sufficient to make the digest highly alkaline. Distillation is allowed to pro-
Deviation from Mean
M1.
% +O.Ol +0.01 $0.01 -0.01
8.65 8.65 8.65 8.63 8.64 8.62 8.65 8.65 8.65 8.65 Mean 8.64 Standard deviation
(0)
$% -
=
0
-0.02 +0.01 +0.01 +O.Ol fO.01
r0.0115
Table 11. Analytical Results N No. of N DeterminaExuected tions -
APPARATUS
Micro-Kjeldahl Distillation Apparatus. This unit may be ob-
Ca.,0.012 M Thiosulfate
Found with Standard Deviationa
Deviation of Averane
'WQ.
Distillation and titration, standard (NH~ZSOI Entire procedure, standard (NH&
so4
10 10
1.00 1,174b 0,899 0.793b 0.1256
Glycine Tyrosine Adenine Egg albumin Frog egg brei 0
0.501 1.00
....
Standard deviation
(0)
=
7 6 6
5 6 10
dgl
0.501 t0.00447 1.00 r0.00469 1.00 * O
1.163 * 0,00480 0.894 * 0.0128 0,801 * 0.00132 0.126 * 0.00322 1.15 *0.00748
0
0 0
-0,991 -0.659 +1.00 +0.800
....
b Values calculated from results of Dumas nitrogen analyses kindly run by W. Saschek, College of Physicians and Surgeons, Columbia University, S e w York, to whom the authors express their thanks.
ceed for 6 minutes (about 15 ml. are distilled over in this time), after which the delivery tip is lifted about 2 cm. above the surface of the receiver contents and washed off into the receiver with a fine stream of distilled water. Distillation is continued for 2 minutes longer. The receiver is then removed. Titration. A small pinch of potassium iodide (about 25 mg.) is added to the receiver contents, and the liberated iodine is titrated with the 0.011 molar thiosulfate. Near the end point one drop of the starch indicator is added. A blank titration is also run. Calculation of results:
A A
X 10 N X 14.008 = mg. of nitrogen in sample
where
A B
ml. of thiosulfate in blank titration ml. of thiosulfate in experimental titration S = normality with respect to hydrogen ions of potassium biiodate = =
RESULTS
The three major phases of the entire procedure-titration, distillation, digestion-were examined in order to determine which step limited the reproducibility of the results obtained. Table I presents data to show that the titrations can be made with a standard deviation of *0.0115 ml. of 0.012 molar thiosulfate, which is equivalent to slightly less than 0.002 mg. of nitrogen. It can be seen from the analyses presented in Table I1 that the introduction of the processes of digestion and distillation hardly increases the magnitude of the standard deviation. Table I1 contains also the results of analyses on various substances, including amphibian embryos, with satisfactory reproducibility and recovery of amounts of nitrogen varying between about 0.125 and 1.2 mg. DISCUSSION.
Digestion. Satisfactory results were not obtained with the Tyong persulfate method as outlined by Peters and Van Slyke ( 7 ) , in which a saturated soIution of potassium persulfate was used as a catalyst. It was almost impomible, for instance, to clear digests
V O L U M E 19, NO. 4, A P R I L 1 9 4 7 of tyrosine with this‘ procedure, possibly because of excess water introduced by the saturated solution, which allows the destruction of the catalyst during heating before the digest becomes concentrated enough for actual oxidation of the tyrosine to occur. By the use of solid potassium persulfate with only a little water this difficulty was avoided. Excess foaming was also much reduced. It was found that complete clearing of the digest was not a criterion of complete oxidation. Tyrosine, for instance, required a t least 5 additional hours in the sand bath a t 375” C. before complete recovery of nitrogen. This agrees with the findings of Miller and Houghton (3) who used a different digestion mixture. A t least 5 hours’ heating after complete clearing is obtained has been made a standard procedure in all the analyses of biological material reported here. After several digestions have been carried out, the Kjeldahl flasks may become etched and scale may form on the bottom. This may interfere somewhat with the transfer of the flask contents to the still. The scale may be largely removed by vigorous scouring with a motor-driven rod tipped with a short length of rubber tubing, using fine sand or other abrasive. Distillation. I n early trials, it was noticed that the first distillation of a series nearly always yielded low ammonia values. This could be prevented by “seasoning” the still by distilling over a sample or two of ammonia from ammonium sulfate before each series of analyses. The reason for this is not completely clear, but it is possible that an equilibrium existed between the ammonia in’the water which always was present in the traps between the still and the condenser, and the ammonia in the still atmosphere. When this equilibrium was established by distilling over ammonia and allowing the water in the traps to absorb its full complement of ammonia, loss of ammonia during subsequent analyses was never observed. The use of 40% sodium hydroxide which is sometimes recommended for neutralizing digests prior to distillation was unsatis-
283 factory because of violent bumping when the strong alkali was added to the concentrated acid of the digest. When heavy bumping occurred low nitrogen values were always obtained, apparently because some volatile hydrogen-ion-yielding material (HIS?) was carried over into the receiver with the consequent liberation of too much iodine when potassium iodide was added. The presence of large amounts of chloride in the digests gave a similar result, but 20% sodium hydroxide was found to be completely satisfactory for neutralizing digests and could be run In freely without fear of bumping. Best results were obtained when every step in the distillation procedure-amount of wash water, time of distillation, etc.-waa standardized and rigidly adhered to in all distillations. Undoubtedly the best conditions for distillation will vary considerably with the particular still, heating system, etc., and this should always be investigated before experiments are begun. The entire analytical procedure should be carefully checked against standard substances. It was not necessary to run blank determinations through the entire procedure, since in the experiments in which standard ammonium sulfate was carried through the whole procedure the expected values for nitrogen were obtained, ihdicating the freedom from nitrogen of the distilled water and other reagents used. LITERATURE CITED
(1) Gregg, J. R., and Ballentine, R., J . E z p t . Zool., 103, 143 (1946). (2) Kassner, J. L., and Kassner, E. E., IND.ENQ.CHEM.,ANAL.ED., 12, 655 (1940). (3) Miller, J., and Houghton, J. A,, J . Biol. Chem., 159, 373 (1946).
(4) Niederl, J. B., and Niederl, V., “Micromethods of Quantitative Organic Analysis”, 2nd ed., p. 54, New York, John Wiley 8: Sons. 1942. (5) Ibid.,-p. 69. (6) Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemistry”, Vol. 11, Methods, p. 355, Baltimore, Williams and Wilkins Co., 1932. (7) Ibid., p. 525. I
~
~~
CORRESPONDENCE Measuring the Distribution of Particle Size in Dispersed Systems SIR: Dotts (1) has recently described his use of a sedimentation technique for the determination of particle-size distribution in emulsion systems. He has apparently overlooked a previous paper (2) by the author, in which improvements such as reported by him are discussed. This note is to point out that the sensitivity of the method can be increased considerably by an added feature which has been successfully used in our laboratories. If a bulb, such as that shown in the accompanying figure, is added to the side arm near its union with the large tube, one can use a different liquid in the side arm from that of the suspension medium. The two liquids should be immiscible. The increase in sensitivity is inversely proportional to the ratio of the density of the side-arm liquid to that of the dispersion-e.g., the use of petroleum ether in100 . creases the sensitivity over the use of water by a factor Per-
0.66 haps a more important factor in the use of petroleum ether is its lower surface tension, as compared to that of water, which reduces the tendency of the side-arm liquid to stick during sedimentation. The large-diameter bulb, where the interface between the two liquids exists, is necessary, so that no appreciable movement of this Side Arm interface need occur if the bore of the side-arm tube is relatively small. RAYMOND H. LAMBERT Eastman Kodak Co. Rochester 4, N. Y .
SIR: The stated aim of the author’s recent paper (1) on particlesize distribution measurement was to establish a foolproof technique suitable for the occasional needs of the majority of investigatora. The apparatus discussed was stripped to its essentials, yet it provided sufficient accuracy and versatility for general use. The time required to set up the apparatus and obtain complete data was minimized by eliminating all calibration and by treating only those sources of error which affect an accuracy of 1%. Experimental considerations yielding greater significance in results were discussed. The author refers investigators of extremely fine dispersions and those desiring optimum sensitivity to a paper ( 2 ) by Lambcrt in which an automatic photographic recorder for sedimentation continuing several days and a method for correcting for lag in the movement of the side-arm liquid are discussed. Lambert has also pointed out that the use of a side-arm liquid specifically lighter than the dispersion medium and immiscible with it yields an increase in sensitivity for any system. Lambert‘s improvements are useful for investigators doing very accurate or specialized n-ork. LITERATURE CITED
(1) Dotts, W.M., IND. ENG.CHEM.,ASAL.ED.,18, 326-5 (1946). (2) Lambert, R. H.,and Wightman, E. P., J . Optical SOC.A m . , 11, 393-402 (1925). WALTERM . DOTTS Quaker Hill, Pawling, N. Y.
