806
ANALYTICAL CHEMISTRY
Figure
3. Automatic Combustion Carriage with Mounted Furnaces
The furnaces of this type were found to have excellent uniformity of temperature in the whole length of the tube. They have working temperatures up to 1250" C. Several furnaces have been heated up to 1350" C. for short times without failing, and some of them have survived such shocks for several years of intermittent use a t 1100' to 1200" C. The cost of the furnaces is only a fraction of that of most earlier described furnaces. Splibtype furnaces based on the principles outlined above hiEve also been developed. They are constructed for working temperatures up to 1150" C. The furnaces can be oontrolled using automatic temperature controllers, input controllers, variable transformers, or resistors. Stepped transformers (Figure 3, H)are especially Convenient, bersuse one transformer e m feed several furnaces and other apparatus. The furnaces and accessories have been commercially available for some time (Nieroma, Klara. Viistra Kyrkogatan 7, Stockholm), and hundreds of them ara in use in Swedish laboratories. Dimensions and capacities of the furnaces shown in Figure 3 are given in Table I. ACKNOWLEDGMENT
I n the first furnaces made, the heating element uras wound around a refractory tube. This was found less convenient because heating the furnaces to working temperature takes a long time, and the arrangement, with the windings rather far away from the center of the furnace, increases the heat losses a t the ends and upscts the uniformity of the furnace tmperature. The method of baking the wire into a refractory cement was, therefore, preferred. Alundum cement (Norton Behr-Manning, Overseas, In?., Worcester, Mass.) wa8 found to be B very con venient cement far Xanthal wire.
The author is much indebted to E. Stenhagen for his interest in the work, to A. Edenstrom, Uppsala, to E. Svanheck, Nicroma, Stockholm, for assistance with the technical designs, and to E. Sepp for drawing the figures. LITERATURE CITED
(1) Kirsten, Wolfgang,ANAL.Cnm., 25,74 (1953). (2) Kirsten, Wolfgang, Mikrochemie DE?. Mikrochim. A d a , 35, 174 (1950). (31 IbkZ., 8.217. (4) Untersctucher. J.. Be.deul. chem. Ges., 73,391 (1910). (5) Unteraeuoher, J., C h a . Iny. Tech.. 22, 39 (1950). ( 6 ) Walden. L., J . Sci.Inst7.. 16,l (1939). Recmvro for review August 18. 1962. Aoaepted December 15, 1852.
Determination of Niitrate in Plant Materials R. E. UNDEKUVWN nwealth Division of Biochemistry and General Nutrition, Commo University of Ade [aide. So
G . B. JONES
AN D
mocedures far the estimation of nitratenitrogen r h i c C dependon the nitration of phenol disulfonicacid in the presenc URREXT
of strong sulfuric acid have proved satisfactory for inorganic s m des. but as chloridesand organic matter interfere, the estimation c
silver sulfate; various measures have been advocated in order to remove the orgenie matter which is extracted from tho plant tissues, and successful procedures have been evolved for the estimation of nitrate in certain types of plant materials. I n this laboratory the problem centered round the nitratenitrogen content of grasses, particularly the young shoots of a perennial grass, Phalaris luberosa. The phenol disulfonic acid methods of Gilbert, Eppson, Bradley, and Beath (5),and Eastowe and Pollard (9) were found to give little consistency between duplicate analyses, and the recovery of added potassium nitrate was incomplete. With mast grasses and with mature P. tubema, the method of Johnson and mrich (7) was much more satisfactory, hut with young shoots of P. luberosa recoveries wem quite low. Modifications of the original xylenol method suggested by Blom and Treschow (8)proved to be more successful. Barnes (I) bas advocated the use of 2.4xvlenol. the nitration product of which is
tion. They found the latter tho more consistent when the nitrs, tian product is distilled in s t e m , and applied the method to the determination of nitrate-nitrogen in soil extracts made d h copper sulfate solution and from which organic matter and excess copper were removed by precipitation with cdcium hydroxide and magnesium carbonate. The method of Piper and Lewis (If) was adapted by us to deal with the greater quantities of organic matter in the extracts from plant material. Activated carbon was added during the clarification stage to produce a colorless extract, and the solutions were chilled in ice water in order to minimize the reduction of nitrate by any orgmio mattor during the addition of the 83% sulfuric acid. This method gave consistent results with 95 to 100% recoveries of added potassium nitrate in the case of rye grass (Loliurn spp.), wild oats (duma saliua), and mature P. luberom. However, with both the phenol disulfanic acid method of Johnson and Ulrich (7) and the modified method of Piper and Lewis (ff), recoveries from young shoots of P. luberosa were consistently in the region of 50%, hut as the growing season progressed, recoveries from more mature plant material improved up to 90%. The young shoots clearly contained substances which interfered seriously with the determination of nitrate. The method described below was evolved to overcome the interference by such substancea. Complete recoveries of nitrate added to all of the grasses tested and consistency of malytical results were achieved by the retention of the nitrate ions ou au anionic exchange resin from which the interfering organic matter was separated by washing with water.
V O L U M E 25, NO. 5, M A Y 1 9 5 3 The nitrate 11-as then eluted nith sodium hydroxide and estimated by the vylenol method. The effect of ascorbic acid a a s investigated, since Lugg (10) found that nitrate, nitrite, and ascorbic acid mal- be incompatible in acid solution due to mutual oxidation and reduction. It was suspected that large quantities of ascorbic acid in thr young shoots of P. tuberosn intcrfered nith the estimation of nitrate. Synthrtic ascorbic acid 15 as added to the samples, and the nitinte-nitrogen n as estimated by both the phcnol disulfoiiic acid method and the iesin-xyienol method. The presence of ascoihic acid (10 ing., resulted in slightly lower results nith the forincr method; the latter method waq unaffected. APPARATUS
Resin column, consisting of a glass tube 50 em. long, with an internal diameter 1.2 to 1.5 em., one end of n.hich is closed by a rubber stopper carrying a small tube fitted with a short piece of rubber tubing and a screlv clip. The long tube is supported vertically with the closed end don-nnard. thin layer of glass wool is then placed on the top of the stopper, and Amberlite IK-4B resin is added t,o provide a depth of 15 em. of resin. d column of these dimensions will hold 50 nil. of solution in the tube above the resin. The resin is converted to the hydroxide form by treatment Tvith 4% sodium hydroxide solution followed by six rinses with water. This leaves it in a suitable condition for anion exchange. Flasks, round-bottomed, 500-ml., fitted with ground glass joints. Adapter to connect the round-bottomed flasks n-ith a condenser set in a vertical position. Any suitable spectrophotometer or colorimeter fitted Tvith filters alloxing it to be used in the range of 410 to 430 m p . REAGENTS
Phosphoric Acid, 1% v./v. Dilute 1.2 nil. of 89% orthophosphoric acid, analytical reagent grade, to 100 ml. with distilled water. Sodium Hydroxide, analytical reagent grade, 4 6 w./v. aqueous solution. Sulfuric Acid, 837,. -4dd 5 volumes of nitrogen-free concentrated sulfuric acid to 1 volume of distilled water. hIix carefully and cool. Xylenol Solution. Dissolve 1 gram of 3,4-xylene-l-ol in 100 nil. of acetone and keep in the dark. Silver Sulfate Solution. Dissolve 5 grams of nitrate-free silver sulfate in 60 ml. of concentrated ammonium hydroside. Boil off the excess ammonia and dilute t o 100 ml. with n-ater. Standard Nitrate Solution. Dissolve 0.T218 grams of potasFium nitrate, dried in a vacuum desiccator, in water and dilute to 1 lit,er. This solution contains 100 micrograms of nitratenitrogen per nil. PROCEDURE
As soon as possible after collection, the sample of grass is dried overnight a t 55 to 60" C. in an oven with good ventilation, and then it is ground in a mill to a fine state of division. The moisture content is determined on another portion of the sample, so that the results may be expressed on a dry weight basis. A portion (0.2 to 0.5 grams) of the sample is transferred t o a 100-ml. flask to xvhirh is added 50 ml. of lY0 phosphoric acid. The flask is stoppered and shaken vigorously for 10 minutes, and the contents are filtered through a fast type of filter paper or by suction into :I c!ry Biichner flask. A measured volume, approximately 40 nil., of the filtrate is added to the resin column tube and is alloned to percolate through the column a t the rate of one drop every 2 seconds. JVhen the level of liquid in the tube has fallen as far as the top of the resin bed, distilled water is added, and the percolation is continued at, the same rate for 10 minutes, after v-hich the drop rate may be increased to two per second. I n order to wash away the organic material in the plant extract, the resin is flushed with six 50-ml. portions of distilled water. Up to this stage the effluent from the column is rejected. When the last of the flushing water has drained down to the level of the resin, a 100-ml. volumetric flask is placed under the outlet of the column, and 50 nil. of 4 % sodium hydroxide solution are added to the tube and allowed to percolate through the resin a t the rate of one drop every 2 seconds. During this elution process the nitrate and phosphate ions on the resin are exchanged for hydroxyl ions, and, a t the same time, the resin is being regenerated for the next exchange reaction.
807
The composition of the effluent is now essentially sodium nitrate, sodium phosphate, and any excess sodium hydroxide. Water is next passed through the column until the volume of the effluent collected in the flask is 100 ml. .4 15-ml. aliquot of this solution, containing not more than I50 micrograms of nitrate-nitrogen, is transferred to a 500-ml., roundbottomed flask fitted x i t h a ground glass joint. Two glass beads in the flask ensure smooth boiling during the distillation. If the volume of this aliquot is less than 15 ml., the deficit should be made up n i t h distilled water. 4 t this stage it is necessary to neutralize the free alkalinity of the aliquot. This is done by transferring a similar quantity to a beaker, and adding coneentrated sulfuric acid dropaise using bromothymol blue as an indicator. The s:me quantity of acid is then added to the aliquot to be analyzed. The authors have tried adding bromothlmol blue directly to the solution, but this introduced an appreciable blank error, presumably due to the presence of nitrate as an impurity in the indicator. Chlorides are removed by adding silver sulfate solution, drop by drop, until the precipitate formed after shaking no longer shows the TI hite color of silver chloride but the yellon color of silver phosphate. The solution is nom chilled in ice water and 50 nil. of 83% sulfuric acid is added in small increments. The solution is mixed and cooled after each portion is added. The temperature should not be alloved to rise above 10" C. After the addition of the sulfuric acid, the mixture is warmed up to 25' C., and 1 ml. of xylenol solution is added. The flask is stoppered and, after being s n irled thoroughly, is alloived to stand for 25 minutes, during n hich time some of the xylenol becomes nitrated in accordance n ith the quantity of nitrate present. Then 150 ml. of distilled water is added, the flask is attached to the condenser system, and the nitroxylenol is distilled into a 100-ml. volumetric, stoppered cylinder containing 2 ml. of 4% sodium hydroxide solution. With samples containing small amounts of nitrate, 50 ml. of distillate may be collected, as it v a s found that all of the nitroxylenol is distilled over in the first 40 ml. of distillate. The distillate is mixed thoroughly and filtered through a Whatman KO.541 filter paper, the first runnings being rejected. The absorption of the clear sdution of the sodium salt of nitroxylenol is determined in a spectrophotometer a t 430-mp wave length and compared Kith a calibration curve prepared from known quantities of nitrate-nitrogen, In preparing the calibration curve it is not necessary to exchange the anions on the resin. Varying quantities of the standard nitrate solution are transferred to the round-bottomed flasks, and the volume is made up to 15 ml. as required prior to the addition of the sulfuric acid and sylenol solution. Blank determinations should be conducted frequently to check the purity of the reagents. DISCUSSION
.4bsorption of the nitrate ion on the anion exchange resin followed by a thorough rinsing with water provides a very convenient means of removing the organic constituents of plant extracts which would othervr-iseinterfere with the estimation of nitrate-nitrogen. A weakly basic resin such as Amberlite IR-4B is suitable for this purpose as it exchanges with strong acids and allows very weak acids and neutral and basic compounds to pass through it. Other resins v,ith corresponding activities are Permutit E, DcXcidite E, Duolite &4-2,Duolite -4-3, and Wofatite, and presumahlv these may be used in the same manner as Amberlitr IR-413. Although cationic exchange resins have been employed frequently in the pact for collecting and concentrating metallic ions prior to their estimation, or for the removal of intcifcring ions, e.g , calcium in phosphstr estimations, anion exchange rcxins have apparently not been used so extensixe'y for the collection of thr anion to be deter~nined. However Egner, Ericksson and Cmanuclsson ( 4 ) reported the use of Amberlite IR-4B for collecting and holding the anions exchanged nith rain water, nhich were later eluted nith sodium carbonate prior to estimation; and Klenient and Dmytruk (8) have advocated the use of Wofatite to remove phosphate ions in the estimation of sodium by thr uranyl acetate method. Kunin and Myers (9) pointed out that anions are held by lite IR-4B in the following order of affinity: sulfate > chromate > citrate > tartrate > nitrate > arsenate > phosphate > molybdate > acetate = iodide = bromide > chloride > fluoride, and that the retention of these ions by the resin is negligible in neutral
ANALYTICAL CHEMISTRY
808,
Under favorable conditions a single estimation may be completed within 110 minutes of which 40 minutes would be Kitrogen, Kitratepersonal-attention time. This compares P P.M. Coefficient Fiducial Limits KO.of (.\lean Standard of Variafavorably with 3 5 hours (with 40 minutes Sample Replicates Yalue) Deviation tiona 5% 1% Phalaris 14-7 8 1160 16 1 39 1147-1173 1140-1180 personal-attention time) for the phenol Phalaris 26-6 15 2517 16 0 62 2508-2526 2505-2529 disulfonic acid method ( 7 ) with samples Wild oats 27-6 a 8896 41 0 46 8862-8930 8845-8947 of high nitrate content involving small 0 Standard Deviation X 100 aliquots and minimal evaporations on the Mean water bath; with samples of lower nitrate content larger aliquots would be required and longer evaporations would solution but is rapid in acid solution. To ensure the complete abincrease the time of operation. With the method described here sorption of nitrate ions, it is thus essential that the reaction of the a large number of estimations may be carried out concurrently extract from the plant material be definitely acid, and that the by setting up a series of resin columns. acidity be provided by an acid of lower activity ton ards the resin Further, the proposed method requires a smaller number of reathan nitric acid; in fact the further down the list the better. gents than does the phenol disulfonic acid method, and is therefore Kere it not for the fact that chloride interferes with the final subject to less chance of reagent blank. It also avoids the use of estimation of nitrate, dilute hydrochloric acid would probably ammonia which is an important consideration in a laboratory such provide a very satisfactory medium for the extraction of nitrate as this where a large number of Kjeldahl determinations are carand its subsequent complete retention by the resin. The next conried out. venient choice falls to acetic acid, as plant extracts with this acid The preposed procedure is thus a high-precision, short-time are very easily filtered and the end point of the silver chloride premethod capable of being used for routine field investigations as well cipitation is readily observable. For some reason, as yet unas for precise laboratory research work. known, recoveries were not so complete when acetic acid was used. Phosphoric acid of 1% v./v. strength was finally chosen as an ACKNOWLEDGMENT extracting medium. When it was used the recoveries of nitrate The authors wish to acknowledge gratefully the guidance and added to P. tuberosa extracts were complete, and analytical results interest of H. R. Marston, F.R.S., Chief of the Division of Biowere very consistent, although phosphoric acid extracts from the chemistry and General Nutrition, C.S.I.R.O. They also wish to grasses do not filter quite as rapidly as those made with acetic thank C. S. Piper for his helpful discussions in the development and acid, and furthermore the end point of the removal of chloride by modifications of the methods employed, and A. E. Cornish for his silver sulfate is slightly obscured by the precipitation of silver phosadvice in the statistical interpretations. phate. But neither of these objections is serious. Sodium hydroxide was chosen for the elution. . 4 strength of LITERATURE CITED 4% w./v. is necessary so that 50 ml. of the solution will neutralize Barnes, H., Analyst, 75, 358 (1950). 50 ml. of 1% phosphoric acid and provide sufficient excess for reBlom, Jacob, and Treschow, Cecil, 2. Pfianz., Dung U . Bodenk., generation. 13A, 159 (1929). A yellow color frequently appears in the eluate. The pigment Eastowe, J. E., and Pollard, A. G., J. Sci. Food Agric., 1, 266 in plant tissues which is responsible is probably held by adsorption (1950). Egner, H., Ericksson, E., and Emanuelsson, A., Ann. Roy. rather than by exchange with the resin. However, its presence is Agric. Coll. Sueden, 16, 593 (1949). no disadvantage subsequently, provided the solution is chilled in Gilbert, C. S., Eppson, H. F., Bradley, W.B., and Beath, 0. A., ice water during the addition of the 83% sulfuric acid. Without Univ. Wyoming, Agr. Ezpt. Sta.. Bull. No. 277 (December chilling, results tend to be slightly lowvvhen this pigment is present. 1946). Holler, A. C., and Huch, R. V.. ANAL.CHEM.,21, 1385 (1949). In Table I are shown the standard deviation, the coefficient of Johnson, C . M., and Ulrich, A,, I b i d . , 22, 1526 (1950). variation, and the fiducial limits a t the 5% and 1% levels for samKlement, R., and Dmytruk, R., 2.ami. Chem., 128, 106 (1948). ples of Phalaris and wild oats which contain varying amounts of Kunin, Robert, and Myers, R. J., J . Am. Chem. Soc., 69, 2574 nitrate-nitrogen. The figures indicate that the proposed method (1947). Lugg, 6. W. H., Med. J . Malaya, 5, 140 (1950). is capable of yielding results of high precision. The final deterPiper, C . S., and Lewis, D. G., unpublished data. minations of the nitroxylenol were made with the aid of a Beckman RECEIVED for review July 31, 1952. Accepted January 5 , 1953. RIodel DU spectrophotometer. Table 1.
Precision of Nitrate Estimation
Colorimetric Determination of Potassium with Dipicrylamine ROGER FABER
AND
THEDFORD P. DIRKSE, Cahin College, Grand Rapids,Mich.
HE fact that the potassium ion forms an insoluble crystalline Tsubstance with dipicrylamine has been suggested as the basis for a gravimetric ( 2 , IO), conductometric (IO), and colorimetric (1, 3, 5, 6) method for the determination of potassium. For this laboratory the convenient and rapid method outlined by Amdur ( I ) seemed best. In this method the potassium is precipitated with a lithium dipicrylaminate reagent, the resulting mother liquor is drawn off and diluted, and its optical transmittance is determined. The larger the amount of potassium, the more dipicrylamine is precipitated. Hence, increasing amounts of potassium bring about an increase in the optical transmittance of the final solution. All the transmittance vaIues in the work re-
ported here were measured with a Coleman Cniversal spectrophotometer, using a wave length of 470 mp, It was soon discovered that there were limits with this method that had not been pointed out in previous reports. Specifically, zinc was a contaminant in solutions in this laboratory, and it was of importance to knoly the extent to which this introduced errors. The work of Kolthoff and Bendix (6) indicated that zinc might be an interfering ion, but the extent of the interference was not known. This paper is a description of some work carried out to determine the extent of zinc interference and also to evaluate other interferences. Any ion which brings about an appreciable precipitation of
