tion and retxoducibilitv are obtained. Figure 1 is the typicrd chromatogram of a 60 hydrogen: 40 helium commercial gas mixture and hydrogen: 2o helium gas mixture. This method has been employed for the determination of helium as low as 3% in hydrogen as observed in Figure 2
LITERATURE CITED
(1) Brenner, N., Coates, V. J., Nature 181, 1401-2 (1958). (2) Janak, J., Krejci, M., Dubsky, H. E., Chem, ~ i52 (6). ~ 1099-1107 t ~ (1958).
( 3 ) Krejci, k., Tesarik, K., Colleciion Czech. Chem. Commun. 25 (3), 691-4 (1960).
(4)Kyryacos, G., Boord, C. E., ANAL. CHEM.29. 787 11957). ( 5 ) Lard, E: W., 'Horn; R. C., Ibid., 32, 878 (1960). ELLIOT H. BAUM
Materials Laboratory Sperry Gyroscope Co. Division of Sperry Rand Corp. Great Neck, N . Y.
Note on a Reversed Oxyacetylene Flame SIR: The June 1963 issue of ANACHEMISTRY 17ontains a paper by Hans F. Loken, James S. Teal, and Eugene Eisenberg (p. 875) describing a modified Beckman flame photometer burner employed for the flame spectrometric determination of calcium. The modification consists essentially of causing the fuel, acetylene, to flow through the central or oxyger tube of the burner LYTICAL
while the oxygen surrounds the burning fuel. This arrangement was used by the writer some years ago, albeit not with a Beckman burner, and referred to as the "oxygen sheath" in U. S.Patent 2,858,729, Kovember 4, 1958, specification and claims. The advantages of the arrangement received comment in the patent specification. Further development and improvements along with the
filing of additional patent applications have been in progress and now the perfected burner forms part of the flame spectrophotometers manufactured and marketed by the Keyes Scientific Corporation, 122 Hampshire Street, Cambridge, Mass. FREDERICK G. KEYES Physical Chemistry Laboratory Massachusetts Institute of Technology Cambridge, Mass.
Determination of Ammonium, Amide, Amino, and Nitrate Nitrogen in1 Plant Extracts by a Modified Kieldahl Method SIR: Studies of nitrogen metabolism in the authors' laboratory required separation and recovery for isotopic analysis of the various nitrogen fractions present in green leaiTes after the addition of either Kr\'150:r or (S15H4)2S04 to the leaves. Quantitative recovery of ammonium and nitrate nitrogen was necessary to prevent contamination of amino and amide nitrogen with unmetabolized tracer. A method for the determination of ammonium, amide, nitrite, and nitrate nitrogen has been described by Varner et al. ( 4 ) . However, two limitations were encountered while trying to apply their method in this laboratory: nitrate reduction was incomplete and amin, nitrogen was not estimated. The modified melhod described in this communication permits the determination of ammonium, amide, amino, and nitrate nitrogen with minimal contamination of amide and amino nitrogen by unmetabolized tracer nitrogen. EXPERlMliNTAL
Apparatus. Semimicro - Kjeldahl Distillation Unit. Reagents. Borate Buffer. Saturated solution of sodium tetraborate adjusted t o p H 10 with XaOH. Mercuric Sulfate Solution. Ten grams of red HgO dissolved in 100 ml. of 4h' H2S04. Sodium Hgdroxidcb-Sodium Thiosul-
fate Solution. Na2S203 (42 grams) dissolved in 1 liter of 14-V NaOH. Boric Acid-Mixed Indicator Solution. Ten grams of boric acid and 5 ml. of mixed indicator of methyl red (2 volumes of solution of 200 mp. of indicator in 100 ml. of water) and methylene blue (1 volume of solution of 100 mg. of indicator in 50 ml. of water) made to 500 ml. with distilled water. Potassium Biiodate Standard Solution. KH(IO& (3.897 grams) dissolved in distilled water and diluted to 1 liter. Procedure. The following procedure gives a n accurate estimate of t h e amounts of ammonium, amide, amino, and nitrate nitrogen present in a plant extract. Ammonium Nitrogen. X portion of a 70y0 ethanol extract of plant tissue is transferred to a 100-ml. semimicro-Kjeldahl flask and evaporated to dryness a t room temperature utilizing a jet of air. The flask is placed in a water bath a t 90" C . and attached t o the distillation unit. A small amount of silicone antifoam agent is added to help prevent foaming. Twenty-five milliliters of water and 5 ml. of borate buffer are added through the distillation head after attaching the 125-m1. receiving flask, containing 15 ml. of the boric acid-indicator qolution. A vacuum is created in the system using a water pump, and distillation is conducted for 30 minutes. Amide Sitrogen. The receiving flask containing the boric acid-indicator solution is replaced, and 5 ml. of 40% potassium hydroxide solution and 25 ml.
of water are added through the distillation head. The vacuum distillation is continued for 30 minutes at 90" C. Sitrate Nitrogen. The receiver is changed again, and a large piece of mossy zinc and 25 ml. of water are added to the Kjeldahl flask. The nitrate reduction process requires 4 hours a t 90" C. for complete reduction of 2 mg. of nitrate nitrogen. Distillation of the ammonia may be accomplished during or after reduction. However, a large volume of water will be collected in the receiver if the vacuum distillation is conducted during the entire 4-hour reduction period. Amino Nitrogen. The distillation head is rinsed into the Kjeldahl flask, and the mossy zinc is rinsed and removed by forceps from the flask. After adding 10 ml. of concentrated sulfuric acid and 0.5 ml. of the mercuric sulfate catalyst, the flask is placed on an electrically heated digestion rack. The water is boiled away slowly before applying full heat for digestion. Upon completion of the digestion, 14,V sodium hydroxide is added to the flask under a cold water tap until most of the sulfuric acid is neutralized as indicated by cessation of the violent reaction and the allpearance of precipitated ?;a2SO4. The flask is attached t o the distillation unit, and the solution is made basic with excess sodium hydroxide-sodium thiosulfate solution. A vacuum distillation is conducted for 15 minutes a t 90" C., collecting the ammonia in boric acid-indicator solution. The ammonia collected in each of the VOL. 36, NO. 2, FEBRUARY 1964
439
Table I.
Recovery of Nitrogen from Known Mixtures of Solutions of Nitrogen Compounds a t
90" C.
Sitrogen, mg. Amide (NHa)oSOa Added Recovered 5.00 5.00
a
b
4.92 4.75
. . .
...
5.00 5.00 ... 5.00 0 0
4.95 4.77
Added
Glutamine Recovered
0.74 0.37 0.37 0.37
0 73 0.34 (0.38)* (0.32)*
Asparagine Added Recovered ,..
...
... 0.42 0.42 0.42
(0'42)b (0.38)* 0.42
, . .
...
... ...
...
...
4.93 0.14 0.07
0.74 0.48 0 96
0 . 7711 ( 0 . 43)b 0.84
... 0 . 5522
3.00 2.00 2.00 ...
...
(0.'4S)b (0 4 8 ) b
...
...
Added
Kitrate Recovered
Added
Amino Recovered
2.90 1.02a 2 12 ...
. . . . . . . .
. . . . . . . .
...
0 38
...
... 0.80
0.76
0.42 1.05
, . .
, . .
...
. .
...
...
...
...
00 1 . 00 0.96
0 87 0.91
0.82
One-hour reduction time; all others 4 hours. Proportioned to glutamine and asparagine according to amounts added.
Table II. Recovery of Nitrate Nitrogen after Reduction by Mossy Zinc a t 90" C. in Presence of Borate Buffer and Potassium Hydroxide
Reduction period, hr.
Total nitrogen recovered, mg. K (2.00 mg. S added as K S G ) ~
1 1
0 97
2 4
2.00
i.48
2 1 24 1.77 2.01
four receiving flasks is titrated with standard potassium biiodate according to the procedure described by McKenzie and Wallace ( 2 ) . DISCUSSION
The vacuum distillation is not conducted in a completely closed system, but rather a capillary leak is provided through the distillation head. The passage of air through this leak prevents bumping, accelerates the distillation of water and ammonia, and provides for the gradual release of vacuum upon completion of the distillation. The vacuum distillation of ammonia a t 53" C., Varner et al. ( 4 ) , was incomplete in our apparatus, rerulting in a recovery of only 90% in 30 minutes. Since the complete recovery of am-
Table 111.
monia added to the leaves was necessary for the studies in this laboratory, some modification of this part of the procedure of Varner et al. (4)was needed. Elevating the temperature of the water bath to 90" C. resulted in nearly 1007, recovery of the ammonia, but caused a loss of 7 to 14% of the amides (column 2, Table I ) . Presumably most of this loss came from glutamine breakdown. The small loss of the amide-nitrogen was more acceptable to us than contamination of the amide fraction with the labeled ammonia added to the leaf. Since 70% ethanol extracts only a small amount of protein (S), the interference with amide determination by proteins yielding ammonia should be small. Arginine and serine, unstable in alkali, occur in considerable amounts in ethanol extracts of some plant tissues (I). N o contamination of the amide fraction was found when these amino acids were present during the amide distillation. -1 solution of potassium hydroxide rather than sodium hydroxide was used to hydrolyze the amides to provide potassium sulfate for the subsequent Kjeldahl determination. The higher solubility of potassium sulfate in concentrated sulfuric acid makes it superior to sodium sulfate for increasing the temperature of the Kjeldahl digestion mixture.
Analyses of Three Extracts from Plant Tissue Grown on Different Sources of Nitrogen
(Recover) of known amounts of nitrogen added as a mixture to the extracts) Sitrogen, mg Xitrogen source Ammonium Amide Nitrate Amino 0 04 0 06 0 56 0 53 Extract 1 0 12 0 25 0 30 1 10 Extract 2 0 07 0 11 0 19 0 79 Extract 3 Known mivture 4 86 0 52 2 00 0 32 Extract 1 + miuture 4 86 0 56 2 84 0 98 Extract 2 + mixture 4 84 0 79 2 36 1 48 Extract 3 0 49 mg K H --S
+
~~
440
ANALYTICAL CHEMISTRY
0 55
0 10
0 25
0 76
Incomplete recovery of nitrate nitrogen was consistently obtained when ferrous sulfate was used a3 a reductant, even in the presence of silver sulfate catalyst. Zinc metal provrd to be an effective reductant. Devarcla'.s alloy, while giving more rapid reduction than zinc, produced an alkali qiray, which often macle it necessarl- to rrdihtill the sample prior to titration. 1 1 o ~ s yzinc was selected as the most suit,ahle reductant since it could be removed prior to digestion, thus keeping the salt content low. Table I1 s h o w that 4 hours are required for the complet'e reduction of 2 mg. of nitrate nitrogen. Approximately 50% of this amount of nitrate is reduced in 1 hour, and 75% in 2 hours (Table I I). Copper wire, copper sulfate. silver sulfate, and manganous sulfate did not accelerate the reduction of nitratrs by mossy zinc. The 4-hour period required for complete reduction of nitrate produces no particular problem since the sample requires no attentioh during reduction, and a number of reductions may be carried out simultaneously. It may be possible to reduce the reduction time when small amounts of nitrate are present, but this is not recommended when complete recovery of added isotopic nitrate nitrogen is desired. Since the reduction of nitrates is followed by a Kjeldahl determination of the amino nitrogen, it is necessary to keep the salt content as low as possible to complete the analysis in the 100-ml. Kjeldahl flask. Too high a salt to sulfuric acid ratio will cause low of ammonia from the digesting material ( 2 ) and insoluble salts causc bumping during dige:tion. I t was imposdile to conduct the digestion when ferrous sulfate was used for nitrate reduction because of bumping. Data showing the application of this procedure to known mixtures of nitrogen compounds are shon-n in Table 1. Recovery of all nitrogen fractions was usually 95% or grrater, and hydrolysis of amides during ammonium-nitrogen
recovery was small. Percentage r e covery of itmino nitrogen was slightly lower than that of the other fractions, but was still good. Best recoveries were obtained in the ammonium and nitrate fractions. Complete recovery of these fractions w:ts one of the main ohjectives Of
the procedure’ ’
Of
the
method for the separation and recovery of the various nitrogen fractions from
Steve, Burrell, R. C., ANAL.CAEM.25, 1528 (1953). A L L EV.~ B m a RICH- J. VO&K LITERATURE CITED Department of Soil Science North Carolina State College (1) ~ ~~ 1kv,,1p h~,~~thesis, , ~~ cornell , University, 1962. Raleigh, N. C. (2) McKenzie, E. A., Wallace, Heather PUBLISHED with the approval of the direcS., Australian J . Chem. 7, 55 (1954). tor as paper No. 1633, Journal Series, (3) Stewart, F. C., Street, H. E., Plant North Carolina Acicultnral Experiment Station. Supported in part by National Science Foundation Grant No. G21454.
plant extracts has proved successful (TableIII).
X-Ray Probe with Slit Aperture re in the Seconc Secondary Beam
.._....
n , . r Eugene P. Bertin, Electron Tube Division, Radio Corporation of America, Harrison, N. J.
x-ray primary emisspectrometers for analyzing sample areas -1 micron in diameter are becoming more available (1) and more widely applied. However, many applications require analysis of areas having diameters of the order of 0.01 to 1.0 mm. Accessories have been described for modifying standard flatcrystal General Electric (2,$, 6, 7, 1316), Philips (9, 12), and Siemens (io,11) x-ray secondary emission (fluorescence) spectrometers for such selected-area operation using pinhole apertures. Such accessories and literature describing them are or soon will be available commercially for all three instruments (4,6, 8). Of course, the smaller the aperture, thelower the intensity, and apertures of diameter
