number of standard samples of ssalio acid, sodium carbonate, p-aminophenol. and ammonium nitrate were then analyzed routinely to check this method, with the results given in Table 11. The relatively large errors for certain runs are probably due to the instability of the neutron beam monitoring system. It is expected t h a t larger counting rates, which could be made available with modification of this experimental arrangement, would reduce the relative error in the 5- to 100-mg. range to *5%. The calibration curve was established at the neutron flux level of lo8 n cmSW2 see.-' from a consideration of target life and sample area. Figure 1 shows that fluxes of this order of magnitude are available for more than 1 hour at the irradiation position. Since the fast neutron fluu varies approximately with l / r 2 in air (where r is the distance from target t o sample), a total sample area of 100 cc. is available for activation analvsis for periods up to 1 hour per targrt with an average flux of lo8 n sec.-' Neutron scattering in large samples (- 100 cc.) varies with the matrix; however, if the sample is homogeneous, this scattering can be determined readily by using thin copper foils. Self-absorption of the 6- to 7m.e.v. 0l6y-ray radiation in the sample is neglected. K o r k is progressing on t h e design of an irradiation facility t o utilize such a volume. It is probable t h a t concentrations as low as 1 mg. of oxygen per 100 grams of oxygen-free matrix can be analyzed with this ac-
celerator, providing the neutron flux is monitored accurately. The only other isotope that produces N16 under 14-m.e.v. neutron bombardment is F19. This is accomplished b y the neutron-alpha reaction F19(n,a)N16. Fortunately, F19 also undergoes a neutron-proton reaction F19(n,p)01Q t o produce 29-second Olg. By establishing calibration curves for both reactions, the interference of fluorine with oxygen analysis can be eliminated. Fluorine-oxygen ratios as large as 10 can be tolerated before the signal-noise ratio becomes too small for accurate analytical determination. ACKNOWLEDGMENT
The authors thank H. W. Kass for helpful discussions of the problem and R. W. Shideler for the design and construction of the transfer and neutron monitoring systems. LITERATURE CITED
(1) Ajzenberg, F., Lauritsen, T., Rev. Modern Phys. 27, 77 (1955). (2) . , Coleman. R. F.. Perkin. J. L.. Analust
84, 233 (1959). ' (3) Ibid., 8 5 , 154 (1960). (4) Elbling, P., Goward, G. IF7., ANAL. CHEM.32, 1610 (1960). (5) Faull, N., Brit. At. Energy Research Establ., Rept. AERE-RJR-1919(1956). (6) Fogelstrom-Fineman, I., HolmHanson, O., Tolbert, B. M.,Calvin, M., Intern. J. Appl. Radiation Isotopes 2,280 (19571. (71 Griffiths, V. S., Jackman, M. I., ANAL.CHEY.31, 161 (1959). (8) Harris, W. F., Hickam, W. M., Ibid., 31, 1115 (1959).
Table 11. Analysis of Known Oxygen Samples by Fast Neutron Activation Analysis Oxygen, RIg. %
Sample Oxalic acid
Added 5.2 5.2 5.2 5.2 5.2 37.0 31.2
Sodium carbonate p-Aminophenol 23.8 Ammonium 10.4 nitrate 40.3 54.0 58.2 58.2
Found 5.0 6.0 5.5 4.9 5.4 37.7 33.0 22.1 11.3 40.6 55.1 58.0 59.1
Error
- 3.8
t-15.4 5.8 - 5.8 3.8 1.9 5.8
+ ++ +
- 7.1 +10.4 0.7 2.0 - 0.3 1.5
++ +
(9) Iiallamann, S., Collier, F., Ibid., 32, 1617 (1960). (10) Leveque, P., Proc. Intern. Conf. Peaceful Uses of Atomic Energy, U.N., Kew York, 1956, Vol. XV, Paper 342. (11) Maddock, R. S., hIeinke, W. W., U. S. At. Energy Comm., Progress Rept. 8, Rept. AECU-4438 (1959). (12) Ibid.,Rept.9,Rept.TID-11009(1960). (13) Ibid., Rept. 10 (Kovember 1961). (14) hIeinke, W.,W., IAEA Conf. Use of Radioisotopes in Physical Science and Industry, Paper RICC/283, Copenhagen, Denmark, 1960. (15) Rleinke, W. W., I17ucZeonics 17, S o . 9, 86 (1959). (16) Osmund, R. G., Smales, A . A., Anal. Chim. Acta 10, 117 (1954). (17) S,ue, P., Compt. rend. 242,770 (1956). 118) Teal. D. J.. Cook. C. F..ANAL.CHEM. 34, 178'(1962). '
RECEIVED for review September 27, 1961. Accepted November 17, 1961.
Nitrogen-13 in Hydrocarbons Irradiated with Fast Neutrons J. T. GILMORE and D. E. HULL California Research Corp., Richmond, Calif.
b The concentration of nitrogen in hydrocarbons can b e measured b y means of the 10-minute nitrogen-1 3 activity produced by irradiation with 14-m.e.v. neutrons. The analysis is simple and gives valid results in the per cent concentration range. Attempts to extend it to the part-permillion range have failed because even the purest hydrocarbons irradiated with fast neutrons give rise to a NI3 activity corresponding to several hundred parts per million of nitrogen. This activity does not arise from nitrogen or any other impurity, but it is produced
inherently in hydrogen-carbon systems a t this energy by the reaction
H'
+ ClS = N13 + n1.
The protons receive the necessary energy b y recoil from collision with fast neutrons.
A
of hydrocarbons for nitrogen in concentrations greater than 0.1% is possible by fast neutron irradiation. Nitrogen-13 is formed b y a n n,2n reaction on the nitrogen-14 in the sample (4) and can be measured b y NALYSIS
counting the positrons or the annihilation photons. The 10-minute halflifeis convenient for irradiation and counting. The positrons are detected with a liquid scintillation counter and photons with a sodium iodide crystal. The neutrons are produced in the H2-H3 reaction in a 150-k.e.v. Texas Nuclear accelerator with energies ranging from 13.3 to 14.9 m.e.v. This energy is well over the 10.5-m.e.v. threshold for the n,2n reaction in N1* but is too low to excite the corresponding reaction in C12 and form 20-minute (211. At a deuteronbeam current of 0.1 ma., the average flux VOL. 34, NO. 2, FEBRUARY 1962
187
is 3.5 X lo7 n/cm2./sec. over a 5-ml. volume; and a 10-minute irradiation of a sample containing 1% nitrogen gives a n initial counting rate of 140 counts per second. Analyses are performed by comparing the activity produced in samples with that produced in a solution of pure acetonitrile or pyridine containing a known concentration of nitrogen. By using a 1-ma. beam current, a larger sample, and a larger scintillation counter, \\e expected that this technique would be sufficiently sensitive to permit analyzing for nitrogen a t the part-permillion level. However, attempts to reach this goal have been thwarted by the finding of nitrogen-13 in every hydrocarbon we have irradiated, a t levels corresponding to several hundred parts per million. Even pure reagent materials and samples that had been crosschecked by different chemical analyses at the part-per-million level showed nitrogen activities hundreds of times higher than expected. I n seeking the cause of this disconcerting discrepancy, the following hypotheses have been tested: The samples contain dissolved nitrogen gas from the atmosphere. They contain an elusive compound of nitrogen which is not easily separated from hydrocarbon and escapes the usual chemical identification. They contain a chemical impurity which gives an activity sufficiently like nitrogen-13 to be confused with it-e.g., copper-62 from copper or magnesium27 from aluminum or silicon. The nitrogen-13, or its counterfeit, is produced in the wall of the container and driven by recoil into the sample Protons are produced in some nuclear reaction and then react with the carbon atom? to produce nitrogen-] 3.
UNRECOGNIZED NITROGEN COMPOUND
A hypothetical nitrogen compound would have to occur in comparable concentrations in liquids of such different volatilities as benzene and high-boiling mineral oils to account for the observed N1* activity. To reduce this probnbility even further, we irradiated liquid butane (Phillips 99%). Both the irradiation tube and the polyethylene counting tube were topped with a dry iceacetone cold finger to retain the liquid activity indicated butane. The "3 460 p.p.m. of nitrogen in this sample, although gas chromatographic analysis of the butane showed no impurity to one-tenth this level. OTHER TEN-MINUTE ACTIVITIES
Copper is a common, low-level contaminant in plant samples that could be mistaken for nitrogen in the n,2n reaction, inasmuch as Cu62 also emits positrons without gamma rays; and its half life (9.8 minutes) is almost identical with that of X13 (10.0 minutes). T o distinguish it from nitrogen, the activities induced in copper by slow neutrons, namely, Cu64 (12.8 hours) and Cu66 (5.2 minutes) could be used. However, the small yields in the absence of a moderator and in a 10-minute irradiation made this unattractive. We chose rather to distinguish the "3 and C d 2 on the basis of their differing beta= 1.24 and 2.91 m.e.v., ray energies, E, respectively. The irradiated samples were counted in a liquid scintillation counter with a multichannel pulseheight analyzer. The beta-ray spectra thus obtained from nitrogen and copper are clearly distinguished, as seen in the normalized curves in Figure 1. A plant sample gave a spectrum coinciding
ATMOSPHERIC NITROGEN
Attempts to remove the dissolved nitrogen by boiling the sample or by bubbling oxygen, argon, or methane through it did not give any large reduction in the nitrogen-13 activity. The solubility of gaseous nitrogen in hydrocarbons is too small, in any case, to account for the observed N13 activity. Thus, iso-octane in equilibrium with air should contain 340 p.p.m. of dissolved nitrogen. Analysis by neutron actiration showed 1040 p.p.m., reduced by sweeping with oxygen to 650 p.p.m. Although the atmospheric nitrogen was eliminated. the source of the greater part of the N13 remained. I n a more severe test, reagent grade iso-ocatane was frozen in a vacuum system, pumped, then melted. This was repeated three times. Xeutron activation showed only a 40% reduction in the W3 activity. These experiments eliminated dissolved nitrogen as the cause of the X13anomaly. 188
ANALYTICAL CHEMISTRY
#NITROGEN-13
I
0 COPPER
- 62
r
z
U W W CL
0
within experimental error with the nitrogen spectrum. There is only one other isotope that might be confused with the 10-minute N13-namely-9.4-minute ?\IgZ7. This could be formed from either aluminum or silicon in the sample. However, this beta decay is accompanied by 0.84- and 1.02-m.e.v. gamma rays; and it was distinguished from nitrogen by counting in a well scintillation crystal with and without a base level discriminator set a t 0.7 m.e.v. Of the NI3 pulses, 10% fell above 0.7 m.e.v., owing to coincidence captures of the two annihilation quanta, but 297, of the 1\IgZ7pulses had the higher energy. The plant sample gave 12 f 3% in the higher bracket. These experiments eliminated the possibility that any other isotope could be responsible for the activity identified as N13. NITROGEN FROM CONTAINER
I n early experiments samples were irradiated in glass cells, and 2.3-minute A P from the n,p reaction on Siz8of glass could be detected in the sample. When the 14m.e.v. neutron reacts with a Si2* nucleus, the momentum transferred to the product nucleus is sufficient to drive it through about 0.1 micron of glass into the sample. To eliminate the ill2*contamination, later irradiations were performed with polyethylene sample cells. If the polyethylene contained nitrogen, the recoil mechanism could transfer XI3 into the sample. T o estimate the magnitude of this effect, we irradiated a polyethylene tube and found S I 3 corresponding to about 1000 p.p.m. of nitrogen. Based on this concentration of nitrogen in polyethylene, calculations showed that the recoil mechanism would account for only 0.1 p,p,m. of nitrogen in the sample. T h a t no large part of the observed N13 came into the samples by recoil was verified b y experiment. One polyethylene tube was cut into four segments and placed inside another tube filled with iso-octane. The segments were arranged in such a way that the surface from which N13 could recoil into the sample was increased by a factor of 3 to 4. When the sample was irradiated and counted, the N13 activity was the same, within experimental error, as the N1* activity from a sample irradiated in a single tube. This result also excludes the possibility that the N13 activity could come from atmospheric nitrogen adsorbed on the container walls. NUCLEAR REACTIONS
0.1
10
20
30
40
CHANNEL NUMBER
Figure 1 . Multichannel analysis of pulse heights of liquid scintillation spectra
Although no simple reaction of carbon with neutrons could increase the nuclear charge to that of nitrogen, one can postulate consecutive reactions which could produce N13. Thus, a fast neu-
Table I. Effect of Nitrogen Concentration
Analysis, P .P.M . Activa- ChemiDiftion cal ference
Sample KO.
+
8-10 140 2 838 440 + 120 74 766 _ ~ --. . 1720 i 130 815 905 2340 i 140 1450 890
1
2 -
~
:$ 4
z!=
140
+ 120 + 130 + 140
tron could ieact with a carbon nucleus giving a proton which could then react with another carbon nucleus to produce K13. el2
+
C12
+ H'
=
Bl2
+ H' Q
=
+E
(1)
~ I
1-13
$1.9
=
-3.0 n1.e.v.
in
(3)
Reaction 1 is energetically possible with 14-n1.e.v. neutrons; it has been studied by Kreger and Kern ( 3 ) . who found a cross section of 1.9 millibarns. Reaction 2, being esothermic, would be possible with the 1-m.e.v. protons from Reaction 1; but the thick-target yield of 7x per proton (6) is calculated to be several orders of magnitude less than Reaction 3 requires the obsened ?;I3. more energy than the protons from Reaction 1 would have; so Reactions 1 and 3 do not provide a route to N13. However, protons of the required energy for Re:ictioii 3 are available through recoil of hydrogen nuclei in elastic scattering of fast neutrons. Recoil protons from 14-m.e.v. neutrons are distributed uniformly in energy up to 14 n1.e.v. with an average energy of 7 1n.e.r. The number of protons produced can be calculated from the cross sections, =
=
YH
~
n
p
pn .
6 5 X loz2atoms/cm.* X 0.7 X 1 0 - 2 4 cm.2/atom X 3 X 107 n./cm.2 - sec.
= 14
x
(4)
106 p / c ~ n .-~ sec.
and is adequate, considering their average range of 0.8 mm. in the liquid, to produce the number of N13atoms observed : Nh13 =
=
=
Nh13
. V (I - e - i f )
=
3 6 a t o m s / ~ r n .~ sec. X 5 ~ r n .X ~0.5
=
4 atoms/sec.
S,
R p
. Nr13 .
Several pieces of experimental evidence support the C13- H1 mechanism as the major source of F 3 in hydrocarbons. I n a series of samplw in which nitrogen had been determined chemically, we found the concentration based on i S 1 3 activity shown in Table
I. The 800- to 900-1i.p.m. excess found b,v activation analysis is approximately that calculated to come from the recoil reaction, and the near constancy of the excess is consistent with its formation from the hydrogen and carbon of the samples. An additional test of the recoil reaction hypothesis n-as the irradiation of a material containing no hydrogen. A sample of irradiated carbon disulfide showed no P3.Small amounts of radioactivity formed from the sulfur of CS2 could have mashed a maximum of about 50 p.p.m. of nitrogen. I n anothei experiment the ?;l3 activity was measured for a series of hydrocarbons containing different proportions of carbon and hydrogen. Esamination of Equations 4, 5, and 6 shows that for equal weights of hydrocarbons irradiated and counted under identical conditions the activity ,
Wc
. Rp
(7)
Here WB and 1l-C are weight fractions of hydrogen and carbon, respectively; and R, is the proton range in milligrams per square centimeter.
0'6
+ HI = NI3 + He4
(8)
The cross section for this reaction is smaller than the cross section for Reaction 3, but the relatively large concentration of 0l6target nuclei results in a larger yield. This reaction would impose a lower limit of several hundred parts per million on the determination of nitrogen in aqueous solutions. The variation of nitrogen activity predicted in Equation 7 was tested over a wider range by diluting a hydrocarbon sample with an inert material. The diluent, chosen to give no large activity of its own, was to be separated from the hydrocarbon before counting. If the nitrogen activity is produced by the hydrogen-carbon mechanism, it will be reduced in this experiment by the square of the dilution ratio, in the factors T R / D Ti'c ' D , then increased by the factor D upon being reconcentrated, leaving a net reduction in a ratio of 1/D If the diluent material also reduces the range of the recoil protons. the activity nill be further reduced in proportion. On the other hand, if the activity arises from nitrogen atoms in the irradiated sample, dilution will be only a firstpouer effect, TJys/D; and it nill be exactly compensated by the reconcentration. R, not coming into consideration, the XI3 produced from S I 4 will not be affected by dilution. Iron ponder was selected as diluent material. The free voluriie in the powder was 52%, and the particle size was much smaller than the average range of protons in the liquid. The calculated R, in the heterogeneous medium was 0.33 of that in the pure hydrocarbon. A benzene sample irradiated by itself showed a n apparent nitrogen concentration of 630 p.p,m. Irradiated in the iron matrix in the same geometrical position, the sample would be expected to show 107 p.p.m, if the N13came from
up,-
1 4 X 106 pjcm.3 - sec. X 0.08 cm. X 3.2 X 1QZo atom~/cm.~ x 1 x 1 0 - 2 6 cm.*/atom (5) 3.6 atoms/cm.'
EXPERIMENTAL PROOF
Ax13 a WH
I n Table 11, N13 activities from four hydrocarbons are compared with the product (abbreviated P ) of TYE, WO, and R,. Within the limit of experimental error and approximations involved in choosing R,, the term P / A N Lis~ constant, as predicted by the hypothesis. I n materials containing oxygen and hydrogen, \ve have found even larger activities of 5 ' 3 . Here recoil protons induce the reaction
(6'
The observed XI3 actix-ity in a sample of 5 cm.3 irradiated for 10 minutes was 5.6 atoms per second. Considering approviniations involved in choosing U~ ,, and R,, the agreement is as good as might be eypectcd.
e.v. ( 2 )
=
+ Q
N,
As13 =
= SI3
Q c 1 3
-12.5 m.e v.
+7
curves ( 1 , 6 )over the energy range to 14 m.e.v. Glickstein and Winter ( 2 ) have also observed W3formed by this series of reactions in polyethylene evposed to fast neutrons in a reactor. The 5 1 3 activity corresponding to Equation 5 is given by
- sec.
The neutron flus, qn, mas calculated from the observed "3 activity produced in a nitrogen sample and the cross section (4) for N14 (n,2n). The value taken for up ,,is averaged from published
Table II.
Hydrocarbon Benzene Mesitylene Iso-octane Pentane
Effect of Proportions of Carbon and Hydrogen WH
wc
RP
P
Ax13
ANla/P
0.077 0.101 0.159 0.168
0.92 0.90 0.84 0.83
68 62 57 57
4.8 5.6 7 6 7.9
89 100 132 126
19 18 17 16
VOL. 34, NO. 2, FEBRUARY 1962
189
the hydrogen-carbon reaction, but 630 p.p,m. if i t came from N14. The experimental result was 160 =t 40 p.p.m., decisively in favor of the H-C mechanism.
this investigation and especially his help in preparing liquid butane for irradiation.
ACKNOWLEDGMENT
(1) Blaser, J. P., el al., Helv. Phys. Acta 24, 465 (1951). (2) Glickstein, S. S., Winter, R. G., Nuclear Instr. & Methods 9, 226 (1960).
I5'e have the suggestions and advice of B. A. Fries throughout
LITERATURE CITED
(3) Kreger, b ' . E., Kern, B. D., Phgs. Rev. '13, (4) Paul, E. B.,(1y5g). Clarke, R. L., Can. J. Phys. 31, 267 (1953). (5) Seagrave, J. D., Phys. Rev. 84, 1219
( 1951), ( 6 ) Khitehead, -4.B., Foster, J. S., Can. J . Phys. 36, 1276 (1953).
RECEIVED for review September 18, 1961 -4ccepted December 4, 1961.
Estimation of Manganese in Biological Material by Neutron Activation Analysis HAMILTON SMITH Departrnenf of Forensic Medicine, The University, Glasgow, Scotland, and Western Regional Hospital Board, Regional Physics Department, Glasgow, Scotland
b Neutron activation analysis combined with chemical separation is a quick, accurate method for the estimation of M n in small samples of biological materials. After nitric-sulfuric acid digestion of the activated sample, a solvent extraction separation is combined with a colorimetric yield determination. Two modified yield determination and counting techniques are examined.
Manganese-56. K h e n the only stable isotope of h l n is irradiated n i t h thermal neutrons, a n unstable isotope is produced b y neutron capture. This isotope has a half life of 2.58 hours and emits @-particles and ?-rays. The capture reaction is represented by:
F
Where possible, AnalaR reagents were used. The complexing agent was tetraphenylarsonium chloride hydrochloride-3.5% in water. All absorbance measurements were made using a 1-em. optical cell in a Hilger absorptiometer with 5450-A. filter. Digestion of Samples. I t was necessary t o have M n in simple ionic form; the samples and 100-pg. carrier M n were therefore heated with a mixture of nitric and sulfuric acids until all the organic material was destroyed. The maximum sulfuric acid permissible in the extraction stage was 2 ml. (see below). A suitable digestion mixture was 2 ml. of 36N sulfuric acid and enough 16N nitric acid to destroy all the organic material. The excess nitric acid was removed by heating the mixture till fumes of sulfuric acid appeared. With normal care, no h l n was lost a t this step. Removal of Interfering Substances. After digestion, t h e sulfuric acid was allowed to cool a n d diluted t o 40 ml. One drop of tetraphenylarsonium chloride solution was added, a n d the mixture was extracted twice with chloroform. I n this way, most of t h e substances which would otherwise follow the M n extraction 'Ir-ere removed without removing a n y of the M n which did not form a complex in the bivalent state. Following this
OUR PROBLEMS in the quantitative determination of h l n in biological tissue were: the destruction of tissue while retaining M n in the reaction medium; the chemical separation; the yield recovery estimation; and the final determination of hln. The following outline covered these points and acted as a basis for the investigation. After neutron activation samples were placed in a beaker with some inactive carrier and digested with a mixture of nitric and sulfuric acids, a preliminary extraction using tetraphenyl arsonium chloride was carried out to remove interfering substances. Manganese was then converted to permanganate and extracted with the same reagent. A yield determination was made, and the activity was measured and compared with a standard. Preparation and Irradiation of Samples. T h e samples, preferably a b o u t 40 mg., were iveighed into polyethylene tubes which were then sealed. A sample (about 0.25 gram) of standa r d M n solution (100 pg. of M n per gram) was weighed into a silica tube n-hich was then sealed. T h e samples a n d standard were packed into a standard aluminum can and irradiated in a reactor at a thermal neutron flux of 1OI2 neutrons per square cm. per second for 2 hours. The unit was returned and processed as described below. The standard was diluted as necessary.
190
ANALYTICAL CHEMISTRY
56RIn (n, y ) 5eRln REAGENTS AND APPARATUS
process, the aqueous layer containing the h l n was evaporated to fumes of sulfuric acid to remove any traces of chloride ion or complexing agent which would interfere in the nest step. Chromium follon ed the h l n through the above and the following oxidation step where it was oxidized to dichromate. This reacted with tetraphenylarsonium chloride and was extracted into the chloroform solution in the final step. The elimination of Cr activity is described under the activity estimation below. Oxidation to Permanganate. T h e cooled sulfuric acid from the last step was diluted t o 40 nil. and transferred to a centrifuge tube where t h e M n 1% as oxidized to permanganate b y stirring with about 2 grams of sodium bismuthate. The suspension formed was centrifuged, and the supernatant liquid was transferred to a separatory funnel. If a n y chlorides or tetraphenylarsonium chloride were accidentally left till this stage, there were large losses of M n due to trapping of the tetraphenylarsonium permanganate in the sodium bismuthate sludge, or reduction of the permanganate b y the chlorides. T h e Extraction. Five drops of tetraphenylarsonium chloride solution were added to the contents of t h e separatory funnel and niived \\-ell. T h e tetraphenylarsonium permanganate was then evtracted b y shaking twice with 8-ml. portions of chloroform. T h e combined extracts were made u p to 20 ml. for the next step. The extraction proved to be sensitive to varying conditions. The following experiments were made to find the n-orking limits of the extraction. Acid Concentration. It was necessary to work from a digestion residue containing about 2 ml. of concentrated sulfuric acid. Using t h e extraction
