cofactors in a phosphate buffer pH 7.4. At the end of a 15 minute incubation period, tetrahydrofuran was added to a concentration of 30% by volume, 10 mg of NaBH4 was added, and the reduction was carried out for 1 5 minutes at 38 OC. The extracted alcohols (from basic solution) were taken up in 30 p1 of benzene, and 10 pl were chromatographed. Thus, if tetrahydrofuran in a concentration of 30% or greater will stop the enzymic reaction, a stop analysis of coenzyme A derivatives can be achieved. Limitations. There are several factors which may limit the overall effectiveness of this method. A number of divalent ions, such as Mg2+ and Mn2+, catalyze the decomposition of NaBH4 preventing effective reduction. If they were present in the reaction mixture, the acylWoA derivatives could be precipitated by the addition of acid and the precipitate washed with dilute acid and resuspended in 30% tetrahydrofuran for reduction.
NaBH4 will reduce two functional groups which could be present in the acid moiety of the acyl-thiolesters. If keto groups are present, diols would be produced which do not chromatograph well on EGS ; thus acetoxy derivatives should at least be made initially on the alcohols produced from CoA esters of biological origin. We have found that A2 acylthiolesters are readily reduced to the saturated alcohol under the conditions used. Interestingly enough, the double bond in A2 acids or methyl esters is not reduced under these conditions. Double bonds in fatty acid moiety of the acyl-thiolester further from the carboxyl group are not reduced with these conditions.
RECEIVED for review February 26, 1968. Accepted June 17, 1968. This work was supported by the Department of Health, Education, and Welfare, Grant AM-07806. A portion of this paper was presented at the 154th Meeting, ACS, Chicago, Ill., Sept. 1967.
Determination of Trace Nitrogen in Organic Materials by a Microcombustion Technique J. P. Wineburg' Eastern Laboratory, Explosives Department, E. I . du Pont de Nemours & Company, Gibbstown, N . J .
A MICROCOMBUSTION METHOD for use in the determination of trace nitrogen in organic materials has been described by Norris and Flynn ( I ) . They burned an organic sample over a platinized asbestos catalyst at 550 "C, in a tube swept with oxygen. Combustion products were scrubbed from the gas stream via a helical absorber containing a sulfanilic acid solution. Nitrite, formed during scrubbing, reacted with the sulfanilic acid to form a diazo compound which was subsequently coupled with a solution of 1-naphthylamine to form a red dye (Griess-Ilosvay reaction). The amount of nitrogen in the sample was then determined spectrophotometrically. This paper describes a study of the microcombustion technique. The unmodified method of Norris and Flynn could not be reproduced to yield satisfactory results ; corresponding revisions were made in the colorimetric reagents used, the combustion catalyst, the combustion procedure, and the method of calibration. Nitrogen contents of 0.0005-1 .OO% as nitro, amide, nitrile, amine, and heterocyclic (nicotinic acid) compounds were determined. Errors in the analysis of known mixtures averaged & l o % of the amount of nitrogen added. EXPERIMENTAL
Apparatus. The apparatus is basically the same as that used by Norris and Flynn ( I ) . Platinum combustion boats were A. H. Thomas Cat. No. 8308M. Reagents. Matheson Extra Dry Grade oxygen was used. Ground glass joints were lubricated as indicated with Airco No. 20 lubricant for oxygen service. All other reagents were AR grade or equivalent. Acetanilide and m-nitrobenzoic acid were used as standard organic nitrogen compounds. Sulfanilic acid reagent was prepared by weighing 0.60 ir 0.01 of a gram of sulfanilic acid into a 100-ml volumetric flask and 1 Present address, Fels Research Institute, Department of Chemistry, Temple University, Philadelphia, Pa. 19122
(1) T. A. Norris and J. E. Flynn, ANAL.CHEM., 37, 152 (1965).
1744
ANALYTICAL CHEMISTRY
dissolving it in 50 ml of water; 20 ml of 3 7 z hydrochloric acid were added, and the solution was diluted to volume with water and mixed thoroughly. The solution was then mixed with 1100 ml of distilled water and 300 ml of 95 ethanol. Naphthylamine hydrochloride reagent was prepared by diluting to volume with water in a 100-ml volumetric 0.60 =k 0.01 of a gram 1-naphthylamine hydrochloride and 1 ml. of 37% hydrochloric acid. Sodium acetate buffer was prepared by weighing 16.4 ir 0.1 grams of anhydrous sodium acetate into a 100-ml volumetric flask, dissolving and diluting to volume with water. Cobalt(I1,III) oxide catalyst was prepared as follows. Eighty-eight grams of oxalic acid dihydrate was mixed with 300 ml of water. This mixture was added slowly with continuous stirring to a solution of 145 grams of cobalt(I1) nitrate hexahydrate in 200 ml of water. The precipitated cobalt oxalate was filtered off using a Buchner funnel and washed thoroughly with water and ethanol. The resulting pasty material was spread over a flat bottom evaporating dish and placed under a stream of air to drive off remaining ethanol. When the odor of ethanol could no longer be detected, the cobalt oxalate was heated in a muffle furnace at 500-600 "C for 2 hours to decompose the oxalate to oxide. The moderately dry powder was then pressed into pellets which were broken carefully to pass through a No. 18 U. S. standard sieve. The cobalt oxide was then transferred to a No. 25 U. S. standard sieve. The combustion tube was packed with the cobalt oxide retained on this sieve (0.7-1.0 mm particles). An 11-cm layer was used. Combustion conditions: long furnace 700 "C, short furnace 600 OC, and oxygen flow 10-25 ml/minute. The packed combustion tube was pretreated by subjecting it to the same procedure described for blanks and samples below but without the absorption coil attached. Blank Determination. Pipet 15 ml of sulfanilic acid reagent into the exit end of the absorption coil and let it drain as far as the coil leading to the inlet joint. Connect the inlet end of the coil to the exit end of the combustion tube without greasing the ball and socket joint. Attach the coil to the flowmeter with rubber tubing. Adjust the oxygen flow rate
z
~~~~~~~~
to 10-15 ml/minute. Insert an empty platinum boat into the combustion tube and push it to within 40 mm of the long furnace. Lubricate the ball and socket joint at the combustion tube entrance. Position the short furnace around the combustion tube so that there is a space of about 65 mm between the two furnaces. Start the short furnace advancing at a rate of 5 mm/minute. When the short furnace has advanced to the long furnace, turn off the motor but keep the short furnace heat supply on and leave the short furnace around the combustion tube at the final position for 7 minutes to ensure complete “rinsing” of combustion products from the catalyst and combustion tube. After the seven minutes, turn off the short furnace heat supply and remove the short furnace from the combustion tube. Drain the coil into a 50-ml volumetric flask. Rinse the coil with about 20 ml of water, adding the rinsings to the flask. Add 1 ml of naphthylamine hydrochloride reagent and 1 ml of sodium acetate buffer solution to the flask. Dilute to volume with water, mix thoroughly, and let stand 10 minutes. Measure the absorbance of the blank us. water at 520 mp, using matched cells (1 cm or 10 cm) of the same length as used for the sample absorbance measurements below. Two blank determinations must be run and check within 0.005 absorbance unit before samples are run. In addition, a blank must be run every fourth determination when samples are being run as a check against contamination of the system. Sample Analysis. Weigh a sample of appropriate size into a platinum boat. Efficient combustion is obtained for any sample weighing 7 mg or less. The sample size must be further restricted so that the resulting sample absorbance does not exceed 0.700 in a 1-cm cell; at higher absorbances Beer’s law is not followed. Combustion procedure is the same as that described above for the blank determination. In measuring absorbance, use the cell path best suited to the color intensity of the sample solution. Deduct blank absorbance from the sample absorbance and read pg nitrogen per 50 ml from the appropriate average calibration curve (see below). Calibration. Weigh three m-nitrobenzoic acid standards : 50, 100, and 150 pg, respectively, into platinum boats and analyze them by the above sample procedure. Plot absorbance in I-cm cells US. pg nitrogen per 50 ml (sample weight in micrograms X 0.0838). Repeat using acetanilide instead of m-nitrobenzoic acid (sample weight in micrograms X 0.1037 = pg nitrogen per 50 ml). When these two curves have been plotted, draw an average curve between them, obtaining the points for the plot by averaging the two absorbance readings at the 5 , 10, and 12 pg nitrogen points. The average curve obtained covers the range 0-17 pg nitrogen. For samples containing less than 100 ppm nitrogen, 10-cm cells must be used to read the absorbance. A new calibration curve is needed for these low levels. It has been experimentally determined that such a curve may be obtained by extrapolation. RESULTS AND DISCUSSION
Colorimetric Reagents for Nitrite Determination. Sensitivity and reproducibility are essential in the microcombustion method. As little as 0.035 f 0.007 pg of nitrogen per 50 ml of solution must be detected. The following reagents were studied for possible adaptation to the microcombustion method: the Griess reagent ( I ) , a modified Griess reagent consisting of a mixture of sulfanilamide and l-naphthylethylenediamine, Saltzman’s reagent (2), and Rider and Mellon’s reagent (3). Rider and Mellon’s reagent, although not the most sensitive of the group, was found to be the most ac(2) B. E. Saltzman, ANAL.CHEM., 26, 1949 (1954). (3) “Colorimetric Determination of Nonmetals, ” D. F. Boltz, Ed., Interscience, New York and London, 1958, pp 124-9.
~
~~~
Table I. Analyses of Known Mixtures. Compound tested
%N
Added
% Error (as a percentage Found of the N added) N
0.064 0.170 0.200 0,119 0.070 0.100 0.155 0.217 0.0025
0.071 +10.9 m-Nitrobenzoic acid 0.185 +8.8 m-Nitrobenzoic acid 0.216 +8.0 m-Nitrobenzoic acid 0.119 0.0 Nicotinic acid 0.063 -10.0 Acetanilide 0.093 -7.0 Acetanilide 0.140 -9.7 Acetanilide 0.203 -6.4 Acetanilide Acetanilide 0.0023 -8.0 0.0028 0.0026 -7.1 Acetanilide 0.0030 0.0027 - 10 2-Naphthylamine 0.0033 0.0033 0.0 2-Naph t h ylamine 0.0049 -3.9 2-Naphthylamine 0.0051 0.200 0.220 +10.0 rn-Nitroaniline 0.217 0.214 -1.3 3-Nitrosalicylic acid 0.204 0.184 1-Naphthylamine -9.8 0.164 0.153 -6.7 2,CDinitrobiphenyl Mixtures contained 7.0-mg matrices of DMT or TPA plus microgram quantities of the indicated standard organic nitrogen compounds.
ceptable for the microcombustion method. It was the most reproducible of the methods studied and easily adaptable to use in the absorption step. Ethanol was added to the sulfanilic acid reagent described above to reduce the surface tension of the coil absorbent, thereby improving its efficiency. Its presence does not affect the determination in any other way. Color development is rapid and the color is stable for hours. The reagent solutions described are stable for weeks. Combustion Catalysts. Erratic blank and sample values were obtained using platinized asbestos as the combustion catalyst. No set of parameters (such as temperature, oxygen flow rate, combustion time, etc.) was found that gave reproducible results, The applicability of cobalt(I1,III) oxide to trace nitrogen work was investigated because it had previously been recommended as the best catalyst for complete combustion in carbon and hydrogen analyses ( 4 ) . Reproducible blank and sample values were readily obtained at any long furnace temperature between 650 and 750 “C following substitution of cobalt(I1,III) oxide for platinized asbestos. An 11-cm layer was found to support efficient combustion and did not require an objectionably long time for the rinsing of adsorbed combustion products. Combustion Products. Norris and Flynn obtained microcombustion results which indicated that organic nitrogen was quantitatively oxidized by platinized asbestos to nitrogen dioxide. However, results obtained during microcombustion with cobalt(I1,III) oxide suggest that a mixture of nitrogen oxides (e.g., NzO, NO, NO*,NzOJ is formed during oxidation of organic nitrogen and that the composition of the mixture depends on the identity of the functional group in which the nitrogen is bound. Basis for this was found when a number of standard organic nitrogen compounds were individually subjected to microcombustion. If the same species of combustion products were formed and absorbed in every case, one would expect to obtain, if the resulting absorbances were plotted us. micrograms nitrogen added, a calibration curve of constant slope no matter what nitrogen-containing functional group was present in the sample being combusted. How(4) M. Vecera, 2.Anal. Chem., 208, No. l, 15-26 (1965). VOL 40, NO. 1 1 , SEPTEMBER 1968
1745
Table 11. Sample Analyses Using Average Calibration Curves
Absorbance at 520 rnp
Length of absorption cells, cm
Micrograms nitrogen from curve
Nitrogen content of sample
7.433 5.034
0.225 0.147
10 10
0.565 0.370
76 ppm 73 PPm
DMT
6.351 4.074
0.222 0.137
10 10
0.560 0.345
88 ppm 85 P P ~
Purge solids
4.400 3.238
0.390 0.295
1 1
9.90 7.45
0.23% 0.23%
DMT-MHT
6.140 4.538
0.465 0.365
1 1
11.75 9.20
0.19% 0.20%
0.577 0.505
0.220 0.197
1 1
5.6 4.9
0.97% 0.97%
Sample weight, mg
DMT
Sample designation
DMT
1.07% N by Coleman Nitrogen Analyzer.
ever, plots of absorbances led to calibration curves of different slope when organic nitrogen compounds of differing functional groups were combusted (see Standards and Samples below) indicating the same combustion products were not being absorbed in each case. Additional consistent evidence was obtained when the applicability of Saltzman’s reagent (2) to the microcombustion method was being evaluated. Saltzman’s reagent is a solution containing a mixture of sulfanilic acid and N-1-naphthylethylenediaminedihydrochloride. Because the coupling solution is present in the reagent, the color develops in the absorption coil. When nitro compounds were combusted, color development was restricted to the first six or seven loops of the coil; on the other hand, when amides and amines were combusted, color diffused through all but the last few loops of the coil. These results again indicate that the same species are not being absorbed in all cases. Absorption of Nitrogen Oxides. In nitrogen dioxide absorption studies using conventional and modified Griess reagents, Patty and Petty (5) and Saltzman (2) had found that recoveries of nitrogen dioxide as nitrite could range from 48 to 72% depending of the composition of the absorbing solution and related treatments. Rider and Mellon’s reagent was not among those studied. Therefore, since the corresponding recovery factor for Rider and Mellon’s reagent was unknown, the calibration curve was prepared empirically by combusting microgram quantities of standard organic nitrogen compounds. Actually, the recovery factor corresponding to Rider and Mellon’s reagent could have been determined by subjecting known quantities of nitrogen dioxide to the colorimetric procedure. But knowledge of this factor would still not permit correct results to be obtained if the combustion products contained a mixture of nitrogen oxides instead of solely nitrogen dioxide. The amount of nitrite formed during the absorption step would then still depend on the composition of the nitrogen oxide mixture. Therefore, the calibration curve was prepared empirically.
( 5 ) F. A. Patty and G . M. Petty, J. Ind. Hyg. Toxicol., 25, 361
(1943).
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ANALYTICAL CHEMISTRY
Standards and Samples. Miscellaneous microgram quantities of standard nitrogen compounds were analyzed by microcombustion. A calibration curve was prepared by plotting absorbances us. micrograms nitrogen added. When the absorbances were collectively plotted, it was discovered that although straight line calibration curves were obtained for each compound investigated, all the curves did not have the same slope. The curves of greatest and least slope, respectively, were obtained by combustion of nitrobenzoic acid and acetanilide standards. By averaging absorbance readings obtained for these two compounds at various nitrogen contents, points are obtained that can be used to construct an average sample curve. Table I shows results obtained when specially prepared mixtures of known nitrogen content were subjected to analysis by microcombustion. Each mixture consisted of a 7.0-mg matrix of either dimethylterephthalate (DMT) or terephthalic acid (TPA) and known microgram quantities of various nitrogen-containing compounds. Absorbance readings obtained in the analysis of these known mixtures were related to the average calibration curve discussed above. Results were obtained in which errors averaged to f10% of the amount of nitrogen added. Table I1 shows the precision obtained in analyses of samples of unknown nitrogen content containing nitrogen believed to be present in those functional groups for which the method has been proved applicable. CONCLUSION
The trace nitrogen procedure described above is simple and rapid, but is not an absolute method for total nitrogen. If a sample is believed to contain nitrogen in a functional group not previously investigated, it is necessary to run standards containing this group to be sure it is determinable by the method. Nitrogen present in an untested functional group unknown to the analyst might be missed entirely or only partly recovered by this procedure. Nitrogen in hydrazine sulfate, for example, was not determinable by this method. RECEIVEDfor review February 15, 1968. Accepted May 27, 1968.
