Anal. Chem. 1984, 56, 1859-1861 1859

Comparison of Kjeldahl and Combustion Methods for Measurement of Nitrogen Isotope Ratios in Organic Matter

Masao Minagawa,' David A. Winter, and Isaac R. Kaplan* Institute of Geophysics and Planetary Physics, University of California, Los Angeles, California 90024

The nHrogen Isotope ratios of several organlc materlals have been analyzed by Kjeldahl dlgestlon and by a combustlon method. Both methods give chemlcal yields close to the theoretical values for organic compounds such as histidine, thlourea, and vallne, as well as for ammonium sulfate. How- ever, for natural blologlcal materlals, such as egg albumln, soy bean nodules, marlne plankton, and kerogen, the chemlcal yleld obtained by the Kjeldahl method Is only 87 to 94% of that obtained by the combustlon method. For these samples, results from the Kjeldahl method are generally depleted In ''N relatlve to those obtalned from the combustlon method. The maximum measured difference In the 6'N value between the techniques Is 0.86%0. An Improved combustion method, uslng a double tube and heating to 850 O C , has been recommended for analyzlng blogeochemlcal samples for nitrogen Isotope measurement.

The nitrogen isotope ratio (16N/14N) of organic materials is generally analyzed by using purified nitrogen gas from ammonium sulfate prepared by the Kjeldahl method (I). A combustion technique which results in the liberation of ni- trogen (2) has been used widely because i t is both more convenient than the former method and also allows the si- multaneous collection of carbon dioxide and hydrogen gas for later 13C/ 12C and D/H measurements. Combustion methods for carbon isotope analyses have been described elsewhere (3-6). Since the initial work by Stuermer et al. (3) there have been numerous published works on combustion methods. However, few data have appeared in the literature which can be used to evaluate combustion as an appropriate approach for measuring the natural abundance of nitrogen isotopes, especially from natural organic material. This report presents a comparative study on the nitrogen content and 15N/14N ratio of materials obtained by the Kjeldahl digestion and by the combustion methods.

EXPERIMENTAL SECTION A double tube method combined with preheating at 500 "C

have been developed to avoid the cracking of quartz tubes due to reaction between SiOz and alkali carbonate salts. An inner (5 cm X 6 mm) and an outer (25 cm X 9 mm) quartz tube are each sealed at one end and baked at 850 "C for 2 h to remove con- taminants.

Analytical grade reagents, valine, histidine, thiourea, and am- monium sulfate were used in the experiments. Kerogen, from Tanner Basin sediments off the southern California coast, was prepared by the method described (3), dried, and homogenized. Agricultural samples, egg albumin, soy bean leaf tissue, and soy bean nodules were supplied by Kohl and Shearer at Washington University, St. Louis. Marine plankton, predominantly zoo- plankton, was collected in the western North Pacific Ocean.

The Kjeldahl method used in this study was mostly the same as described by Bremner ( I ) . The catalyst used was suggested by Wada (7) and is composed of mercurous oxide, selenium

'Permanent address: Mitsubishi-Kasei Institute of Life Sciences, 11 Minami-Oya, Machida-Shi, Tokyo, Japan.

powder, and potassium sulfate in the ratio 3:1:4, respectively. Samples were digested at 200 "C and heating was continued for at least 5 h after the solution became discolored. Ammonium sulfate generated during digestion was distilled by steam and trapped in 0.5 N sulfuric acid solution using an apparatus similar to that described by Bremner and Edwards (8). Concentrated ammonium ion was converted to Nz by reaction with sodium hypobromide. Further purification was accomplished by circu- lating the gas sample with a Toepler pump in a vacuum line fitted with copper and cupric oxide furnaces. Both furnaces were heated to 700 "C. After cycling for 20 min, nitrogen gas was collected in the manometer connected to a Toepler pump and then transferred into a glass tube following the method described in Des Marais and Hayes (9). The Toepler pump manometer system was calibrated by using known weights of ammonium sulfate.

The following procedure is used in the combustion method: an organic sample of approximately 10-15 mg is put into the inner quartz tube and weighed. One gram of cupric oxide wire pieces is placed into the inner tube followed by a piece of silver foil (0.05 mm thick) measuring 4 mm X 50 mm. This tube is then inserted into the outer quartz tube into which has been placed ca. 1 g of copper metal granules. This outer tube is connected to a vacuum line via a Cajon fitting and evacuated overnight to remove air and water and is then sealed at 18 cm length. The tubes are preheated in a muffle furnace to 500 OC for h and then elevated to 850 "C for 2 h. The furnace is then switched off and the samples are allowed to cool in the furnace for 18 h. After combustion, the outer tube is placed in a vacuum line and cracked to introduce all the gas by using the metal fitting described in ref 9. Nz, COz, and HzO are separated cryogenically with liquid nitrogen and dry iceacetone slush. During collection, the combusted tube is heated to 120 "C by an electric heater. All gases are collected in a 6-mm glass tube with a Toepler pump, and the amount of each gas is measured manometrically.

Copper oxide wire is purified prior to use by firing it in an electric furnace for 2 h at 900 "C. It is sieved through No. 42 mesh before firing. Copper granules are heated at 500 "C for 2 h under vacuum to remove surface oils and other organic matter, reduced by dry hydrogen and kept in a sealed glass tube with hydrogen until used.

As most of the natural samples have mixed chemical properties and compositions, there is a possibility that the combustion conditions could affect the isotopic ratio. We have examined the relationship between combustion temperature and the isotope ratio and have compared the nitrogen isotope ratios of organic materials obtained by the combustion method with those obtained by the Kjeldahl digestion method, respectively.

Isotope ratio measurements were performed with a Nuclide RMS 6-60 dual-collecting mass spectrometer and a Varian MAT 250 triple-collecting mass spectrometer. The results are presented in the conventional notation

(1) where R, and R, are atomic ratios, such as 15N/14N or 13C I l2 c, of samples and references, respectively. Atmospheric nitrogen and Chicago Pee Dee Belemnite are used for nitrogen and carbon isotope standards.

RESULTS AND DISCUSSION The effects of temperature of combustion on the isotope

ratios and yields of Nz and COZ are given in Table I, together with results of paired t tests. Nitrogen content of histidine was found to be the same for all three combustion tempera- tures (550,750, and 850 "C), and the overall yields were found

S(%O) = [ (R , - R,)/R,] x 1000

0003-2700/84/0358-1859$01.50/0 0 1984 American Chemlcal Society

1860 ANALYTICAL CHEMISTRY, VOL. 56, NO. 1 1 , SEPTEMBER 1984

Table I. Effect of Combustion Temperature on Isotope Ratios and Elemental Composition

combustion tempb

550 "C 750 "C 850 O C

Kjeldahl t (550:850)c t(7f~0:850)~ t(K.D:850)e

550 "C 750 "C 850 "C Kjeldahl t (550:850y t(750:850)d t(K.D:850)e

550 O C

750 O C

850 O C

Kjeldahl t (E150850)~ t(750850)d t(K.D:850)e

550 "C 750 OC 850 OC Kjeldahl t(E150850)~ t (7E~0850)~ t(K.D:850)'

P N , %o

-5.37 f 0.04 -5.16 f 0.06 -5.27 f 0.14 -5.37 f 0.10 0.94h 1.01h 0.86h

+7.20 f 0.13 +7.17 f 0.04 +7.37 f 0.06 +7.16 f 0.14 1.68h 3.925

+7.16 f: 0.14 +7.33 * 0.09 +7.52 i 0.04 +7.11 f 0.34 4.96 3.36 2.68'

+6.09 f 0.57 +6.57 f 0.31 +6.47 f 0.09 +5.81 f 0.26 1.14h 0.54h 4.331

nitrogen," n' % ( A 3 j 613C, Yw

Histidine hydrochloride monohydrate

20.7 f 1.1 2 21.1 f 0.6 2 20.0 f 0.9 3 20.4 f 0.3 2 0.7gh (3) 1.48h (3) 0.37h (3)

Tanner Basin Kerogen

5.21 f: 0.13 2 5.46 f 0.04 2 5.37 f 0.06 2 4.73 f 0.03 3 1.58h (2) 1.76h (2) 16.525 (3)

Egg Albumin

10.2 f 1.3 2 12.4 f 0.3 3 13.0 f 0.1 6 11.2 f 1.1 4 4.371 (6) 0.38h (7) 2.71f (8)

Marine Zooplankton

7.51 f: 0.37 3 8.03 f 0.04 3 8.03 f 0.02 3 7.55 f 0.39 2 2.438 (3) O.Oh (4) 2.33h (3)

-8.85 f 0.05 -8.88 f 0.04 -8.78 f 0.07

1.26h 1.84h

-21.60 f 0.11 -21.52 f 0.01 -21.59 i 0.0

0.13h 9.6

-17.00 f 0.03 -17.02 f 0.06 -17.01 f 0.06

0.74h 0.24h

-20.84 f 0.04 -20.71 f 0.03 -20.77 f 0.03

2.438 2.08h

carbon," %

35.1 f 0.5 35.4 f 0.1 35.2 f 0.2

0.42" 1.30h

47.0 f 2.9 52.4 f 0.4 52.3 f 0.0

2.588 0.35h

36.8 f 5.3 44.8 f: 0.8 45.6 f 1.5

3.4J 0.83h

28.0 f 2.5 32.8 f 0.3 32.7 f 0.2

3.29 0.48h

"Percent based on dry materials. bTemperature of maximum heating. cgdreStudent's t test for 850 "C combustion to 550 and 750 "C and Kieldahl method, respectively. f P < 0.05. 8P < 0.1. h P > 0.1. 'The number of measurements. jThe number of degrees of freedom.

Table 11. Comparison 6I6N Values Obtained by Kjeldahl and Combustion for Pure and Natural Organic Matter

sample

valine

thiourea

histidine

ammonium sulfate

ammonium sulfate

ammonium sulfate

egg albumin

soy bean leaf

soy bean nodule

Tanner Basin kerogen

marine zoo- plankton

hydrochloride H 2 0

(NBS N-1)

(NBS N-2)

6I5N, Ym

+11.16 f 0.19

-0.89 f 0.10

-5.36 f 0.13

+0.24 f 0.07

+0.32 f 0.04

+19.68 f 0.18

+7.11 f 0.34

-1.14 f 0.20

+6.94 f 0.28

+7.16 f 0.13

+5.81 f 0.26

nitrogen, %

12.1 f 0.2 ( 100.8)"

37.4 f 0.6 (101.6)"

20.4 f 0.3 (102.0)O

21.1 f 0.4 (99.5)"

21.4 f 0.9 (100.9)"

21.2 f 0.2 (100.0)"

11.2 f 1.1 (86.5)b

3.66 f 0.2 (92.3)*

5.75 f 0.13

4.73 f 0.03 (88.1)b

7.55 f 0.39 (94.0)b

(93.7)b

616N, %o

+11.35 f 0.23

-0.96 f 0.07

-5.27 f 0.14

+0.34 f 0.12

+0.41 f 0.14

+19.81 f 0.04

+7.52 f 0.04

-1.20 i 0.25

+7.46 f 0.36

+7.37 f 0.06

+6.47 f 0.09

nitrogen, %

12.1 f 0.5 (100.8)"

36.9 f 1.2 (100.3)"

20.0 f 0.9 (100.0)"

21.3 f 0.5 (100.5)"

21.8 f 0.2

21.6 f 0.2

12.9 f 0.2

3.97 f 0.04

6.14 f 0.10

5.37 f 0.06

8.03 f 0.02

(102.8)"

(100.9)"

Kieldahl method combustion method difference:

%O

-0.19

+0.07

-0.09

-0.10

-0.09

-0.13

-0.41

+0.06

-0.52

-0.21

-0.66

t value 615N N, % ne

1 . 2 h O.Oh 5

1.2h 0.8h 7

0.7h 0.6h 3

1.4h 0.6h 5

l.lh O A h 4

1.2h 2.48 4

2.19 2.6f 5

0.3h 2.18 4

2.39 4.81 6

2.0h 16.g 3

4.3f 2.58 3

a Percent of yield relative to theoretical content. Percent relative to content obtained by combustion. Difference given by subtracting 615N by combustion from 615N by the Kjeldahl method. dThe number of measurements. eThe number of degrees of freedom. f P < 0.05. 8P < 0.1. hP > 0.1.

ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984 1861

to be 103 f 3% relative to the theoretical value (20.0%). The 615N values are reproducible and agree to within 0.1% of the result obtained by the Kjeldahl method. The volume of Nz and C02 obtained by 850 "C combustion is larger than that obtained by 550 "C combustion for egg albumin and marine zooplankton, and a significantly lower 615N was obtained when egg albumin combustion was performed at 550 "C. There is no difference in the yield N2 and COz from combustion at 750 "C and that a t 850 "C. However, the 616N obtained at 750 "C is slightly lighter than that obtained at 850 "C for Tanner Basin kerogen and egg albumin and shows a large fluctuation for the marine zooplankton.

The Kjeldahl method gave slightly lighter 615N values than those obtained by 850 "C combustion, while the chemical yield was equal to or lower than that obtained by combustion at 850 "C. These results suggest that incomplete digestion or combustion may cause a loss of some 15N. Thus, these results show that heating to 850 "C provides the best yield and precision for total carbon and nitrogen for the samples tested here and the most reliable 15N/14N isotope ratio values.

The 613C values do not demonstrate such a temperature dependence, even when yields of C02 are not quantitative. This observation agrees with the previous results for com- bustion of petroleum fractions and biological samples (4 ,5 ) .

Further comparisons were made among laboratory sub- standards and selected samples analyzed by the Kjeldahl method and the combustion method at 850 "C. Table I1 shows that the 615N values for pure valine, thiourea, histidine, and ammonium sulfate, obtained by the combustion method are identical with results obtained by the Kjeldahl method. The regression curve calculated from the six samples analyzed yields

615N (by combustion) = 1.001 615N (by Kjeldahl) - 0.067

r = 0.99996 (2)

The chemical yield compared with theoretical values was close to 100% for both methods, indicating that both methods are reliable. However, significant differences in 615N between the two methods have been found for naturally occurring complex organic substances such as egg albumin, soy bean nodules, Tanner Basin kerogen, and plankton. The nitrogen content obtained by the Kjeldahl method was lower than that obtained by combustion, ranging from 86.5 to 94.0%. Low yields of nitrogen do not always result in light isotope values, as is the case for soy bean leaf (Table 11), and a quantitative relationship between chemical yields and isotope ratios has not yet been recognized. We believe that the isotope change related to incomplete recovery of nitrogen is probably not caused by isotope discrimination but may be due to the heterogeneous distribution of 15N in natural organic materials.

CONCLUSIONS The effect of combustion temperature has been investigated

in the measurement of stable nitrogen isotope ratio in organic materials. Sealed tube combustions at 550 "C provided a lower chemical yield of nitrogen and carbon atoms than the 750 "C and 850 "C combustions. Incomplete combustion resulted in a shift of nitrogen isotope ratios toward light values for egg albumin and marine zooplankton, but the carbon isotope ratio remained unchanged. The 615N obtained at 750 "C com- bustion is slightly lower or more variable than that obtained by 850 "C combustion, whereas, the volume of N2 and C02 showed little significant change. Hence, combustion at 850 "C is recommended as the method for nitrogen isotope ratio measurement of naturally occurring organic substances.

Selected samples have been analyzed by the Kjeldahl me- thod to compare with the combustion method. Both methods gave identical 615N values for ammonium sulfate and amino acids. However, for complex organic matter, Kjeldahl di- gestion did not provide complete chemical yields. In this study, the maximum measured difference of 6I5N between the two methods is 0.66 per mil for a marine zooplankton sample. The deviation of measured nitrogen isotope ratios depends on the heterogeneity of samples and the recovery of nitrogen during sample preparation. Accordingly, the combustion method at 850 "C described here is recommended as the appropriate technique for preparing biological and geochemical samples for stable nitrogen isotope ratio analysis.

ACKNOWLEDGMENT We thank D. Kohl and G. Shearer, Washington University,

Registry No. N2, 7727-37-9; 15N, 14390-96-6. for providing their agricultural samples.

LITERATURE CITED Bremner, John M. Agronomy 1965, 1256-1286. Stump, Ronald K.; Frazer, Jack W. Nucl. Sci. Abstr. 1073, 28, 746. Stuermer, Daniel H.; Peters, Kenneth E.; Kaplan, Isaac R. Geochim. Cosmochim. Acta 1078, 42, 989-997. Sofer, Zvl Anal. Chem. W80, 52, 1389-1391. Boutton, Thomas W.; Wong, Wllllam W.; Hachey, David L.; Lee, Lucin- da S.; Cabrera, Mercedes P.; Kleln, Peter D. Anal. Chem. 1063, 55, I 832- I 833. Schoell, Martin; Faber. Eckhard; Coleman, Max L. Ora. Geochem. 1083, 5 , 3-6. Wada, Eiatro, Mlsubishi-Kasei Institute of Life Sciences, Tokyo, 1978, personal communlcatlon. Bremner, J. M.; Edwards, A. P. Soil Sci. Proc. 1965, 504-507. Des Marals, David J.; Hayes, John M. Anal. Chem. 1976, 48, 1651-1652.

RECEIVED for review January 27, 1984. Accepted April 24, 1984. This work was supported by a contract from the U.S. Department of Energy, No. DOE DE-AT03-76EV70134 Mod. No. 29. This paper is publication number 2498 of the Institute of Geophysics and Planetary Physics, University of California a t Los Angeles, Los Angeles, CA.