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Quantitative determination of particulate organic N and C in marine-phytoplankton samples using mass-spectrometer signals from isotope-ratio analyses in 15N- and 13C-tracer studies
.I. Exp. Mar, Biol. Ecui., 1988, Vol. 115, pp. 187-195 Elsevier
187
JEM 01014
Qu~titativ~ determination of p~ti~ulat~ organic N and C in marine-phytoplankton samples using mass-spectrometer signals from isotope-ratio analyses in “N- and X3C-tracer studies
Abstract: A gas-sampling device between an oven for Pumas combustion and an isotope mass spectrometer enables the de~rrn~~at~on of particulate organic N and C ~OnceRtrations during isotopic “N and ‘%Z analysis of p~~topl~kto~ samples from d~-~~bel~~g experiments. A descriptionrtfthe interface is given with details of the operating procedure. The precision ofthe method was comparable with an elemental C-N analyser with a C.V. of 2.7Opi, at the level of 5.17 I.tmol for N and 7.26”/, at the level of 48.77 pmol for C. Key words: C; 13C; Mass spectrometry; N; “N; Phytoplankton
Mass-spectrometric analyses of phytuplankton samples experiments yield specific uptake rates of N (V,) and C (V,) 2% Goering, 1967; SEayk et LA, 1377, 1979). Transport compounds (pi and pc) in units of mass *volume- I - time-
in 15N- and *%-uptake in units of time (Dugdale rates of the two latter ’ are calculated by:
p,=V;,xPON, pc = V, x POC,
(1) (2)
where PQN and POC are the particulate organic matter coxlcentrations for N and C, respectively, In most marine investigations, samples for PON and POC were collected before incubation in parallel with samples for ’ 5N and 13Clabelling and initial concentrations were determined on an elemental analyser. However, in order to match the correct values of particufates and specific uptake rates and to gain time in processing samples Correspondence address: G. Siawyk, Centre d’Oc&mologie de &farseille (U.A. 41), Facult6 des Sciences de Luminy, Case 901, 13288 Marseille Ckdex 9, France. OO22-0981/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
G.SLAWYK
188
ETAL.
and accuracy in estimating 15N and 13Cincorporation, it is preferable to measure PON and POC concentrations as well as “N- and 13C-isotope enrichments at the end of incubation on the same particulate fraction during the mass spectrometry procedure (Dugdale & Wilkerson, 1986). Mass spectrometry was frst applied to the analysis of total N content and ‘5N-isotope enrichment in marine phytoplankton by Wada et al. (1977). Several combined systems of a mass spectrometer (MS) with an automatic elemental analyser have been developed for simultaneous analysis of PON and “N (Barsdate & Dugdale, 1965; Pavlou et al., 1974; Tsuji et al., 1975; Preston & Owens, 1983; Barrie & Workman, 1984), for POC and 13Cby Preston & Owens (1985), and for both (PON, POC, “N, and 13C)by Otsuki et al. (1983), but only Otsuki et al. (1983) and Barrie & Workman (1984) used the MS for total elemental analysis in addition to isotope analysis. This paper describes a convenient gas-sampling device which allows the simultaneous measurement of PON and POC concentrations and 15N and 13Cenrichments of marine phytoplankton on a single sample from monitoring MS signals only. Our system offers the advantage over previous methods in that it obviates the need for a commercial C-N analyser. APPARATUS
AND
METHODS
CONVERSION OF ORGANIC MATTER
PON and POC of samples were processed by the dry-combustion technique of Dumas to CO, and N,. The conversion was performed in a modified Coleman (U.S.) 29A N analyser. He (He > 99.9999x, N, < 0.6 ppm; L’Air Liquide, France) was used as a carrier gas at a sweep rate of 60 ml * min- I. The CuO-packed quartz-oxidation reactor and the CuO/Cu-packed quartz-reduction reactor were heated at 840 and 520 “C, respectively. CuO wire (0.65 x 6 mm; surface, CuO; core, Cu,O) was purchased from Merck (F.R.G.) and Cu (60-100 mesh) was obtained from Perkin-Elmer (France). All solenoid valves were removed from the analyser to ensure that the combustion train was under constant pressure (0.8 bar) of He during the analytical cycle of the instrument. The means of the converting system blanks were 0.21 (SD = 0.01) pmol and 0.44 (SD = 0.02) pm01 for N and C, respectively. Particulate matter samples containing between 3.00 (20.00) and 7.00 (40.00) pmol of PON (POC) were run for total elemental and isotopic analyses. Under these typical operating conditions, ion currents ranged from ~2.0 to 5.0 x 10-‘” A for N$ and from 28.0 to 55.0 x lo- lo A for CO; (Fig. 2). SAMPLE-INLET SYSTEM The
vacuum
manifold
used
shown in Fig. 1. Essentially,
for the preparation
this is a batch-sampling
of CO,-
and N,-gas
samples
is
system in which the sample gas
DETERMINATION
OF N AND C IN MARINE PHYTOPLANKTON
189
is admitted to an expansion-reservoir
volume of 500 ml (R, for N,, R, for CO,) until suitable pressure conditions are achieved to admit the sample via a 900 x 0.2%mm i.d. stainless-steel capillary to the analyser tube of the MS. Watts the PON and POC working range mentioned above, no significant mass discrimination could be detected, S-SC
He
V
Fig. 1. Gas-sampling device for “N and 13C, and total N and C de~~rm~nati~ns. CNA, Coleman nitrogen analyser; G/M, glass-metal seal; T,, T2, and T,, traps; R,, expansion reservoir for N2; R,, expansion reservoir for CO,; S-SC, stainless-steel capillary to MS; PG, Pirani gauge; MoS, molecular-sieve trap; V, to vacuum pumps; l-13, high-vacuum glass-metal valves @VT, France).
thus indicating that the gas Row in the capillary was predom~~tly viscous in character and the isotope fractionation was minimized. The complete assembly was built of Pyrex-glass tubing (7 mm o.d., 4 mm i.d.). T,, Tz, and T, are cold traps, each consisting of a Pyrex-glass tube (6 mm o.d., 3 mm i.d., % 1 m long) wound into a helix. T, and T, are inserted in the part of the vacuum line for C&-gas preparation. T, is immersed in liquid N just before the gaseous combustion products, by-products (N2, CO,, and H,O), and carrier gas enter the manifold, its purpose being to condense the CO, of the sample. CO, is removed from the trap by heating the helix at 70 “C with a hairdryer. T, is immersed in cold acetone ( - 60 “C, coldfinger) and serves to eliminate water vapour when the CO, gas is removed from trap T, and admitted to the analyser tube of the MS. T3 is immersed in liquid N and ensures minimum pressure in the part of the vacuum iine for N,-gas prep~ation. The molecular sieve was made from 200 mm in length Pyrex-glass tubing in “U” form (7 mm o.d., 4 mm id.) and filled with x 30 beads (2 mm d.) of 5-A molecular sieve (Merck), At liquid-N temperature, the sieve adsorbs
190
G. SLAWYK
ETAL.
N,, leaving the He carrier gas which is pumped out. N, is deadsorbed from the sieve by being heated at 220 “C with a heat gun. The manifold is evacuated to 0.005 mbar (measured by Pirani gauge), by combined use of a mercury-diffusion pump and a rotary pump. MASS
SPECTROMETER
(MS)
Isotopic analyses of the samples were performed in a V.G. Micromass (U.K.) MM 601 isotope MS fitted with a double collector. The MS vacuum was obtained with a water-cooled 150-l * s- ’ oil-vapour diffusion pump (Edwards, U.K.; Model E02). The source settings were the following: filament current 200 PA, electron energy 70 V, repeller t 1.2 and 1.9 V for N, and CO,, respectively. Major and minor ion currents were monitored on two chopper amplifiers (Model CA3). As N, and CO, were to be measured, the accelerating voltage was switched manually between m/z 29 and m/z 45 (minor peaks). Analyser pressure, measured by the ionisation gauge of the MS, ranged from 6.0 x 10-‘” mbar (background pressure) to 5 x lop9 and 2.5 x lo-’ mbar for N, and CO,, respectively. PRECISION
OF ISOTOPE
ANALYSES
The C.V. for seven unenriched samples (0.361 atom% 15N, sample size = 5.17 pmol N; 1.161 atom% 13C sample size = 48.77 pmol C) was 0.55 and 0.26% for “N and 13C, respectively. Foi six urea standards (5.52 atom% i5N, sample size = 4.11 pmol N) and 5 NaHi3C0, standards (2.45 atom% 13C, sample size = 33.00 pmol C), the C.V. was 1.10%. The latter performance is adequate for isotope-tracer experiments in primary-productivity studies (Harrison, 1983; Dugdale & Wilkerson, 1986). OPERATING
PROCEDURE
Particulate organic matter from sea water was collected on precombusted (400 “C for 5 h) Whatman GF/C glass fiber filters (47 mm d.). Each filter was ground with x 30 mg of CuO in a mortar and the resulting powder was poured into a precombusted (400 ’ C for 5 h) aluminium boat. The loaded sample boat was folded and then introduced into the oxidation reactor of the Coleman N analyser. Samples of glycine for calibrations were mixed with 30 mg of finely ground CuO and then treated in the same way as ground filters. In field studies, PON and POC concentrations are roughly estimated from chl a survey before doing the tracer experiment, and volumes of sea water used are then adjusted to obtain z 5.00 pmol of PON and 35.00 pmol of POC after filtration. N- and C-uptake rates are calculated from the rate of increase of i5N and 13Catom% excess over time; therefore, two isotopic analyses are necessary: one at “time zero” and the other after some incubation time. For the measurement at “time zero”, a sample (same volume as incubated sample) is filtered within minutes after isotope addition. This allows to remove pressure effects (variations of measured isotope ratio with sample
DETERMINATION
OF N AND C IN MARINE PHYTOPLANKTON
191
pressure) since the “time-zero” sample and the incubated sample give rise to almost the same gas pressure in the MS. The sequence of valve operations and trap manipulations on the vacuum manifold during a complete analytical cycle is given in Table I. The run of one sample, including sample preparation and MS analyses, takes ~20 min.
RESULTS
DISCUSSION
AND
Since the response of the MS to a constant amount of N, or CO, was found to be reproducible, we decided to use the sum of major and minor ion currents (m/z 28 + m/z 29, m/z 44 + m/z 45) as an alternative to the determination of PON and POC concentrations on an elemental C-N analyser. Appropriate standards, prepared from unenriched glycine, were used to establish a calibration curve (Fig. 2). The relationship between ion currents and concentration is linear up to at least 20 pmol of N and 40 pmol of C. The reproducibility of the proportionality factors (slope of the calibration
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,
Ok Am
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I
I
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10
20 ~.~rnolN
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I
30
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40
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Fig. 2. Sum of ion currents at masses 28 (14Ni4N) and 29 (14N’sN), and at masses 44 (“COJ and 45 ( ‘3C0,) against N and C content, respectively, for samples of glycine at natural abundances. Continuous line applies to A( x 10-i’) = 0.677 (pm01 N) + 0.052 (95% C.I. of slope = +0.015; 95% C.I. of intercept = _+0.122; r = 0.999; n = 18). Dashed line applies to A ( x lo- lo) = 1.429 (pm01 C) - 0.425 (95 y0 C.I. of slope = k 0.027; 95 y0 C.I. of intercept = + 0.430; r = 0.999; n = 18). Arrows indicate range of typical ion currents obtained from PON and POC contents in marine-phytoplankton samples.
TABLE I
(2) Combustion (3) N,, CO, trapping (4) N,, CO, removal and expansion (5) Admission of N, to mass spectrometer (6) Evacuation of Nz (7) Admission of CO, to mass spectrometer (8) Evacuation of CO,
( 1) Purge
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MoS
Trap temperatures (“C)
1
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2 OS
2
6 2
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Duration (min)
Valve positions and temperatures (“C) of trap T, and molecular sieve (MoS) in vacuum line of Fig. I during different steps of N,- and CO,-gas preparation for mass spectrometry. a Valve open, b valve closed, ’ room temperature.
k .
$
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2
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DETERMINATION
OF N AND C IN MARINE PHYTOPLANKTON
193
lines) was tested by running three series of glycine standards during a 6-month period. A C.V. of 0.45 and 2.32% for N and C, respectively, was obtained, indicating a high degree of stability in the analysing system. The precision of the MS procedure for replicate PON and POC determinations was compared with the conventional method using an elemental C-N analyser. Two sets of aliquots of coastal sea water (subsampies from a 30-l Niskin bottle sample) were filtered and analysed in parallel by the two methods. Statistical analyses of the data (Table II) reveal very similar performances by the two techniques and a t test indicates
TABLE II Comparison of POC and PON determinations (in pmol .I - ‘) obtained from C-N elemental analyser (Perkin-Elmer 240) and MS. a Obtained from subsamples taken in sample of coastal sea water off Brest, France. Sample
C-N analyser POC
Replicates of particulate matter= F & C.I. C.V. (7;)
MS
PON
POC
2.48 0.06 kO.14 2.42
24.39 1.77 k4.19 1.26
n=9 25.94 1.94 f 4.39 7.48
PON n=l 2.59 0.07 +0.17 2.70
that at a level of 1y0 the means for PON and POC obtained with the MS procedure are not significantly different from those obtained with the C-N analyser. It should be pointed out that the automated Dumas procedure (Barsdate & Dugdale, 1965) as well as vacuum-combustion techniques (Gunther et al., 1965; Wada et al., 1977; Grunseich et al., 1980) are still in use for “N-isotope analyses in a number of laboratories that might want to modify their systems according to our method to include PON, POC, and i3C-isotope analyses. We have used the Coleman N analyser for Dumas combustion in this study, but note that, as all solenoid valves were removed from this instrument, any tuuular oven will be adequate for the purpose of heating the combustion and reduction columns. Since the gas separation occurs in the glassware interface, the use of an elemental analyser, such as in other studies (Otsuki et al., 1983; Preston & Owens, 1983, 1985), is not required. Therefore, the construction costs of our apparatus are much lower than any of the commercial elemental-analyser/continuousflow MS systems which are now available for N as well as C analyses. On the other hand, the need for continual intervention of a skilled operator for each analysis is a major drawback compared with the latter completely automated systems. However, we would like to emphasize the advantages of doing a N- and C-uptake experiment in the same container (dual-labelling experiment) since incubation conditions are then the same for both measurements. With our manual technique we are now able to run
194
G. SLAWYK ETAL.
samples from such dual-labelling experiments, i.e., to analyse simultaneously PON and POC contents and “N and 13C enrichments on a single filter. Presently available commercial automated systems have not fully solved yet the problem of simultaneous 15N and 13Canalyses and still select N or C analyses by using a switching on (r5N mode) and switching off (13C mode) procedure of the CO, trap in the Dumas sample converter. Thus, they need two filters for a dual-labelling experiment instead of only one with our apparatus. Several solutions to the latter problem have been recently proposed by Preston & Owens (1985) which should allow the development of a completely automated system for simultaneous analysis of total N/C and 15N/13C.
ACKNOWLEDGEMENTS
Two anonymous reviewers who provided constructive criticisms on earlier drafts of the typescript are gratefully acknowledged. We thank M. C. Bonin for drafting. This work was supported by the Centre National de la Recherche Scientifique (U.A. 41, GRECO 130034, and ER 256).
REFERENCES BARRIE,A. & C.T. WORKMAN,1984. An automated analytical system for nutritional investigations using lSN tracers. Spectrosc. Int. J., Vol. 3, pp. 439-441. BARSDATE,R. J. & R.C. DUGDALE, 1965. Rapid conversion of organic nitrogen to N, for mass spectrometry: an automated Dumas procedure. Anal. Biochem., Vol. 13, pp. 1-5. DUGDALE, R.C. & J.J. GOERING, 1967. Uptake of new and regenerated forms of nitrogen in primary productivity. Limnol. Oceanogr., Vol. 12, pp. 196-206. DUGDALE,R. C. & F. P. WILKERSON,1986. The use of ‘sN to measure nitrogen uptake in eutrophic oceans, experimental considerations. Limnol. Oceanogr., Vol. 3, pp. 673-689. GRUNSEICH,G. S., R. C. DUGDALE,N. F. BREITNER& J. J. MACISAAC, 1980. Sample conversion, mass spectrometry, and calculations for 15N analysis of phytoplankton nutrient uptake. Coast. Upwelling Ecosyst. Anal. Tech. Rep., No. 44, pp. l-34. GUNTHER,H., H. G. FLOS & H. SIMON,1965. Ein vereinfachtes Verfahren zur ‘sN-Bestimmung. Z. Analyt. Chem., Vol. 218, pp. 401-408. HARRISON,W.G., 1983. Use of isotopes. In, Nitrogen in the marine environment, edited by E.J. Carpenter & D.G. Capone, Academic Press, New York, New York, pp. 763-807. OTSUKI,A., Y. INO & T. FUJII, 1983. Simultaneous measurements and determinations of stable carbon and nitrogen isotope ratios, and organic carbon and nitrogen contents in biological samples by coupling of a small quadrupole mass spectrometer and modified carbon-nitrogen elemental analyser. Int. J. Muss Spectrom. Ion Phys., Vol. 48, pp. 343-346. PAVLOU, S. P. G., G. FRIEDERICH& J. J. MACISAAC, 1974. Quantitative determination of total organic nitrogen and isotope enrichment in marine phytoplankton. Anal. Biochem., Vol. 61, pp. 16-24. PRESTON,T. & N. J. P. OWENS,1983. Interfacing an automatic elemental analyser with an isotope ratio mass spectrometer: the potential for fully automated total nitrogen and nitrogen-15 analysis. Analyst (London), Vol. 108, pp. 971-977. PRESTON,T. & N. J. P. OWENS, 1985. Preliminary ‘% measurements using a gas chromatograph interfaced to an isotope ratio mass spectrometer. Biomed. Muss Spectrom., Vol. 12, pp. 510-513. SLAWYK,G., Y. COLLOS& J.C. AUCLAIR,1977. The use of the i3C and ‘sN isotopes for the simultaneous measurement of carbon and nitrogen turnover rates in marine phytoplankton. Limnol. Oceanogr., Vol. 22, pp. 925-932.
DETERMINATION
OF N AND C IN MARINE PHYTOPLANKTON
195
SLAWYK,G., Y. COLLOS & J.C. AUCLAIR, 1979. Reply to comment by Fisher eral. Limnol. Oceanogr., Vol. 24, pp. 595-597. TSUJI, O., M. MASUGI & Y. KQSAI, 1975. An instruments method for ~tr~gen-15 and total nitrogen by combined system of mass spectrometer and automatic nitrogen analyzer. Anal. Biochem., Vol. 65, pp. 19-25. WADA, E., T. TSUJI, T. SAINO & A. HATTORI, 1977. A simple procedure for mass spectrometric microanalysis of “N in particulate organic matter with special reference to “N-tracer experiments. Anal. Biochem., Vol. 80, pp. 312-318.
187
JEM 01014
Qu~titativ~ determination of p~ti~ulat~ organic N and C in marine-phytoplankton samples using mass-spectrometer signals from isotope-ratio analyses in “N- and X3C-tracer studies
Abstract: A gas-sampling device between an oven for Pumas combustion and an isotope mass spectrometer enables the de~rrn~~at~on of particulate organic N and C ~OnceRtrations during isotopic “N and ‘%Z analysis of p~~topl~kto~ samples from d~-~~bel~~g experiments. A descriptionrtfthe interface is given with details of the operating procedure. The precision ofthe method was comparable with an elemental C-N analyser with a C.V. of 2.7Opi, at the level of 5.17 I.tmol for N and 7.26”/, at the level of 48.77 pmol for C. Key words: C; 13C; Mass spectrometry; N; “N; Phytoplankton
Mass-spectrometric analyses of phytuplankton samples experiments yield specific uptake rates of N (V,) and C (V,) 2% Goering, 1967; SEayk et LA, 1377, 1979). Transport compounds (pi and pc) in units of mass *volume- I - time-
in 15N- and *%-uptake in units of time (Dugdale rates of the two latter ’ are calculated by:
p,=V;,xPON, pc = V, x POC,
(1) (2)
where PQN and POC are the particulate organic matter coxlcentrations for N and C, respectively, In most marine investigations, samples for PON and POC were collected before incubation in parallel with samples for ’ 5N and 13Clabelling and initial concentrations were determined on an elemental analyser. However, in order to match the correct values of particufates and specific uptake rates and to gain time in processing samples Correspondence address: G. Siawyk, Centre d’Oc&mologie de &farseille (U.A. 41), Facult6 des Sciences de Luminy, Case 901, 13288 Marseille Ckdex 9, France. OO22-0981/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)
G.SLAWYK
188
ETAL.
and accuracy in estimating 15N and 13Cincorporation, it is preferable to measure PON and POC concentrations as well as “N- and 13C-isotope enrichments at the end of incubation on the same particulate fraction during the mass spectrometry procedure (Dugdale & Wilkerson, 1986). Mass spectrometry was frst applied to the analysis of total N content and ‘5N-isotope enrichment in marine phytoplankton by Wada et al. (1977). Several combined systems of a mass spectrometer (MS) with an automatic elemental analyser have been developed for simultaneous analysis of PON and “N (Barsdate & Dugdale, 1965; Pavlou et al., 1974; Tsuji et al., 1975; Preston & Owens, 1983; Barrie & Workman, 1984), for POC and 13Cby Preston & Owens (1985), and for both (PON, POC, “N, and 13C)by Otsuki et al. (1983), but only Otsuki et al. (1983) and Barrie & Workman (1984) used the MS for total elemental analysis in addition to isotope analysis. This paper describes a convenient gas-sampling device which allows the simultaneous measurement of PON and POC concentrations and 15N and 13Cenrichments of marine phytoplankton on a single sample from monitoring MS signals only. Our system offers the advantage over previous methods in that it obviates the need for a commercial C-N analyser. APPARATUS
AND
METHODS
CONVERSION OF ORGANIC MATTER
PON and POC of samples were processed by the dry-combustion technique of Dumas to CO, and N,. The conversion was performed in a modified Coleman (U.S.) 29A N analyser. He (He > 99.9999x, N, < 0.6 ppm; L’Air Liquide, France) was used as a carrier gas at a sweep rate of 60 ml * min- I. The CuO-packed quartz-oxidation reactor and the CuO/Cu-packed quartz-reduction reactor were heated at 840 and 520 “C, respectively. CuO wire (0.65 x 6 mm; surface, CuO; core, Cu,O) was purchased from Merck (F.R.G.) and Cu (60-100 mesh) was obtained from Perkin-Elmer (France). All solenoid valves were removed from the analyser to ensure that the combustion train was under constant pressure (0.8 bar) of He during the analytical cycle of the instrument. The means of the converting system blanks were 0.21 (SD = 0.01) pmol and 0.44 (SD = 0.02) pm01 for N and C, respectively. Particulate matter samples containing between 3.00 (20.00) and 7.00 (40.00) pmol of PON (POC) were run for total elemental and isotopic analyses. Under these typical operating conditions, ion currents ranged from ~2.0 to 5.0 x 10-‘” A for N$ and from 28.0 to 55.0 x lo- lo A for CO; (Fig. 2). SAMPLE-INLET SYSTEM The
vacuum
manifold
used
shown in Fig. 1. Essentially,
for the preparation
this is a batch-sampling
of CO,-
and N,-gas
samples
is
system in which the sample gas
DETERMINATION
OF N AND C IN MARINE PHYTOPLANKTON
189
is admitted to an expansion-reservoir
volume of 500 ml (R, for N,, R, for CO,) until suitable pressure conditions are achieved to admit the sample via a 900 x 0.2%mm i.d. stainless-steel capillary to the analyser tube of the MS. Watts the PON and POC working range mentioned above, no significant mass discrimination could be detected, S-SC
He
V
Fig. 1. Gas-sampling device for “N and 13C, and total N and C de~~rm~nati~ns. CNA, Coleman nitrogen analyser; G/M, glass-metal seal; T,, T2, and T,, traps; R,, expansion reservoir for N2; R,, expansion reservoir for CO,; S-SC, stainless-steel capillary to MS; PG, Pirani gauge; MoS, molecular-sieve trap; V, to vacuum pumps; l-13, high-vacuum glass-metal valves @VT, France).
thus indicating that the gas Row in the capillary was predom~~tly viscous in character and the isotope fractionation was minimized. The complete assembly was built of Pyrex-glass tubing (7 mm o.d., 4 mm i.d.). T,, Tz, and T, are cold traps, each consisting of a Pyrex-glass tube (6 mm o.d., 3 mm i.d., % 1 m long) wound into a helix. T, and T, are inserted in the part of the vacuum line for C&-gas preparation. T, is immersed in liquid N just before the gaseous combustion products, by-products (N2, CO,, and H,O), and carrier gas enter the manifold, its purpose being to condense the CO, of the sample. CO, is removed from the trap by heating the helix at 70 “C with a hairdryer. T, is immersed in cold acetone ( - 60 “C, coldfinger) and serves to eliminate water vapour when the CO, gas is removed from trap T, and admitted to the analyser tube of the MS. T3 is immersed in liquid N and ensures minimum pressure in the part of the vacuum iine for N,-gas prep~ation. The molecular sieve was made from 200 mm in length Pyrex-glass tubing in “U” form (7 mm o.d., 4 mm id.) and filled with x 30 beads (2 mm d.) of 5-A molecular sieve (Merck), At liquid-N temperature, the sieve adsorbs
190
G. SLAWYK
ETAL.
N,, leaving the He carrier gas which is pumped out. N, is deadsorbed from the sieve by being heated at 220 “C with a heat gun. The manifold is evacuated to 0.005 mbar (measured by Pirani gauge), by combined use of a mercury-diffusion pump and a rotary pump. MASS
SPECTROMETER
(MS)
Isotopic analyses of the samples were performed in a V.G. Micromass (U.K.) MM 601 isotope MS fitted with a double collector. The MS vacuum was obtained with a water-cooled 150-l * s- ’ oil-vapour diffusion pump (Edwards, U.K.; Model E02). The source settings were the following: filament current 200 PA, electron energy 70 V, repeller t 1.2 and 1.9 V for N, and CO,, respectively. Major and minor ion currents were monitored on two chopper amplifiers (Model CA3). As N, and CO, were to be measured, the accelerating voltage was switched manually between m/z 29 and m/z 45 (minor peaks). Analyser pressure, measured by the ionisation gauge of the MS, ranged from 6.0 x 10-‘” mbar (background pressure) to 5 x lop9 and 2.5 x lo-’ mbar for N, and CO,, respectively. PRECISION
OF ISOTOPE
ANALYSES
The C.V. for seven unenriched samples (0.361 atom% 15N, sample size = 5.17 pmol N; 1.161 atom% 13C sample size = 48.77 pmol C) was 0.55 and 0.26% for “N and 13C, respectively. Foi six urea standards (5.52 atom% i5N, sample size = 4.11 pmol N) and 5 NaHi3C0, standards (2.45 atom% 13C, sample size = 33.00 pmol C), the C.V. was 1.10%. The latter performance is adequate for isotope-tracer experiments in primary-productivity studies (Harrison, 1983; Dugdale & Wilkerson, 1986). OPERATING
PROCEDURE
Particulate organic matter from sea water was collected on precombusted (400 “C for 5 h) Whatman GF/C glass fiber filters (47 mm d.). Each filter was ground with x 30 mg of CuO in a mortar and the resulting powder was poured into a precombusted (400 ’ C for 5 h) aluminium boat. The loaded sample boat was folded and then introduced into the oxidation reactor of the Coleman N analyser. Samples of glycine for calibrations were mixed with 30 mg of finely ground CuO and then treated in the same way as ground filters. In field studies, PON and POC concentrations are roughly estimated from chl a survey before doing the tracer experiment, and volumes of sea water used are then adjusted to obtain z 5.00 pmol of PON and 35.00 pmol of POC after filtration. N- and C-uptake rates are calculated from the rate of increase of i5N and 13Catom% excess over time; therefore, two isotopic analyses are necessary: one at “time zero” and the other after some incubation time. For the measurement at “time zero”, a sample (same volume as incubated sample) is filtered within minutes after isotope addition. This allows to remove pressure effects (variations of measured isotope ratio with sample
DETERMINATION
OF N AND C IN MARINE PHYTOPLANKTON
191
pressure) since the “time-zero” sample and the incubated sample give rise to almost the same gas pressure in the MS. The sequence of valve operations and trap manipulations on the vacuum manifold during a complete analytical cycle is given in Table I. The run of one sample, including sample preparation and MS analyses, takes ~20 min.
RESULTS
DISCUSSION
AND
Since the response of the MS to a constant amount of N, or CO, was found to be reproducible, we decided to use the sum of major and minor ion currents (m/z 28 + m/z 29, m/z 44 + m/z 45) as an alternative to the determination of PON and POC concentrations on an elemental C-N analyser. Appropriate standards, prepared from unenriched glycine, were used to establish a calibration curve (Fig. 2). The relationship between ion currents and concentration is linear up to at least 20 pmol of N and 40 pmol of C. The reproducibility of the proportionality factors (slope of the calibration
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TABLE I
(2) Combustion (3) N,, CO, trapping (4) N,, CO, removal and expansion (5) Admission of N, to mass spectrometer (6) Evacuation of Nz (7) Admission of CO, to mass spectrometer (8) Evacuation of CO,
( 1) Purge
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DETERMINATION
OF N AND C IN MARINE PHYTOPLANKTON
193
lines) was tested by running three series of glycine standards during a 6-month period. A C.V. of 0.45 and 2.32% for N and C, respectively, was obtained, indicating a high degree of stability in the analysing system. The precision of the MS procedure for replicate PON and POC determinations was compared with the conventional method using an elemental C-N analyser. Two sets of aliquots of coastal sea water (subsampies from a 30-l Niskin bottle sample) were filtered and analysed in parallel by the two methods. Statistical analyses of the data (Table II) reveal very similar performances by the two techniques and a t test indicates
TABLE II Comparison of POC and PON determinations (in pmol .I - ‘) obtained from C-N elemental analyser (Perkin-Elmer 240) and MS. a Obtained from subsamples taken in sample of coastal sea water off Brest, France. Sample
C-N analyser POC
Replicates of particulate matter= F & C.I. C.V. (7;)
MS
PON
POC
2.48 0.06 kO.14 2.42
24.39 1.77 k4.19 1.26
n=9 25.94 1.94 f 4.39 7.48
PON n=l 2.59 0.07 +0.17 2.70
that at a level of 1y0 the means for PON and POC obtained with the MS procedure are not significantly different from those obtained with the C-N analyser. It should be pointed out that the automated Dumas procedure (Barsdate & Dugdale, 1965) as well as vacuum-combustion techniques (Gunther et al., 1965; Wada et al., 1977; Grunseich et al., 1980) are still in use for “N-isotope analyses in a number of laboratories that might want to modify their systems according to our method to include PON, POC, and i3C-isotope analyses. We have used the Coleman N analyser for Dumas combustion in this study, but note that, as all solenoid valves were removed from this instrument, any tuuular oven will be adequate for the purpose of heating the combustion and reduction columns. Since the gas separation occurs in the glassware interface, the use of an elemental analyser, such as in other studies (Otsuki et al., 1983; Preston & Owens, 1983, 1985), is not required. Therefore, the construction costs of our apparatus are much lower than any of the commercial elemental-analyser/continuousflow MS systems which are now available for N as well as C analyses. On the other hand, the need for continual intervention of a skilled operator for each analysis is a major drawback compared with the latter completely automated systems. However, we would like to emphasize the advantages of doing a N- and C-uptake experiment in the same container (dual-labelling experiment) since incubation conditions are then the same for both measurements. With our manual technique we are now able to run
194
G. SLAWYK ETAL.
samples from such dual-labelling experiments, i.e., to analyse simultaneously PON and POC contents and “N and 13C enrichments on a single filter. Presently available commercial automated systems have not fully solved yet the problem of simultaneous 15N and 13Canalyses and still select N or C analyses by using a switching on (r5N mode) and switching off (13C mode) procedure of the CO, trap in the Dumas sample converter. Thus, they need two filters for a dual-labelling experiment instead of only one with our apparatus. Several solutions to the latter problem have been recently proposed by Preston & Owens (1985) which should allow the development of a completely automated system for simultaneous analysis of total N/C and 15N/13C.
ACKNOWLEDGEMENTS
Two anonymous reviewers who provided constructive criticisms on earlier drafts of the typescript are gratefully acknowledged. We thank M. C. Bonin for drafting. This work was supported by the Centre National de la Recherche Scientifique (U.A. 41, GRECO 130034, and ER 256).
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OF N AND C IN MARINE PHYTOPLANKTON
195
SLAWYK,G., Y. COLLOS & J.C. AUCLAIR, 1979. Reply to comment by Fisher eral. Limnol. Oceanogr., Vol. 24, pp. 595-597. TSUJI, O., M. MASUGI & Y. KQSAI, 1975. An instruments method for ~tr~gen-15 and total nitrogen by combined system of mass spectrometer and automatic nitrogen analyzer. Anal. Biochem., Vol. 65, pp. 19-25. WADA, E., T. TSUJI, T. SAINO & A. HATTORI, 1977. A simple procedure for mass spectrometric microanalysis of “N in particulate organic matter with special reference to “N-tracer experiments. Anal. Biochem., Vol. 80, pp. 312-318.