RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom.

2007; 21: 41354141 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/rcm.3304

Improved isotope ratio measurement performance in liquid chromatography/isotope ratio mass spectrometry by removing excess oxygen
Elena Hettmann, Willi A. Brand and Gerd Gleixner*
Max Planck Institute for Biogeochemistry, P .O. Box 100164, 07701 Jena, Germany Received 18 June 2007; Revised 26 September 2007; Accepted 2 October 2007

A low dead volume oxygen scrubbing system was introduced in a commercially available liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) interface to enhance the analytical capability of the system. In the LC/IRMS interface carbon from organic samples is converted into CO2 inside the mobile phase by wet chemical oxidation using peroxodisulfate (Na2S2O8). After passing the hot reaction zone, surplus oxygen (O2) remains dissolved in the liquid phase. Both CO2 and O2 diffuse through a transfer membrane into the helium carrier and are transferred to the mass spectrometer. The presence of O2 in the ion source may have detrimental effects on measurement accuracy and precision as well as on lament lifetime. As a remedy, a new on-line O2-removing device has been incorporated into the system. The new O2 scrubber consists of two parallel hot copper reduction reactors (0.8 mm i.d., active length 120 mm) and a switch-over valve between them. One reactor is regenerated using He/H2 while the other is actively scavenging O2 from the gas stream. The capacity of each reduction reactor, expressed as usage time, is between 40 and 50 min. This is sufcient for a single LC run for sugars and organic acids. A further increase of the reduction capacity is accompanied by a peak broadening of about 100%. After switching to a freshly reduced reactor the oxygen background and the d13C values of the reference gas need up to 500 s to stabilize. For repeated injections the d13C values of sucrose remain constant (W0.1%) for about 3000 s. The long-term stability for measurements of sucrose was 0.11% without the reduction oven and improved slightly to 0.08% with the reduction oven. The lament lifetime improved by more than 600%, thereby improving the long-term system stability and analytical efciency. In addition the costs per analysis were reduced considerably. Copyright # 2007 John Wiley & Sons, Ltd.
Isotopic discrimination in metabolic reactions is a consequence of kinetic isotope effects generating products with characteristic isotopic signatures and leading to marked variations in the isotope ratios of source and product pools. This offers the possibility to use the natural isotope abundance of carbon in metabolic compounds to study metabolic pathways,13 source-sink relations,4 and effects of climate variability5,6 and stress7 on plant physiology and metabolism. The analysis of the natural isotopic abundance in non-labeled compounds requires a very high analytical precision and consequently demands quantitative sample preparation procedures. Most biological materials are non-volatile. In general, derivatization of the components is needed to make the material amenable for gas chromatography/mass spectrometry (GC/MS) and on-line isotopic analysis. The derivatization step unavoidably adds carbon atoms to the target compounds, thereby altering the original
*Correspondence to: G. Gleixner, Max Planck Institute for Biogeochemistry, P.O. Box 100164, 07701 Jena, Germany. E-mail: ggleix@bgc-jena.mpg.de Contract/grant sponsor: Max Planck Society.
Copyright # 2007 John Wiley & Sons, Ltd.

isotopic signature. In addition, isotopic fractionation may accompany the derivatization process. If the reaction has no isotope effect a mass balance approach yields the correct isotopic composition.8 However, both factors can reduce the achievable precision of the measurements. For most biological molecules (sugars, nucleotides, etc.) derivatization is not feasible. Their isotopic composition is better determined by HPLC (high-performance liquid chromatography) separation. Individual components are collected, solvent is evaporated or freeze-dried and the remaining solids are analyzed using an elemental analyzer linked on-line to an isotope ratio mass spectrometry (IRMS) system (see, for instance, Refs. 4, 7, 9 and 10, and references cited therein). This procedure yields very reliable results. It is, however, rather labor-intensive and time-consuming and therefore not practical for day-to-day routine measurements. An alternative for measuring such components is an on-line coupling between HPLC and IRMS. The rst strategies to develop such a coupling were attempted in

4136 E. Hettmann, W. A. Brand and G. Gleixner

Figure 1. Schematics of the LC/IRMS system with integrated reduction reactor (modied from Krummen et al.13). Rectangular components are parts of the original system, rounded rectangular components have been added. The shown schematics was used during the experiments; recently we positioned the water trap downstream of the reduction reactor as this new conguration is regarded as slightly more benecial. the early 1990s.11,12 Based on a moving wire technique, they comprised a transport wire (or belt) where the solvent is evaporated and the sample is transported on the wire through a combustion unit for CO2 production and subsequent isotope ratio analysis. Because of the complexity of the system it was not robust enough for routine analysis and consequently has not been made commercially available. A new concept for coupling liquid chromatography (LC) on-line to IRMS was introduced in 2004.13 The commercially available ThermoFinnigan IsoLink system (Thermo Electron, Bremen, Germany) is designed for the isotopic analysis of a broad spectrum of non-volatile compounds with a wide range of applications for industrial, medical and scientic purposes.1418 The IsoLink interface technique is based on chemical oxidation of the organic compounds inside the liquid phase. The CO2 produced during the combustion reaction is subsequently separated from the mobile phase, dried and enters the ion source of the mass spectrometer in a helium stream via the usual open split interface.19 Oxidation occurs by reaction of peroxodisulfate and the organic analyte inside the liquid. Hence, the solvent must be pure water, free of any organic contaminant. However, for quantitative and fractionation-free conversion of organic compounds into CO2 the reaction must be carried out with an excess of oxygen (O2). Unfortunately, the commercial design of the IsoLink interface does not include a removal step for unused O2. As a consequence, the gas mixture reaching the mass spectrometer ion source is composed of CO2 in helium plus a large and varying amount of O2. The presence of O2 in the mass spectrometer ion source implies a number of potential experimental problems. Like all ions in the ion source (normally mainly He and CO 2) oxygen ions enhance the space charge effects in the source. Furthermore, the oxygen background is not stable. Inevitable uctuations are determined by the oxidation efciency in relation to sample amounts. O2 is consumed when oxidizing individual components eluting from the LC system. Between sample peaks the O2 background increases back to the initial high level. Such variations lead to uncontrolled oxidative reactions with the hot lament and other surfaces in the source and, as a consequence, to ion source tuning problems and CO2 background uctuations. Thus the presence of O2 in

Figure 2. Schematics of the reduction reactor. (A) Eight-port valve, (B) ceramic tubes lled with copper wire, (C) heating reactor. Gas mixtures (He CO2 O2 from gas dryer; He H2 from lab gas tank) pass through the reactor at 5808C, causing oxidation/reduction of copper wire. Switching the valve changes the ow directions.
Copyright # 2007 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2007; 21: 41354141 DOI: 10.1002/rcm

Improved isotope ratio measurement performance in LC/IRMS

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Figure 4. Changes in the oxygen background during oxidation of eluting organic compounds (no reduction reactor installed). The three larger negative peaks (o) result from oxygen consumption during the combustion step.Three small positive peaks (x) at the start and at the end are observable when reference gas enters the ion source. The possible reason for these changes are the adsorbtion/desorbtion on the surfaces in the ion source and competition between O2 and CO2.

EXPERIMENTAL
As a model compound we used sucrose (Merck, Darmstadt, Germany, supra pure 99.9%) samples ranging in sample size between 210 ng and 2940 ng carbon. Sucrose was chosen because within the order organic acids monosaccharide disaccharide it requires the highest oxidation capacity for quantitative, fractionation-free conversion into CO2. For isotopic analysis an HPLC system (Finnigan Surveyor HPLC system, Thermo Electron, Bremen, Germany) coupled to the LC/IRMS interface (IsoLink) and subsequently to the isotope ratio mass spectrometer (DeltaXP, Thermo Electron, Bremen, Germany) was used. All solutions (oxidation reagent, phosphoric acid, water) were degassed under vacuum in an ultrasonic bath; HPLC-grade water was used for sample preparation. A schematic representation of the LC/IRMS interface is presented in Fig. 1. The interface can be used in two operational modes: (1) HPLC mode for compound-specic analysis of individual peaks, separated via a HPLC column; and (2) ow injection mode for bulk sample analysis. For the experiments described in this paper we used the ow injection mode in order to avoid possible effects of a column Table 1. Time required to stabilize the oxygen background after switching between reactors. Data are given in V (as measured by MS); 1 V 3.3 nA
Initial background (V) 3.6 4.0 6.7 8.0 8.8 9.6 10.2 15.0 20.0 Time needed to reach 50 mV (s) 2 2 3 3 3 3 3 8 9 Time needed to reach 20 mV (s) 28 316 189 312 312 122 259 414 165

Figure 3. (a) d13C values of reference gas pulses as a function of the oxygen background. Plotted are two independent sequences (oxygen background and d13C values of reference gas), injections were made manually. The larger standard deviation of reference gas d13C values between 50 and 1000 s is not necessarily for the system and occurred because of the startup effects. (b) d13C values of reference gas pulses as a function of the oxygen background. The ve points are the means of each level of oxygen background or d13C values of reference gas presented in (a). the source can reduce the reliability of the measured isotope relations. The second major problem generated by O2 in the ion source is oxidation of tungsten and tantalum which are major parts of the lament. Formation of tungsten and tantalum oxides changes the physical properties of the lament resulting in a markedly reduced lament lifetime. Fast deterioration of the lament increases the cost per analysis, makes the whole system less stable over time, and leads to a requirement for frequent maintenance, which in turn hampers routine measurement performance and renders analysis of a large number of samples cumbersome. Removal of O2 from the helium carrier is possible using oxidation/reduction reactions of copper at elevated temperatures (6008C) without affecting isotopic delity. The technique is widespread in CHN analyzers coupled to isotope ratio mass spectrometers and has been introduced for similar reasons to those for the GC/IRMS systems in the early 1990s.8,20 Here we investigated if the O2-free atmosphere in the mass spectrometer source corresponding to O2 removed from the system contributes to (a) better controlled measurement conditions and (b) to a markedly increased lifetime of the lament.
Copyright # 2007 John Wiley & Sons, Ltd.

Rapid Commun. Mass Spectrom. 2007; 21: 41354141 DOI: 10.1002/rcm

4138 E. Hettmann, W. A. Brand and G. Gleixner

Figure 5. Oxygen background as a function of time after switching between the reduction reactors at a temperature of 5808C (switched at 50 s). On the left Y-axis are shown the changes of oxygen background between 0 and 25 V, on the right Y-axis the changes of oxygen background during the same run with zooming. on the d13C values of compounds and their precision. For combusting organic compounds eluting from the HPLC unit a constant mixture of two reagents, sodium peroxodisulfate (0.55 M) and phosphoric acid (1.7 M), is added at the T-piece. The reagents are pumped separately by two-head pumps with 75 mL/min and 50 mL/min ow rates. Chemical oxidation in the mobile phase takes place in the oxidation reactor at 99.98C. Afterwards, the formed individual CO2 peaks together with other gases (water vapor, oxygen, argon, etc.) are removed from the cooled liquid phase by the membrane separation unit. The gas mixture immersed in the helium carrier stream (2 mL/min) is dried on-line using two Naon gas dryers (selective removal of water vapor) and then directly introduced into the isotope ratio mass spectrometer via an open split. Water vapor remaining in the gas phase behind both Naon gas dryers is further removed from the system with an additionally installed cold trap in order to avoid peak broadening this water trap is realized by simply immersing the long fused-silica capillary

normally connecting the last Naon trap with the open split unit into a dry ice/ethanol slush. The surface of the fused silica is large enough for the quantitative removal of the residual water. On the other hand, the partial pressure of water vapor at this location is low enough to avoid a larger accumulation of ice. Hence, clogging is not a problem. To quantitatively remove the excess O2, we installed the reduction reactor immediately before the open split, i.e. behind the dry-ice trapa (Fig. 2). In the rst series of experiments, two Al2O3 ceramic tubes (1.5 mm o.d., 0.8 mm i.d., Friatec, Mannheim, Germany) lled with 110 mg elemental copper (d 0.125 mm, purity 99.9%, Goodfellow GmbH, Bad Nauheim, Germany), enclosed in a heating oven (temperature controller Eurotherm 2132, Carbolite, UbstadtWeiher, Germany), were connected by an eight-port two-position valve (VICI-Valco, Schenkon, Switzerland). For the second series of experiments two ceramic tubes (2.5 mm o.d., 1.3 mm i.d., Friatec) lled with 1.05 g copper wire (0.5 mm o.d., 99.9%, Goodfellow GmbH) were used. In one of the two positions of the eight-port valve the helium stream from the gas dryer, containing CO2 and O2, ows through one of the reactor tubes (Fig. 2). Here, a temperature of 5808C is sufcient for copper oxidation and quantitative removal of free oxygen via reaction (1) (oxidation of copper): 2 Cu O2 ! 2 CuO (1)

While on one side copper is oxidized and oxygen is scavenged from the analyte stream the other reactor is ushed with a helium/hydrogen mixture (97:3) at 3 mL/min (Fig. 2). The previously oxidized copper wire is reduced again via reaction (2) (regeneration of copper): CuO H2 ! Cu H2 O (2)

The reducing hydrogen ow has been adjusted to match the expected exhaustion of the active reactor. If required, the hydrogen concentration in the helium/hydrogen mixture can be raised to more than 5%. The maximum amount of O2 which can be bound by the reactor depends on the copper surface and mass. Switching between the two valve positions is possible either manually or automatically using the two-position valve actuator (VICI-Valco, high speed EH actuator, Schenkon, Switzerland) (Fig. 2). The latter has been connected to the LC autosampler so that a trigger signal actuates the valve within a time control sequence.

RESULTS AND DISCUSSION

To test the system properties and the associated changes with and without the reduction reactor, a number of experiments was performed. The inuence of the variable O2 background in isotope measurements was quantied and the efciency and capacity of the O2-removal system were determined for small and large reactors. The stability and accuracy of d13C results for CO2 reference gas pulses and for sucrose were used as a test for the system performance and lament lifetime was measured in analytical time.
a

Figure 6. Reduction capacity measured as duration in which O2 background stays below 50 mV. Plotted is the time of a repeated full oxidation cycle for a 0.8 mm i.d.reduction reactor lled with 105 mg of copper (lower X-axis) or the time of a repeated full oxidation cycle for a reduction reactor 1.3 mm i.d. lled with 1051 mg of copper (upper X-axis).
Copyright # 2007 John Wiley & Sons, Ltd.

Please note that the water trap was repositioned later, i.e. after the experiments described in this contribution.
Rapid Commun. Mass Spectrom. 2007; 21: 41354141 DOI: 10.1002/rcm

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12 to 17 V of O2 background. The mean values and standard deviations are improved signicantly with the installed reduction reactor. Upon exhaustion, the oxygen background starts rising at a relatively low rate, taking about 300 to 500 s until reaching the initial background again. This is understood in terms of the high reduction capacity at the beginning and a slow deterioration of the exhausting copper with elemental copper inside the wires being oxidized last. The active, usable lifetime (from O2 background stabilization to the rst signs of exhaustion) of about 2000 s or 30 min is sufcient for at least one complete chromatogram in the case of separation of plant sugars and organic acids. Figure 7. Simultaneously monitored changes in oxygen background and d13C values of injected reference gas pulses. Comparison of new and used reduction system (always with the same lament).

Capacity of oxygen removal

The reductive capacity of a copper reactor can be estimated from the amount of copper placed into the reactor and the effective length of the hot zone of the furnace. Eight Cu wires of 0.125 mm o.d. have been twisted together on a small lathe to t into one reactor. The heated length of the copper wire is approximately 12 cm, which is equivalent to 1.64 mM Cu. Hence, the maximal O2 consumption amounts to about 0.82 mM O2. For a typical sodium peroxodisulfate concentration (0.55 M) at the usual ow rate of 75 mL/min this number translates into a reactor lifetime of about 41 min. For the initial runs similar results regarding the rising O2 background and related isotopic signature of CO2 were observed (Fig. 6, lower x-axis, tubes 1 and 2, 0.8 mm i.d.). Further oxidation/regeneration runs led to a subsequent decline in reducing capacity, possibly owing to an alteration of the Cu surface properties, or losses of the Cu from the reactor surface as well as formation of chemical compounds, probably halides, blocking regeneration with hydrogen. The overall lifetime of a reduction reactor depends on the quality of the Cu, unaccounted trace gases in the helium stream and the time elapsing for a single average run. We have used a reactor for about 200 oxidation/reduction cycles before a new lling was required, which is still a considerable improvement over the necessity to change broken laments frequently with its requirement to break the vacuum, retune the ion source, and check all other instrument performance parameters before the next analysis can be made.

Effect of variable O2 background on the d13C values of reference gas

As a rst test the temperature of the oxidation reactor in the commercial system was varied to produce different O2 concentrations in the mobile phase. Every 25 min a new temperature point was set manually: 608C 808C 908C 958C 99.98C. In a rst sequence the O2 background on m/z 32 was monitored, and in an analogous second sequence CO2 reference gas pulses of 20 s length were repeatedly injected two times per minute and their d13C values measured (Fig. 3(a)). The observed d13C results exhibited a signicant correlation with the measured O2 background (R2 0.999; p < 0.0001, Fig. 3(b)). This linear relationship can affect the analytical results when the O2 background in the ion source varies during elution of CO2 generated from combusting the organic compounds at the end of the LC separation. O2 is consumed to form CO2 during combustion. Every CO2 peak, hence, is accompanied by a decreasing background of O2, as depicted in Fig. 4. The size of this decline depends on the amount of the target compound. It reached about 35% in the example given where the m/z 32 ion current was monitored during three sucrose injections and combustions. Summarizing both experiments it can be concluded that d13C measurements in the LC/IRMS system either need a constant O2 level (which obviously is not feasible due to O2 consumption during combustion) or they must be made at a very much reduced O2 background level, thus eliminating any interference resulting from variations of O2.

Increasing the reduction capacity and the effect of peak broadening with larger reduction reactors
As described above, the maximum reducing capacity of copper as given by the concentration of O2 in the gaseous phase and the copper mass in the active segment of the reduction reactor amounts to about 50 min in our system. This time can be enhanced by increasing the length of the reactor and/or mass of copper. Increasing the copper mass requires changes in the geometry of the tubes and the copper wires. However, a larger tube diameter can lead to substantial peak broadening. On the other hand, the time for a single oxidation theoretically can be increased up to several hours. Assuming that the oxidation reaction takes place for the full mass of copper (not just the surface) we installed a new set of reduction reactor tubes with 1.3 mm i.d., packed with ve twisted copper wires (d 0.5 mm). The calculated theoretical time for a full oxidation of the copper is about 7 h for 0.02 mmol O2/min. The observed time at the beginning was between 42 and 59 min and increased to 90 min after nine
Rapid Commun. Mass Spectrom. 2007; 21: 41354141 DOI: 10.1002/rcm

Variation in O2 background
Once a temperature of 5808C in the reduction reactor is reached, the O2 background falls from the initial several volts to 50 mV within 2 to 9 s, with a small dependence on the initial background (Table 1, Fig. 5). A further decline of the O2 background from 50 mV to 20 mV can take up to 400 s, with the rate being largely independent of the initial background. Finally, the O2 background stabilizes between 7 and 20 mV with a variation of between 2 and 6 mV and remains at this level until exhaustion of the copper capacity (Fig. 5). It is noteworthy that the O2 background uctuation without a reduction reactor installed in average amounts to 85 mV (n 139) at
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4140 E. Hettmann, W. A. Brand and G. Gleixner

Table 2. Peak broadening with tubes of 1.3 mm i.d. compared with tubes of 0.8 mm i.d. Peak width has been standardized as the relation of the peak base width to the peak amplitude. The peak base width is a very important parameter in on-line isotope ratio measurements for accurate peak integration and background subtraction. Compared with an unmodied system, peak broadening in the modied system (0.8 mm i.d. reactors) was negligible
Standardized peak width (s/V) Concentration (ng C/10 mL) 2940 1680 420 210 Tube 0.8 mm i.d. 4.3 5.8 19.5 35.0 Tube 1.3 mm i.d. 8.5 10.8 37.0 70.6 Peak width (%) 197.6 185.5 190.0 201.6

Stability and accuracy of sucrose d13C values

To test the reproducibility of d13C values of organic compounds eluting from the LC column, we started measurement of sucrose using conditions similar to those employed for the previous O2 background and reference gas pulse experiments. 1680 ng C aliquots of sucrose were injected via the 10 mL loop (concentration 168 mg C/mL) in ow injection mode (n 14). The length of the run was scheduled with respect to the copper capacity so that the last injections coincided with the new increase of the O2 background. The pre-run reference gas peaks were placed correspondingly to the previous experiments at 590 s shortly after stabilization, and the last reference peak was injected at the end of the O2 rise. The measurements were repeated four times with a time gap of between 1 week and 1 month. The SD over these four measurements was 0.03% of the mean, although within the individual runs the SD was 0.090.11% (Table 3). The sucrose d13C variability in single runs is slightly better than that described by Krummen et al.13 It is, however, comparable with our own results, measured previously without a reduction reactor. The reproducibility was also determined for a range of sucrose amounts between 210 and 1680 ng (Table 4). The peak area (given in Vs) is linearly correlated with the amount of sucrose (R2 0.9995). High precision quantication has always been a challenge in LC separations, and this method may be very well suited for closing this gap in analytical capabilities and techniques. It is interesting to note that for long-term measurements (sequences, measured over more than 1 month) the precision of mean d13C values for sucrose within a concentration range of 294 ng to 1680 ng carbon amounts to 0.11% without and 0.08% with the reduction reactor. These results demonstrate that similar or even slightly better quality of measurements can be obtained with the reduction reactor installed.

oxidation/reduction runs (Fig. 6, upper x-axis, tubes 1 and 2, 1.3 mm i.d.). Between runs 9 to 12 no further improvement in oxidation time was observable. The large difference between the theoretically predicted and the measured capacity can be explained by a non-quantitative oxidation of the copper with preferential oxidation of the surface which can be activated slowly by re-crystallization effects. However, the stabilization of the reactor lifetime at 90 min offers an improvement of about 50 additional minutes compared with the lifetime of the small reactors. Further improvement of the reducing capacity may be reached by also using thinner copper wires (e.g. 0.125 mm) in the larger tubes, thereby increasing the copper surface several times. The effects of the larger tube diameter upon peak broadening were estimated by relating the standardized peak width of compounds measured with the larger reactor to the standardized peak width measured with the smaller tubes (Table 2).b For all measured concentrations the peak broadening was roughly a factor of two, with the peak width ranging from 185 to 202%. Despite the signicant peak broadening the benet of tubes with larger diameter is clear for long chromatograms with isolation of few peaks or peaks with initially good separation. The addition of the make-up gas can be also used to reduce peak broadening.

Filament lifetime
The normally observed O2 background adequate for the oxidation of organic compounds amounts to 1935 V Table 3. d13C values of 1680 ng C sucrose measured by LC/ IRMS with installed reduction oven (0.8 mm i.d.) (n 14 for each run)
Run 1 2 3 4 Mean (runs) Average d13C sucrose [% 26.65 26.63 26.61 26.69 26.65
V-PDB]

Alteration of reference gas d13C values

The d13C value of the reference gas takes much longer to stabilize than the O2 background. Only after 500 s following a switchover to a new reactor does the d13C value nally settle to a stable value (standard deviation (SD) 0.05%, Fig. 7). While the reasons for this behavior have not been investigated separately we speculate that the chemical conditions in and around the hot lament (>22008C) are still changing rapidly and that some of the CO2 reacts with the hot lament to form tungsten carbide. Towards the end of the useful lifetime of the reactor the d13C of the reference gas became more positive at one time and slightly more negative or less stable the second time. This time window does seem to produce erratic d13C results and must be avoided.
b Full-width at the base of the peak instead of the more commonly used full-width at half maximum (FWHM) was used because of the relevance of peak base for peak integration and background substraction. At the FWHM the calculated peak broadening was similar.

SD [%] 0.11 0.10 0.10 0.09 0.03

Table 4. d13C values and accuracy of measurements of sucrose with installed reduction oven (n 24 for each concentration)
Sucrose (ng) 210 420 1680 Mean Peak area (Vs) 3.4 0.4 6.44 0.08 25.7 0.4 d13C values [% 26.67 26.61 26.53 26.60
V-PDB]

SD [%] 0.04 0.02 0.15 0.07

Copyright # 2007 John Wiley & Sons, Ltd.

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Table 5. Lifetime of laments. The oxygen background is shown as the mean of all sequences for the respective lament. For the sequences with the installed reduction reactor oxygen was measured before the reduction oven was turned on. Data are given in Volt units (as measured by the mass spectrometer software). 1 V 3.3 nA
Filament number 1 2 3 4 5 6 7 8 9 10 11 12 Measurements time (h) 220 240 520 240 220 200 180 200 120 420 500 1560 Concentration oxidation reagent (g/l) 200 200 200 200 200 200 200 200 200 130 130 130 Approximate oxygen background (V) 17.9 24.6 24.6 24.5 22.0 19.7 25.0 25.2 25.0 12.0 11.2 17.0 Reduction reactor installed No No No No No No No No No No No Yes

(corresponding to m/z 32 ion currents of 62.7115.5 nA in the mass spectrometer) when no O2 scrubber is in place. Lower amounts of O2 during the chemical oxidation result in non-quantitative combustion and consequently unstable, erroneous measured isotopic values. With a large excess of O2 the negative effects in the source are magnied (as described above), in particular regarding the lament lifetime. At a mean measured O2 background of about 23 V a lament on average lasted for 12 analytical sequences, each one taking about 20 h (Table 5). The lower concentration of the oxidation reagent (0.55 M sodium peroxodisulfate instead of 0.8 M) and differing liquid phase and carrier gas uxes are responsible for the improved (declining) total O2 concentration in the system and the enhance lifetime for laments number 10 and 11 in the table. In these cases, however, the low concentration of O2 leads to a reduced oxidation capacity, not sufcient for the fractionationfree oxidation of organic compounds and restricting the measurement range to values between 120 and 1200 ng carbon (sucrose). The installation of the reduction reactor has removed the problem of surplus O2 in the source, increased the lament lifetime and even enabled us to increase the amount of available O2 in the liquid phase during the oxidation.

through the same analytical steps (IT-principle9). However, accuracy has been a problem and the removal of O2 from the helium carrier case has also removed its inuence upon measured d13C values. Moreover, the increased lament lifetime leads to a considerably enhanced stability in the whole system and thus can reduce both the cost per analysis and a good deal of grief for the operator.

Acknowledgements
We thank Frank Voigt and Bernd Schlo ffel for manufacturing some of the reduction system components and especially Steffen Ru hlow for assisting in system installation and for many helpful hints. This project was funded by the Max Planck Society.

REFERENCES
1. Schmidt HL, Kexel H. Isot. Environ. Health Stud. 1998; 34: 19. 2. Tcherkez G, Nogues S, Bleton J, Cornic G, Badeck F, Ghashghaie J. Plant Physiol. 2003; 131: 237. 3. Nogues S, Tcherkez G, Cornic G, Ghashghaie J. Plant Physiol. 2004; 136: 3245. 4. Gleixner G, Scrimgeour C, Schmidt HL, Viola R. Planta 1998; 207: 241. 5. Scartazza A, Lauteri M, Guido MC, Brugnoli E. Aust. J. Plant Physiol. 1998; 25: 489. 6. Scartazza A, Mata C, Matteucci G, Yakir D, Moscatello S, Brugnoli E. Oecologia 2004; 140: 340. 7. Ghashghaie J, Duranceau M, Badeck FW, Cornic G, Adeline MT, Deleens E. Plant Cell Environ. 2001; 24: 505. 8. Brand WA. J. Mass Spectrom. 1996; 31: 225. 9. Werner RA, Brand WA. Rapid Commun. Mass Spectrom. 2001; 15: 501. 10. Jamin E, Naulet N, Martin GJ. Plant Cell Environ. 1997; 20: 589. 11. Caimi RJ, Brenna JT. Anal. Chem. 1993; 65: 3497. 12. Brand WA, Dobberstein P. Isot. Environ. Health Stud. 1996; 32: 275. 13. Krummen M, Hilkert AW, Juchelka D, Duhr A, Schluter HJ, Pesch R. Rapid Commun. Mass Spectrom. 2004; 18: 2260. 14. Hilkert AW, Krummen M. Application Note, Thermo Electron Corporation, 2004; AN30024_E. 15. Hilkert AW, Krummen M. Application Note, Thermo Electron Corporation, 2004; AN30025_E. 16. Hettmann E, Gleixner G, Juchelka D. Application Note, Thermo Electron Corporation, 2005; AN30075. 17. Heuer V, Elvert M, Tille S, Krummen M, Mollar XP, Hmelo LR, Hinrichs KU. Limnol. Oceanography Methods 2006; 4: 346. 18. McCullagh JSO, Juchelka D, Hedges REM. Rapid Commun. Mass Spectrom. 2006; 20: 2761. 19. Merritt DA, Brand WA, Hayes JM. Org. Geochem. 1994; 21: 573. 20. Brand WA, Tegtmeyer AR, Hilkert A. Org. Geochem. 1994; 21: 585.
Rapid Commun. Mass Spectrom. 2007; 21: 41354141 DOI: 10.1002/rcm

CONCLUSIONS
The presence of large amounts of oxygen in the ion source of the mass spectrometer caused a number of analytical difculties for LC/IRMS including uncontrollable variations in the CO2 and O2 background ion currents, potential decline of measurement accuracy and an intolerably short lament lifetime. The installation of a copper-lled reduction reactor presented for the rst time in this contribution enables complete removal of excess O2 from the LC/IRMS system following oxidation. A considerable advantage of the new device is that the oxidation capacity of the liquid can be adjusted according to the analytical requirements and the dynamic range of the sample amount is no longer limited by the oxygen tolerance of the mass spectrometer ion source. The new reactor does not immediately offer a signicant improvement in the precision of the measured d13C values of the organic compounds, provided that the results have been properly standardized through reference materials passing
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