Compound-Specific Stable Nitrogen Isotope Measurements Using a Stable Isotope Ratio Mass Spectrometer

Compound-specific stable nitrogen isotope measurements of amino acids, for example, can supply a wealth of valuable information for use in biological, ecological, archaeological and environmental research

The analysis of compound specific δ15N has traditionally been a lot more demanding than that of compound specific δ13C analysis. This is because of the system made up of a two-stage conversion of organic nitrogen in the precursor molecule to gaseous N2 via oxidation and subsequent reduction.

Each organic molecule inside the oxidation furnace tube is changed into a mixture of CO2, N2, N2O, NO and H2O, therefore requiring the combustion products to pass through a secondary reduction furnace tube where all nitrogen compounds are reduced to N2 before passing into the IRMS for δ15N analysis.

The difficulty with the traditional setup is that the reduction furnace tube is in line with the oxidation furnace tube (which is rich in oxygen). The oxidation furnace tube will ‘bleed’ some oxygen when the furnaces reach operating temperature, in turn that oxygen will partially oxidize some of the copper in the reduction furnace tube. This then disables the reduction furnace from providing satisfactory reduction conditions.

Recently, isoprime application specialists developed a simplified solution in which the sample components are combusted and reduced using a single stage furnace. This article gives an overview of this system and its advantages for easier GC-δ15N analysis.

Nitrogen Conversion Optimization

It is expected that the reaction will generate several oxidation states of N within a single combustion phase. The amount of available oxygen in the system is clearly a crucial factor affecting the output of the reaction.

A balance of both good combustion to NOx and subsequent reduction to N2 must be ensured for successful GC-δ15N analyses. Therefore, an oxygen balance is sought, too little oxygen would result in less efficient combustion to NOx and too much oxygen would lead to the system being less efficient at reducing NOx to N2 (see Figure 1).

The single furnace tube solution for δ15N analysis from Elementar is made up of a micro-bore quartz tube with eight metal wires braided together; three nickel-chrome (80/20) wires, three copper wires, and two platinum wires, which behave as a catalyst. The furnace is kept at 950 °C.

Periodic re-oxidation of the furnace environment is needed because over time the nickel-chrome and copper wires will lose their surface oxides and its combustion ability will decrease. Reoxidation is performed using a computer-controlled injection of O2 into the helium carrier gas stream.

Scheme of the oxygen balance in a combined combustion and reduction furnace.

Figure 1. Scheme of the oxygen balance in a combined combustion and reduction furnace.

Proving Reduction Efficiency

25 μl pure NO gas (N oxidation state +2) has been injected and analyzed in the GC-δ15N system with the furnace at room temperature and also at 950 °C in order to show the reduction efficiency of the single furnace tube solution. The target masses analyzed were m/z 28 (N2), 30 (NO, oxidation state +2) and 44 (N2O, oxidation state +1). In addition, m/z 29, 45 and 46 were observed.

The average percentage peak areas for the signals at m/z 28, 30 and 44 when the furnace was at 950°C (red) and at room temperature (blue) can be observed in Figure 2. Where there should be no reduction of the NO gas (at room temperature) predominantly m/z 30 (i.e. NO) was seen, but also a large amount of m/z 28.

The m/z 28 is likely to be from atmospheric N2 taken up in the syringe. A small amount of m/z 44 is also present, which could have derived from either or both N2O or CO2. The m/z 30 signal fell to 0.04 % after heating the furnace to 950 °C but the m/z 28 rose to 96.4%, showing the efficiency of reducing NO to N2.

The signal at m/z 44 rose slightly to 3.6%, yet initial concerns that this could be because of the partial reduction of NO (N oxidation state +2) to N2O (N oxidation state +1) were quashed by the 45/44 ratio establishing that it was CO2 derived (Table 1).

Table 1. Theoretical relative isotope abundance of pure CO2 and N2O.

RELATIVE ISOTOPE ABUNDANCE RATIO
45/44
MASS 44 MASS 45
CO2 98.416 1.176 0.0119
N2O 99.032 0.763 0.0077

Satisfied by the adequate reduction of NO to N2, a laboratory nicotine standard was analyzed on the same day as the NO gas injections to examine the precision of the δ15N analyses and potential m/z 30 formation. As previously considered, m/z 30 will grow if NOx does not completely reduce to N2.

Figure 3 shows that the nicotine standard was analyzed six times with a standard deviation of 0.07 ‰. The very low 30/28 ratio suggests that there was negligible mass 30 formation.

Comparison of the reduction of pure NO gas at room temperature and at 950 °C.

Figure 2. Comparison of the reduction of pure NO gas at room temperature and at 950 °C.

δ15N analysis of a nicotine standard using the single tube GC-δ15N system. The blue markers depict the individual analyses, the solid line the mean value of all analyses and the dotted line the standard 2-sigma expected precision of δ15N isotope ratio analyses.

Figure 3. δ15N analysis of a nicotine standard using the single tube GC-δ15N system. The blue markers depict the individual analyses, the solid line the mean value of all analyses and the dotted line the standard 2-sigma expected precision of δ15N isotope ratio analyses.

Application to Amino Acids

δ15N analysis of amino acids provides helpful knowledge of a number of applications of the GC-δ15N system. A standard amino acid mixture comprising serine (SER), isoleucine (ILE), and lysine (LYS) was examined as a demonstration of the single furnace solution.

To make amino acid molecules GC amenable, before analysis and for particular amino acids the amine (-NH2) and the acid group (-COOH) must be derivatized, the R-group also needs derivatization (see Corr et al. 2007). Five later analyses of the standard were carried out with the single furnace tube combustion system. The precision obtained from the replicate measurements was better than 0.2 ‰ (see Table 2).

Table 2. δ15N analyses of an amino acid standard.

ILE SER LYS
SAMPLE HEIGHT (NA) δ15N (‰) HEIGHT (nA) δ15N (‰) HEIGHT (nA) δ15N (‰)
AA1 4.61 -5.12 1.76 -0.94 4.15 0.40
AA2 4.34 -5.24 1.68 -1.17 3.83 0.56
AA3 4.99 -5.18 1.89 -0.85 4.43 0.32
AA4 5.05 -5.07 1.92 -0.70 4.44 0.60
AA5 5.79 -4.85 2.19 -0.66 4.98 0.70
AVERAGE -5.09 -0.86 0.52
SD 0.15 0.20 0.15

An excellent correlation was seen between the known values and the measured δ15N values, as exhibited by a slope of 1.007 and R2 value of 0.996 when plotting the calibration curve (see Figure 4). A calibration curve of this high standard shows that the single furnace tube system converts organic N-containing molecules to N2 with no isotopic fractionation, providing robustly accurate results.

Another benefit of the single furnace tube solution is that it possesses a particularly shorter path length than a dual furnace solution. This decreases the possibility of peak broadening and dead volumes, providing superior peak shapes at the mass spectrometer. The excellent chromatography of a standard amino acid mixture is displayed in Figure 5.

Calibration curve obtained from the analysis of an amino acid standard on the GC-δ15N system.

Figure 4. Calibration curve obtained from the analysis of an amino acid standard on the GC-δ15N system.

Mass 28 and 29 signals of GC-δ15N analyses of an amino acid standard. For quality control reasons, three pure nitrogen monitoring gas peaks are analyzed before the sample peaks.

Figure 5. Mass 28 and 29 signals of GC-δ15N analyses of an amino acid standard. For quality control reasons, three pure nitrogen monitoring gas peaks are analyzed before the sample peaks.

Conclusion and Summary

The single furnace tube solution shown here gives a considerably easier and more robust compound specific δ15N isotope analysis. Detailed investigations employing pure NO gas show the excellent reduction efficiency of the single tube furnace. Analyses of different amino acids and nicotine reveal the high accuracy and precision of the GC-δ15N system.

References

Corr, L.T., R. Berstan and R.P. Evershed, Optimisation of derivatisation procedures for the determination of δ13C values of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry, Rapid Communications in Mass Spectrometry, 3759–3771, 2007.

This information has been sourced, reviewed and adapted from materials provided by Elementar Analysensysteme GmbH.

For more information on this source, please visit Elementar Analysensysteme GmbH.

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