How to Get Compound Specific d15N Measurements

Compound-specific stable nitrogen isotope measurements of amino acids and other compounds can act as a rich source of data for research in the fields of ecology, biology, archeology and the environment.

In the past, it has been significantly more challenging to analyze compound-specific δ15N than compound-specific δ13C. This is the result of a system made up of a two-step conversion of organic nitrogen in the antecedent molecule to gaseous N2 via oxidation and the ensuing reduction.

All of the organic molecules are transformed into a mixture of CO2, N2, N2O, NO and H2O within the oxidation furnace tube. This necessitates that the combustion products travel through an additional reduction furnace tube, in which all nitrogen compounds are reduced to N2, before being directed into the IRMS for δ15N analysis.

Historically, this has been tricky, as the oxidation furnace tube, which is heavily oxygenated, is parallel to the reduction furnace tube. This means that when the furnaces are heated for operation, a number of oxygen leaks from the oxidation furnace tube, which partly oxidizes a quantity of the copper in the nearby reduction furnace tube, thereby restricting the ability of the reduction furnace to provide appropriate reduction conditions.

A new streamlined solution to this issue has been designed by Isoprime application specialists, in which the parts making up the sample are combusted and reduced in a single-stage furnace. A summary of the system and the advantages it can offer for the easiest GC-δ15N analysis are detailed below.

Nitrogen Conversion Optimization

Within an individual stage of combustion, it should be anticipated that the reaction will generate a number of oxidation states of N. The level of available oxygen in the system is evidently a vital factor affecting the yield of the reaction. For effective GC-δ15N analyses, an equilibrium of both high-quality combustion to NOx and subsequent reduction to N2 must be ensured.

With this in mind, an oxygen balance is required, as an excess of oxygen would lead to the system losing efficiency in reducing NOx to N2, while low quantities of oxygen would lead to reduced efficiency of combustion to NOx (Figure 1).

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.

Elementar’s single furnace tube solution for δ15N analysis is made up of a micro-bore quartz tube with eight interwoven metal wires, three copper wires, three nickel-chrome (80/20) wires, and two platinum wires, which perform as catalysts. The temperature of the furnace is held at 950 °C.

It is necessary to re-oxidize the furnace environment at regular intervals, as over time the copper and nickel-chrome wires will drop their surface oxides and efficiency of combustion will, therefore, be reduced. Re-oxidation is carried out through a computer-controlled boost of O2 into the helium carrier gas stream.

Proving Reduction Efficiency

In order to illustrate the reduction efficiency of the single furnace tube solution, 25 μl of pure NO gas (N oxidation state +2) was added to and analyzed within the GC-δ15N system, both with the furnace at room temperature and at 950 °C. The target masses examined were m/z 28 (N2), 30 (NO, oxidation state +2) and 44 (N2O, oxidation state +1). M/z 29, 45 and 46 were monitored alongside this.

The average percentage peak areas for the signals at m/z 28, 30 and 44 when the furnace was at room temperature (blue) and at 950 °C (red) can be seen in Figure 2. At room temperature, where there should be no decrease in the NO gas, mainly m/z 30 (i.e. NO) was observed, but also an ample quantity of m/z 28.

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.

It is most likely that the latter stems from atmospheric N2 brought in via the syringe. A lesser quantity of m/z 44 is also detected, which may have stemmed from either or both N2O or CO2.

Following the warming of the furnace to 950 °C, the m/z 30 signal fell to 0.04%, while the m/z 28 increased to 96.4%, indicating the effectiveness of decreasing NO to N2. The signal at m/z 44 grew marginally to 3.6%, but early worries that this could be a result of the partial reduction of NO (N oxidation state +2) to N2 O (N oxidation state +1) were discharged by the 45/44 ratio showing that it was derived from CO2 (Table 1).

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

Theoretical relative isotope abundance of pure CO2 and N2O.

Content with the acceptable reduction of NO to N2, a laboratory nicotine standard was examined during the same period as the NO gas injections to order to examine and investigate the accuracy of the δ15N analyses and possible m/z 30 formation. As noted earlier, m/z 30 will rise if NOx does not entirely reduce to N2.

After 6 analyses, the nicotine standard demonstrated a standard deviation of 0.07‰ (Figure 3). The very low 30/28 ratio demonstrates that there was a negligible mass 30 formation.

d15N analysis of a nicotine standard using the single tube GC-d15N 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 d15N 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

The δ15N analysis of amino acids increases the awareness of multiple applications of the GC-δ15N system. A standard amino acid mixture made up of isoleucine (ILE), serine (SER) and lysine (LYS) was analyzed to exhibit the single furnace solution.

The amine (-NH2) and the acid group (-COOH) must be derivatized before analysis to make amino acid molecules GC responsive, and for particular amino acids, the R-group also needs derivatization (Corr et al. 2007).

Five successive analyses of the standard were executed with the single furnace tube combustion system. The precision attained through the replicate measurements offered an improvement on 0.2‰ (Table 2).

Table 2. δ15N analyses of an amino acid standard.

d15N analyses of an amino acid standard.

A strong correlation was shown between the measured δ15N values and the known values, as shown by a slope of 1.007 and the R2 value of 0.996 when marking the calibration curve (Figure 4).

Calibration curve obtained from the analysis of an amino acid standard on the GC-d15N system.

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

Such a high-quality calibration curve indicates that the single furnace tube system transforms organic N-containing molecules into N2 without any isotopic fractionation, offering vigorously precise results. A further benefit of the single furnace tube solution is that it has a considerably shorter path length than a two-furnace solution.

This decreases the risk of dead volumes and peak broadening, offering better quality peak shapes at the mass spectrometer. Figure 5 shows the first-rate chromatography of a standard amino acid mixture.

Mass 28 and 29 signals of GC-d15N 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.

Summary and Conclusions

The single furnace tube solution demonstrated in this piece offers a compound-specific δ15N isotope analysis that is greatly simplified and sturdier than alternatives. Comprehensive studies using pure NO gas demonstrate the outstanding reduction effectiveness of the single tube furnace. Studies of nicotine and varied amino acids display the great precision and accuracy of the GC-δ15N system.

References and Further Reading

  1. 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

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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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