Many previous studies reported severe problems with LC-O-IRMS, e.g. the impos-sibility of measuring muramic acid in non-spiked samples due to very low peak areas or the requirement for time-consuming purging steps to maintain performance of the PA 20 column (Bode et al., 2009). The absence of these issues in the currently proposed method can mainly be attributed to the advantages of IC over HPLC. Ion chromatographs are free of metals: all elements that are in contact with sample or eluents are made from polyether ether keton (peek). Thus, metal contamination can originate only from the sam-ple. However, our method contains iron and salt precipitation steps, removing all (poten-tially column destroying) cations. This not only reduces measurement time but also re-duces costs as, e.g., in-line high pressure filters protecting the column from colloids and metal ions are not needed. Even after 600 injections, no decrease in performance of the PA 20 column was detected and the pre-column did not need to be exchanged.
In addition, the CO2-tight construction of Ion Chromatographs is a great advantage of δ13C determination as no shifts in the δ13C value due to increasing carbonate back-ground occurred. Therefore, even CO2-binding eluents, like NaOH, do not cause prob-lems for chromatography and isotope ratio mass spectrometry. In addition, Ion Chro-matographs are routinely equipped with a degasser, which keeps the eluents and oxidiz-ing reagents of the Isolink CO2-free. Thus, although acquisition costs may be higher, the improved performance, higher sample throughput and lower follow-up costs reflect the clear advantages of ion chromatographs for improving LC-O-IRMS.
2.2.4 Conclusions
Amino sugars are important biomarkers for research on bacterial and fungal contri-bution to SOM. This new method enables parallel quantification and δ13C determination of the most frequent amino sugars in soils and thus sets the preconditions for wider adop-tion of δ13C amino sugar determination in soil science.
The combination of iron and salt removal from gas chromatography protocols with purification via cation exchange resins adapted from liquid chromatography methods proved to be an optimal sample preparation for ion chromatography including chroma-tographic separation, system stability and longevity of system components. In addition, using ion chromatograph sets clear advantages over HPLCs as metal and carbonate exclusion from the system avoids column contamination as well as disturbance of δ13C determination by a carbonate background.
These improvements over previous methods enabled parallel quantification and δ13C determination of high-concentrated basic amino sugars and low-concentrated mu-ramic acid. Recoveries ranged from 57 to 66% and could be corrected by using methyl-glucamine as the first internal standard. The quantification limit of muramic acid, the compound with the lowest concentration, was around 0.05 mg per vial for quantification and for isotope measurement. When muramic acid exceeded these values, glucosamine, the most concentrated compound, was still in a linear range for quantification and δ13C measurement. The accuracy of IC-O-IRMS was better than 1‰ for basic amino sugars and better than 1.5‰ for muramic acid compared to calibrated EA-IRMS values. Preci-sion was amount-dependent and less than 0.5‰ over a comparatively broad range of areas. However, the dependence on the matrix and the ratio of muramic acid to gluco-samine in individual samples necessitates adjustment in soil amount or injection volume to achieve the optimal accuracy and precision of δ13C.
The quality of the quantification and δ13C determination as well as sample through-put of this method should enable this method to be used routinly in soil science. The ad-vantages of IC-O-IRMS compared to HPLC-O-IRMS are evident and might also bring advantages for analysis of other biomarkers.
Acknowledgments
We thank the DFG for financing the IC-O-IRMS instrument and the project DFG KU 1184 19/1.
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Supplementary Data
Figure Supplementary A1: Scheme of the instrument coupling: Ion Chromatograph is shown on the left side with pump, autosampler and detector-chromatography compart-ment. Connection to isolink occurs via a peek capillary with interposed colloid filter.
Scheme of LC Isolink is adapted from Krummen et al. (2004).
Figure Supplementary A2: Standard addition line of the quantified amino sugars: quanti-fied amount per g soil is plotted against the amount of spiked amino sugar. Slope repre-sents recovery of the individual analytes and y-axis gap reprerepre-sents soil content without recovery correction. Regression parameters are shown in Table 2.
Supplementary Figure A3: measured δ13C values of spiked samples are plotted against the percent of peak area, which is derived from the added standard: y-intercept of the fitted linear regression reflects the fitted value of soil whereas δ13C-value at 100% stan-dard reflects the δ13C value of the added standard substance
Figure Supplementary A4: area-dependant error terms of equation 6: left side shows the standard error of the measurement repetition of soil samples and right side shows the area-dependant error of the calibration/correction function from the external standard line
Supplementary Table A1: Solvent gradient and flow conditions of the IC-O-IRMS system time 20 mM NaOH 200 mM
Na-OH H2O 0.01 M
NaNO3
flow (ml min-1)
-25 min 0% 100% 0% 0% 0.400
-10 min 8% 0% 92% 0% 0.325
11 min 40% 0% 35% 25% 0.400
15 min 45% 0% 45% 10% 0.400
18 min 25% 25% 50% 0% 0.380
35 min standby