Elemental analyzer

In order to determine the δ¹³C values, the samples (0.060 mg) were loaded into zin capsules and combusted at a temperature of 1100 °C, and then, CO₂ was separated in gas chromatography column in the elemental analyzer (EuroVector). In order to determine the δ¹⁸O values, the samples (0.095 mg) were loaded into silver capsules. To displace moisture-containing air in the glucose samples, the samples were heated over 24 h in a vacuum line (60 °C), and after that, they were put into a special air-filled homemade dry box before stable isotope ratio determination (Sensuła et al. 2011b (link)). The samples were converted to CO by pyrolysis at a temperature of 1350 °C and separated in gas chromatography column in the elemental analyzer (EuroVector).
The stable oxygen and carbon isotope compositions of the samples were determined using an Isoprime continuous flow isotope ratio mass spectrometer (GV Instruments, Manchester, UK) at the Mass Spectrometry Laboratory of the Silesian University of Technology.
The relative deviation of the isotopic composition is expressed, in parts per thousand (‰), as $$\delta =(R_{\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}}/R_{\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{a}\mathrm{r}\mathrm{d}}-1)\times 1000,$$
where R_sample and R_standard are the ratios of the heavy to the light isotope concentration in the sample and in the standard, respectively. The δ¹³C results are reported in values relative to VPDB, whereas the δ¹⁸O results are reported in values relative to VSMOW.

δ¹³C values, a measure of the carbon isotope composition, were determined at Washington State University on leaf samples taken from plants using a standard procedure relative to PDB (Pee Dee Belemnite) limestone as the carbon isotope standard (Bender et al., 1973 (link)). Plant samples were dried at 80 °C for 24 h, milled to a fine powder and then 1–2 mg were placed into a tin capsule and combusted in a Eurovector elemental analyzer. The resulting N₂ and CO₂ gases were separated by gas chromatography and admitted into the inlet of a Micromass Isoprime isotope ratio mass spectrometer (IRMS) for determination of ¹³C/¹²C ratios. δ¹³C values were calculated where δ¹³C=1000×(R_sample/R_standard)–1, where R=¹³C/¹²C.

I assessed individual long‐term diet variation using stable nitrogen isotope values that have previously been used to examine the correlation between relative trophic position and reproductive success (Griffen 2014). Here, I use these same data to examine the relationship between crab size (carapace width, CW) and relative trophic position, which has not previously been explored. δ¹⁵N values generally increase with trophic position and can therefore accurately be used to assess relative trophic position between individuals (Post 2002). δ¹⁵N values were obtained from muscle tissue taken from a single walking leg of each crab and were measured using an Isoprime mass spectrometer connected via continuous flow to a EuroVector Elemental Analyzer. Three internal standards were run approximately every 40 samples to calibrate the system and to compensate for potential drift over time (USGS40, N1 and N2). I tested the hypothesis that relative trophic position would increase with crab size using a linear model with δ¹⁵N as the response variable and crab CW as predictor variable.