Approximately 1 mg of dried root and leaf tissues was transferred into a tin capsule for measurement of δ15N in one run. The analysis was carried out using a Delta V isotope ratio mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with a Flash EA 1112 Elemental Analyser (Thermo Scientific, Bremen, Germany). The isotope ratios were expressed in δ ‰ versus air for δ15N according to the following formula: δ ‰ = [(Rsample–Rstandard)/Rstandard]⋅ 1000 where Rsample is the isotope ratio measured for the sample and Rstandard is the isotope ratio of the international standard. R is the abundance ratio of the minor, heavier isotope of the element to the major, lighter isotope, as 15 N/14 N. The isotope values were calculated against international reference materials: L-glutamic acid USGS 41, ammonium sulphate IAEA-N-2 (IAEA-International Atomic Energy Agency, Vienna, Austria) and urea 33802174IVA (IVA Analysentechnik e.k.). The uncertainty of the nitrogen isotopic determination was ± 0.3‰.
Flash ea 1112 elemental analyser
Manufactured by Thermo Fisher Scientific
Sourced in Italy
The Flash EA 1112 Elemental Analyser is a laboratory instrument designed to determine the elemental composition of solid, liquid, and gaseous samples. It utilizes combustion analysis to measure the content of carbon, hydrogen, nitrogen, and other elements with high accuracy and precision.
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Lab products found in correlation
Delta 5 isotope ratio mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Nicolet is50 ftir spectrometer, by Thermo Fisher Scientific (1 mentions)
Sx20 stopped flow spectrometer, by Applied Photophysics (1 mentions)
6130 mass spectrometer, by Agilent Technologies (1 mentions)
S500 spectrometer, by Agilent Technologies (1 mentions)
Lambda 35 uv vis spectrometer, by PerkinElmer (1 mentions)
Melting point b 540 apparatus, by Büchi (1 mentions)
6 protocols using flash ea 1112 elemental analyser
1
Nitrogen Isotope Analysis of Plant Tissues
2
Characterization of Novel Organic Compounds
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All reagents and solvents were obtained from commercial sources and used without further purification. All aqueous solutions were prepared using either deionized or distilled water. 1H-NMR (500 MHz) spectra were recorded on the Varian S500 spectrometer. Elemental analyses were performed using the Thermo Scientific FlashEA 1112 elemental analyser. Mass spectra were obtained using the Agilent 6130 mass spectrometer. Fourier-transform infrared (FT-IR) spectra were recorded on the Thermo Scientific Nicolet iS50 FT-IR spectrometer. Potentiometric measurements were carried out in the pKa mode using the Metrohm 808 Titrando titrator with the Tiamo 2.3 software and Pt1000 pH electrode. Kinetic studies were carried out in the monochromator mode using the Applied Photophysics SX20 stopped-flow spectrometer equipped with a thermoelectric temperature controller (±0.5°C).
3
Elemental Composition Analysis Protocol
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Prepared samples were sent to an external laboratory (MEDAC Ltd) for CHNS analysis (with oxygen determined by difference). The C, H, N analysis was carried out according to ASTM E777 and ASTM E778. The sulphur content was measured according to ASTM E775. The original analytical method (using the Thermo FlashEA ® 1112 Elemental Analyser) was based on the complete and instantaneous oxidation of the sample by "flash combustion" which converts all organic and inorganic substances into combustion products. The resulting combustion gases passed through a reduction furnace and were swept into the chromatographic column by the carrier gas (He); where they were separated and detected by a thermal conductivity detector (TCD), which gave an output signal proportional to the concentration of the individual components of the mixture.
4
Stable Isotope Analysis of Syngnathids
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For δ13C and δ15N analysis in syngnathids, the samples were rinsed with distilled water, transferred to tin capsules, dried in oven at 60 °C for 24 h and weighted (± 1 μg). Due to the low lipid content in fin samples conserved in ethanol (< 5% lipids, C/N < 3.56) [74 (link)], further full defatting was not necessary [72 (link), 93 (link)]. Samples were analysed at SAI (University of A Coruña) by continuous flow isotope ratio mass spectrometry using a FlashEA1112 elemental analyser (Thermo Finnigan, Italy) coupled to a Delta Plus mass spectrometer (FinniganMat, Germany) through a Conflo II interface. Isotopic values are expressed as permil (‰) in conventional delta relative to VPDB (Vienna Pee Dee Belemnite) and Atmospheric Air. The precision (standard deviation) for SIA of the laboratory standard (acetanilide) was ± 0.15‰ (1-sigma, n = 10).
5
Characterization of Organic Compounds
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Melting points were measured on a Buchi B-540 melting point apparatus. TLC plates silica 60F 254 (Merck, Darmstadt), detection with ultraviolet light (254 nm). Elemental analyses were performed on a Thermo Finnigan Flash EA 1112 Elemental analyser. IR spectra were recorded in KBr pellets in Nujol mulls on a PerkinElmer, Inc. Precisely Spectrum One FT-IR spectrometry. UV spectra in CHCl 3 were recorded on PerkinElmer, Inc. Lambda 35 UV/VIS Spectrometer. 1 (link) H-and 13 C-NMR spectra were recorded on VarianUNITYINOVA operating at 500 MHz. Mass spectra were obtained on a Thermo Finnigan LCQ Advantage MAX LC-MS/MS spectrometer according to ESI probe. Products were isolated by column chromatography on Silica gel (Fluka Silica gel 60, particle size 63-200 µm). All chemicals were reagent grade and used without further purification. Moisture was excluded from the glass apparatus using CaCl 2 drying tubes. Solvents, unless otherwise specified, were of reagent grade and distilled once prior use.
6
Stable Isotope Analysis of Syngnathids
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For δ 13 C and δ 15 N analysis in syngnathids, the samples were rinsed with distilled water, transferred to tin capsules, dried in oven at 60 °C for 24 h and weighted (± 1 μg). Due to the low lipid content in n samples conserved in ethanol (<5% lipids, C/N <3.56) (Post et al. 2007 ), further full defatting was not necessary (Valladares and Planas 2012) . Samples were analysed at SAI (University of A Coruña) by continuous ow isotope ratio mass spectrometry using a FlashEA1112 elemental analyser (Thermo Finnigan, Italy) coupled to a Delta Plus mass spectrometer (FinniganMat, Germany) through a Con o II interface. Isotopic values are expressed as permil (‰) in conventional delta relative to VPDB (Vienna Pee Dee Belemnite) VP: Field sampling, manuscript review. PD: Field sampling, manuscript review. JC: Field sampling design, manuscript review BMN: Field sampling design, manuscript review. GenBank reference sequences for S. abaster (AF356060_S23; JX228141_SCA1), S. typhle (AF356042_S4; AF356059_S22; JX228148_KLU1), S. acus (AF356040), S. rostellatus (AF356041_S3) and S. taenionatus (AF356061_S24; JX228146_VEN89) are provided (see Mwale et al. 2013 . JFB, 82, 2045 -2062 , doi:10.1111/jfb.12130) .
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