The identity and quantity of the amino acids were assessed by comparison with the retention times and peak areas of standard amino acids (Sigma–Aldrich) using norvaline and sarcosine as internal standards.
Trypthophan and cysteine were quantified in , 5 min; 3K30 Sigma Laborzentrifugen Gmb H, Osterode am Harz, Germany) until phase separation.
Sampling was performed at low tide, in the intertidal zone, once every 2 months, from April 2010 to February 2011.
The moisture content of the upper phase was removed using anhydrous sodium sulfate (Panreac).
A 2 µL aliquot from the upper phase was then injected onto a gas chromatograph (Varian Star 3800 Cp, Walnut Creek, CA, USA) equipped with an autosampler and fitted with a flame ionization detector at 250 °C for FAME analysis.
Finally, thawed samples were filtered (0.2 µm pore size) and separation was performed with high-performance liquid chromatography (Agilent 1100 HPLC) using precolumn o-phthalaldehyde and 3-mercaptopropionic acid in borate buffer (OPA, Agilent Technologies) and 9-fluorenylmethylchloroformate in acetonitrile (FMOC; Agilent Technologies) derivatization, a Phenomenex Gemini ODS C18 guard column (4 mm × 3 mm) and a Phenomenex Gemini ODS C18 110A column (4.6 mm × 150 mm, 5 µm).
The solvents and gradient conditions were those described by Henderson et al. Detection wavelengths were set at UV 338 and 262 nm and fluorescence 340/450 and 266/305 nm.
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