Sp 2330 column

Methylation analysis of PLAP was conducted according to the method of previous reports [40 (link),41 (link)] with some modifications. The partially methylated alditol acetates (PMAA) of PLAP were taken for linkage analysis using a 7890-7000A GC-MS system (Agilent, Santa Clara, CA, USA) equipped with a SP-2330 column (Supelco, Bellefonte, PA, USA; 30 m × 0.25 mm, 0.2 μm film thickness). Individual peaks of the PMAA and fragmentation patterns were identified by their mass spectra and relative retention times in the GC traces.

100 μg of the sample was placed in a quartz tube, and 4.5 μL of a diluted, aqueous solution of tetramethylammonium hydroxide was added. The samples were subsequently pyrolyzed at 450°C for 10 s with a CDS 5250 pyrolysis autosampler attached to a Thermo Trace GC Ultra/MD 800 gas chromatography/mass spectrometry system. Volatile products were separated on a Supelco SP 2330 column (30 m, ID 0.32 mm, 0.2 μm film thickness) with helium 4.6 as carrier gas (2 mL·min⁻¹) and identified by comparison to reference compounds as well as interpretation of their EI mass spectra and comparison to NIST 2002, Wiley, and NBS electronic libraries. The pyrolysis interface was kept at 300°C and the GC/MS interface at 280°C; the GC was programmed from 100°C (1 min) to 230°C (5 min) at a rate of 10°C min⁻¹. The mass spectrometer was operated in EI mode (70 eV) at a source temperature of 200°C [35 ].

A methylation exploration was carried out according to Singthong et al. [10 (link)]. First, the dried samples were dissolved in anhydrous DMSO for 2 h at 85 °C. Also, the solution was constantly stirred and further sonicated for 2 h to ensure that the samples were completely dissolved. Next, the 20 mg of dried sodium hydroxide powder was added to the DMSO and stirred for 3 h at room temperature. Then, the methyl iodide (0.3 mL) was added and stirring continued for 2.5 h. Methylene chloride was used to extract partly methylated polysaccharides, which were then put down a sodium sulfate column to remove water and dried under a nitrogen stream. The samples were also hydrolyzed in 4 M trifluoroacetic acid (0.5 mL) in a sealed test tube at 100 °C for 6 h before being dried in a nitrogen stream. Before acetylation with acetic anhydride, the hydrolyzed acid was reduced with deuterated sodium borohydride (0.5 mL). The partly methylated alditol acetates (PMAA) in aliquots were then injected into a GC-MS apparatus (ThermoQuest Finnigan, San Diego, CA, USA) fitted with an SP-2330 column (Supelco, Bellefonte, PA, USA) (30 m × 0.25 mm, 0.2 μm film thickness, 160–210 °C at 2 °C/min, and then 210–240 °C at 5 °C/min) equipped with an ion trap MS detector.