Ionpac as11 hc column

2 mg EPS was hydrolyzed in 2 M TFA for 2 h in a sealed tube at 121°C. The hydrolysis products were vacuumly dried and then solubilized with H₂O. The presence of sulfate was attested by using a high-performance liquid ion chromatography system (ICS-2100, Thermo Scientific) equipped with an IonPac AS11-HC column. A solution of 30 mM NaOH was used as eluent at a flow rate of 1 mL/min. The column temperature was kept at 30°C. A calibration curve prepared with Na₂SO₄ as a standard was used to calculate the sulfate content in the EPS.

Monocytes (1 × 10⁶/700 µl) were stimulated with 100 ng/ml LPS in the presence of 5 mM glucose or in glucose-deficient media on agarose-coated wells. Cells were harvested and centrifuged (350 × g, 5 min) 1, 3, and 6 h after stimulation and washed one time with PBS. The pellet was dissolved in 500 µl 45% (v/v) methanol/5% (v/v) chloroform. After the addition of 500 µl water the suspension was mixed 30 min at 4°C. The solution was centrifuged at 500 × g for 10 min, the upper phase was transferred and vacuum dried. For ion chromatography-tandem mass spectrometry (IC-MS/MS)-based analysis of the metabolites, dried extracts were redissolved in 300 µl water. Samples were afterward up to another 10-fold diluted, and a total volume of 25 µl was analyzed on an ICS-5000 (Thermo Fisher Scientific, Dreieich, Germany) coupled to an API 5500 QTrap (AB Sciex) as described elsewhere (26 (link)). Separation was achieved on an IonPac AS11-HC column (2 mm × 250 mm, Thermo Fisher Scientific) with an increasing potassium hydroxide gradient. MS analysis was performed in multiple reaction monitoring (MRM) mode using negative electrospray ionization and included organic acids and carbohydrates involved in central metabolite pathways. Metabolites were considered to be detectable above a signal-to-noise ratio (S/N) of three within a retention time window of 0.5 min.

The volatile fatty acids below C6, oxalate, and inorganic anions were detected using an ion chromatography system equipped with an IonPac AS11-HC column (ICS-1000, Thermo Fisher Scientific Inc., Waltham, MA, USA). Further, the elute flow rate was set to 1 mL·min⁻¹ at 35 °C with 4 mol·L⁻¹ of hepta-fluoro-butyric acid for the organic acids and 4 mol·L⁻¹ of KOH for the inorganic anions.

The O-acetyl content of O25B-EPA samples was determined by high performance anion exchange chromatography with conductivity detection (HPAEC-CD) following release of the O-acetyl groups by use of mild alkaline hydrolysis. Conjugate samples (saccharide concentration ~ 100 μg/mL) were desalted on a Zeba™ Spin Desalting column featuring a 7 kDa cut off (Thermo Fisher Scientific) and hydrolyzed in 10 mM NaOH (4 h at 37 °C) in the presence of propionate serving as internal reference standard. The resulting samples were loaded to centrifugal filter devices with a MW cut off of 3 kDa (Merck, Darmstadt, Germany) and spun once at 16,000×g for 15 min. Permeates containing released acetate were analyzed by HPAEC-CD on a Summit HPLC system (Thermo Fisher Scientific) equipped with an ED50 conductivity detector and an auto-suppression recycling suppressor ASRS 300. Chromatography was performed on an IonPac AS11-HC column (Thermo Fisher Scientific) by isocratic elution with 1 mM NaOH over 15 min at a flow rate of 1.5 mL/min. Chromeleon V.6.80 SR9 software was used for data analysis. The amount of acetate was determined via a standard curve of 1–10 μg/mL acetate and the extent of O-acetylation in conjugate samples calculated based on the mass proportion of acetate to the polysaccharide RU.