Ionpac as11

Manufactured by Thermo Fisher Scientific

Sourced in United States

The IonPac AS11 is an ion exchange chromatography column designed for the separation and analysis of inorganic anions. It features a high-capacity, low-capacity ion exchange resin that provides efficient separations of common inorganic anions, including chloride, nitrate, and sulfate.

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IonPac 4x250 mm AS11-HC ion-exchange analytical column Dionex IonPac AS11-HC Dionex IonPac AS11 IonPac AS11 column IonPac AS11 250×2 mm 4 μm column IonPac AS11-HC column (4 × 250 mm) IonPac AS11-HC analytical column IonPac AS11-HC-4 μm RFIC&HPIC IonPac AS11-HC analytical IonPac AS11 analytical column

12 protocols using ionpac as11

Glycan Separation and Characterization by HPLC

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Separation of PA-labeled
glycans was carried out on a Shimadzu HPLC system equipped with a
fluorescence detector (RF 10 AXL; 320/400 nm). In the case of RP-HPLC,
a Hypersil ODS column (C18; Agilent) was used with 100 mM ammonium
acetate, pH 4.0 (buffer A) and 30% (v/v) methanol (buffer B); a gradient
of increasing buffer B (1% per minute) was programmed. The column
was calibrated daily in terms of glucose units (g.u.) with a pyridylaminated
partial dextran hydrolysate. For 2D-HPLC, either normal-phase HPLC
(Tosoh TSKgel Amide-80) with an inverse gradient of acetonitrile in
10 mM ammonium formate, pH 7, or combined hydrophobic-interaction
anionic-exchange HPLC (HIAX, Dionex IonPac AS11) with an inverse gradient
of acetonitrile in 800 mM ammonium acetate, pH 3, was applied as previously
described.^{4 (link),27 (link)}

Yan S., Jin C., Wilson I.B, & Paschinger K. (2015). Comparisons of Caenorhabditis Fucosyltransferase Mutants Reveal a Multiplicity of Isomeric N-Glycan Structures. Journal of Proteome Research, 14(12), 5291-5305.

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Chromium Reduction Kinetics in Balanced and Acceptor-Limited Media

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Lactate and acetate concentrations were measured by HPLC (Dionex) with an Aminex HPX-87H ion exclusion column. Sulfate concentrations were measured by ion chromatography (Dionex) with an IonPac AS11 column (Dionex). Cell viability was measured via the most probable number (MPN) method at 3 and 48 h. Washed cells were inoculated into fresh LS4D medium (~ 0.07 OD₆₀₀) that was electron donor to acceptor balanced or electron acceptor-limited and contained 0 or 50 μM K₂CrO₄. Subsamples were removed and serially diluted with LS4D medium that contained sodium sulfide as a reducing agent. MPN cultures were incubated at 30 °C for 3 weeks at which point the MPN for each condition was calculated (Jarvis et al. 2010 (link)).

Franco L.C., Steinbeisser S., Zane G.M., Wall J.D, & Fields M.W. (2018). Cr(VI) reduction and physiological toxicity are impacted by resource ratio in Desulfovibrio vulgaris. Applied Microbiology and Biotechnology, 102(6), 2839-2850.

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CCRF-CEM Metabolome Analysis

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CCRF-CEM cells exposed to compounds (dT (200 µM) and/or dC, OR0642, DI-87 and DI-39 (1 µM)) for 0 and 24 h were washed, counted and pelleted. Pellets were extracted with the cold sampling solution (acetonitrile (4) / methanol (4)/125 mM formic acid (2)), vortexed and placed 1 h at −20 °C. Then centrifuged and evaporated. Central metabolites were separated on an ionic chromatography column IonPac AS11 (250 × 2 mm i.d.; Dionex). Solvent used was KOH at a flow rate of 350 µl/min. The column was then equilibrated for 6 min at the initial conditions before the next sample was analyzed. The volume of injection was 15 µl. High-resolution experiments were performed with an ICS5000+, ion chromatography system (Dionex) system coupled to an Orbitrap Qexactive+ mass spectrometer (Thermo Fisher Scientific, Waltham) equipped with a heated electrospray ionization probe. MS analyses were performed in negative FTMS mode at a resolution of 140,000 (at 400 m/z) in full-scan mode, with the following source parameters: 325 °C for the capillary temperature, 380 °C for the source heater temperature, 50 a.u. (arbitrary unit) for the sheath gas flow rate, 5 a.u. for the auxiliary gas flow rate, 50% for the S-Lens RF level, and 2.75 kV for the source voltage. Metabolites were determined by extracting the exact mass with a tolerance of 5 ppm. Each experiment was performed three times.

Saez-Ayala M., Hoffer L., Abel S., Ben Yaala K., Sicard B., Andrieu G.P., Latiri M., Davison E.K., Ciufolini M.A., Brémond P., Rebuffet E., Roche P., Derviaux C., Voisset E., Montersino C., Castellano R., Collette Y., Asnafi V., Betzi S., Dubreuil P., Combes S, & Morelli X. (2023). From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia. Nature Communications, 14, 3079.

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Quantification of Phosphorylated Metabolites

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Other phosphorylated metabolites (e.g., sugar phosphate, 2PG, and PEP) and nucleotide sugars (ADPG and UDPG) were analyzed using an anion-exchange LC-MS/MS method described in Alonso et al. (2010) (link) with slight modifications. Metabolites were reconstituted in 100 μL of water from lyophilized extract, and 10 μL of extracts was injected into an ACQUITY UPLC pump system (Waters, Milford, MA, USA) coupled with a Xevo ACQUITY TQ Triple Quadrupole Detector (Waters, Milford, MA, USA). Metabolites were separated by an IonPac AS11 analytical column (2 × 250 mm, Dionex) equipped with an IonPac guard column AG11 (2 × 50 mm, Dionex) at a flow rate of 0.35 mL min⁻¹. A multi-step gradient was applied with mobile phase A (0.5 mM KOH) and mobile phase B (75 mM KOH): 0–2 min, 100% A; 2–4 min, 100%–93% A; 4–13 min, 93%–60% A; 13–15 min, 0% A; 15–17 min, 100% A. The KOH concentration was suppressed by a post-column anion self-regenerating suppressor (Dionex ADRS 600, Thermo Scientific), with a current of 50 mA and flow rate of 3.5 mL min⁻¹. An IonPac ATC-3 Anion Trap Column (4 × 35 mm), conditioned with 2M KOH, was used to remove contaminant ions from KOH solvents. Mass spectra were acquired using MRM in negative ESI mode. Parent-product ion transitions for metabolites were described in Supplemental Table S2.

Xu Y., Fu X., Sharkey T.D., Shachar-Hill Y, & Walker A.B. (2021). The metabolic origins of non-photorespiratory CO2 release during photosynthesis: a metabolic flux analysis. Plant Physiology, 186(1), 297-314.

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Chromatographic Analysis of PA-Labeled Glycans

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Separation of PA-labeled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector. In case of RP-HPLC, a Hypersil ODS column (C18; Agilent) was used with 100 mM ammonium acetate, pH 4.0, and 30% (v/v) methanol; a gradient of the latter (1% per minute) was programmed. The column was calibrated daily in terms of glucose units (g.u.) with a pyridylaminated partial dextran hydrolysate. For 2D-HPLC, selected fractions were reapplied to a combined hydrophilic-interaction anionic-exchange HPLC (HIAX, Dionex IonPac AS11) with an inverse gradient of acetonitrile in 800 mM ammonium acetate, pH 3.85, as previously described.32 (link) For further details refer to the Supporting Information.

Yan S., Vanbeselaere J., Jin C., Blaukopf M., Wöls F., Wilson I.B, & Paschinger K. (2017). Core Richness of N-Glycans of Caenorhabditis elegans: A Case Study on Chemical and Enzymatic Release. Analytical Chemistry, 90(1), 928-935.

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Quantification of Colonic Short-Chain Fatty Acids

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SCFAs (i.e., lactate, acetate, propionate, butyrate) in colonic contents were quantified the procedures of Wang et al. [29 (link)]. Briefly, pig samples were thawed on ice and approximately 0.5-g samples were added to 8 mL of deionized water (For mouse samples: 50 mg of samples was added to 1 mL of deionized water). The mixture was thoroughly homogenized by vortexing for 1 min and centrifuged at 13,000 × g for 5 min. The supernatant was diluted 50-fold for pig samples or 25-fold for mouse samples and filtered through a 0.22-μm filter (Millipore, USA). The solution was transferred into the vial for quantitative determination using ion chromatography (ICS-3000, Dionex, USA) with a Dionex Ionpac™ AS11 analytical column (4 × 250 mm). To determine SCFA concentrations of the bacterial media, 1 mL media was collected at each time point and centrifuged at 13,000 × g for 5 min. The supernatant was diluted 100-fold and filtered through a 0.22-μm filter for ion chromatography analysis.

Wen Y., Yang L., Wang Z., Liu X., Gao M., Zhang Y., Wang J, & He P. (2023). Blocked conversion of Lactobacillus johnsonii derived acetate to butyrate mediates copper-induced epithelial barrier damage in a pig model. Microbiome, 11, 218.

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Sensitive Separation of Glycan Derivatives

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Separation
of PA-labeled
glycans was carried out on a Shimadzu HPLC system equipped with a
fluorescence detector. In case of RP-HPLC, a Hypersil ODS column (C18;
Agilent) was used with 100 mM ammonium acetate, pH 4.0, and 30% (v/v)
methanol; a gradient of the latter (1% per minute) was programmed.
The column was calibrated daily in terms of glucose units (g.u.) with
a pyridylaminated partial dextran hydrolysate. For 2D-HPLC, selected
fractions were reapplied to a combined hydrophilic-interaction anionic-exchange
HPLC (HIAX, Dionex IonPac AS11) with an inverse gradient of acetonitrile
in 800 mM ammonium acetate, pH 3.85, as previously described.^{32 (link)} For further details refer to the Supporting Information.

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Anion Analysis by Ion Chromatography

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IC mobile phase A (MPA; weak) was water, and mobile phase B (MPB; strong) was water containing 100 mM KOH. A Thermo Scientific Dionex ICS-6000+ system included a Thermo IonPac AS11 column (4 μm particle size, 250 × 2 mm) with column compartment kept at 35°C. The autosampler tray was chilled to 4°C. The mobile phase flow rate was 360 μL/min, and the gradient elution program was: 0–5 min, 1% MPB; 5–25 min, 1–35% MPB; 25–39 min, 35–99% MPB; 39–49 min, 99% MPB; 49–50, 99–1% MPB. The total run time was 50 min. To assist the desolvation for better sensitivity, methanol was delivered by an external pump and combined with the eluent via a low dead volume mixing tee.

Mahmud I., Wei B., Veillon L., Tan L., Martinez S., Tran B., Raskind A., de Jong F., Akbani R., Weinstein J.N., Beecher C, & Lorenzi P.L. (2024). An IROA Workflow for correction and normalization of ion suppression in mass spectrometry-based metabolomic profiling data. Research Square.

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Quantification of IP6 and IP5 by Ion Chromatography

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A Dionex ICS‐5000 ion chromatograph (Thermo Scientific, Hemel Hempstead, UK) equipped with a KOH eluent generator, ion suppressor and conductivity detector was used for chromatographic separation, identification and quantification of reference standards IP6 and IP5. The compounds were separated using an Ionpac AS11 column (2 × 250 mm; Thermo Scientific) with an AS11G guard column (2 × 50 mm). The flow rate was set to 0.25 ml.min⁻¹, and the column temperature to 30 °C. The elution gradient included a 10 min equilibration at 4 mM KOH, followed by: 0 min: 4 mM KOH, 19 to 24 min: 70 mM KOH, 29 to 30 min: 4 mM KOH. Eluate fractions were collected post‐detection at 30 s intervals in glass vials for HRMS analysis. Chromatograms were analysed in Chromeleon (Thermo Scientific).

McIntyre C.A., Arthur C.J, & Evershed R.P. (2017). High‐resolution mass spectrometric analysis of myo‐inositol hexakisphosphate using electrospray ionisation Orbitrap. Rapid Communications in Mass Spectrometry, 31(20), 1681-1689.

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Quantify FAD and FMN in Mouse Liver

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To determine the relative abundance of FAD and FMN in mouse liver tissue, extracts were prepared and analyzed by high-resolution mass spectrometry (HRMS). Approximately 20–30 mg of tissue were pulverized in liquid nitrogen then homogenized with a Precellys Tissue Homogenizer. Metabolites were extracted using 80/20 (v/v) methanol/water with 0.1% ammonium hydroxide. Samples were centrifuged at 17,000 × g for 5 min at 4°C, supernatants were transferred to clean tubes, followed by evaporation under vacuum. Samples were reconstituted in deionized water, then 10 μl was injected into a Thermo Scientific Dionex ICS-5000+ capillary ion chromatography (IC) system containing a Thermo IonPac AS11 250 × 2 mm 4 μm column. IC flow rate was 360 µl/min (at 30°C), and the gradient conditions are as follows: initial 1 mM KOH, increased to 35 mM at 25 min, increased to 99 mM at 39 min, and held at 99 mM for 10 min. The total run time was 50 min. To increase desolvation for better sensitivity, methanol was delivered by an external pump and combined with the eluent via a low dead volume mixing tee. Data were acquired using a Thermo Orbitrap Fusion Tribrid Mass Spectrometer under ESI negative mode and imported to Thermo Trace Finder software for final analysis. Relative abundance was normalized by tissue weight.

Masschelin P.M., Saha P., Ochsner S.A., Cox A.R., Kim K.H., Felix J.B., Sharp R., Li X., Tan L., Park J.H., Wang L., Putluri V., Lorenzi P.L., Nuotio-Antar A.M., Sun Z., Kaipparettu B.A., Putluri N., Moore D.D., Summers S.A., McKenna N.J, & Hartig S.M. (2023). Vitamin B2 enables regulation of fasting glucose availability. eLife, 12, e84077.

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