The supernatant samples were filtered and 0.2 μL was injected into a high efficiency liquid chromatography (HPLC) system (Jasco, Tokyo, Japan) equipped with a UV-975 detector and automatic injector. The SCFAs were separated on a Rezex ROA Organic Acids column (300 × 7.8 mm) (Phenomenex, Macclesfield, UK) with a thermostat at 50 °C. The mobile phase was a linear gradient of 0.005 M sulfuric acid in HPLC-grade water at 0.6 mL/min, and the elution curves were monitored at 210 nm. A calibration curve for SCFA was established in the concentration range of 1–100 mM.
Rezex roa organic acids column
Manufactured by Phenomenex
Sourced in United Kingdom, United States
The Rezex ROA Organic Acids column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of organic acids. It features a specialized stationary phase that facilitates the effective separation of various organic acids, including lactic, acetic, and formic acids, among others.
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7 protocols using rezex roa organic acids column
1
HPLC Analysis of Short-Chain Fatty Acids
2
HPLC Analysis of Short-Chain Fatty Acids
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Samples from the R1, R2, and R3 compartments were centrifuged (10,000 × g, 10 min) and the supernatants analyzed by HPLC as described earlier (Barroso et al., 2015 (link)). Briefly, samples (20 μL) were injected on a HPLC system (Jasco, Tokyo, Japan) equipped with a UV-975 detector. SCFA were separated using a Rezex ROA Organic Acids column (Phenomenex, Macclesfield, United Kingdom) using 5 mM sulphuric acid as mobile phase. The elution profile was monitored at 210 nm and the identification of the peaks was carried out by comparing the retention times of target peaks with those of the standards: acetic, propionic, butyric, formic, succinic, and lactic acids. Calibration curves of these acids were carried out in the concentration range from 1 to 100 mM. Ammonium was determined using the Nessler’s reagent (Sigma) as previously described (Doo et al., 2017 (link)).
3
HPLC Analysis of Glycerol in Hydrolyzed Fat Media
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Glycerol concentration in media with hydrolyzed fat was analyzed by high performance liquid chromatography (HPLC) with refractive index detection, according to Reference [18 (link)]. The HPLC/refractive index (RI) analysis of glycerol was carried out on a Dionex Ultimate 3000 (Thermo Fischer Scientific, USA) apparatus using a Rezex ROA Organic acids column (Phenomenex; 300 × 7.8 mm). The sample (20 μL) was applied into the column and equilibrated by an elution mixture (0.005 mol/L sulfuric acid) at 60 °C. Separation was performed under isocratic elution (flow 1.0 mL/min) for 15 min. Chromatography data were evaluated using the Chromeleon software.
4
BFBL Gut Model SCFA Quantification
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The BFBL gut model samples (1 mL) were collected daily from each reactor (R1, R2, and R3) for SCFAs analysis during each period. SCFAs were quantified by a method reported by Barroso et al. [26 (link)]. In short, the filtered (0.22 μm) samples were analyzed by an HPLC system equipped with Rezex ROA-Organic Acids column (300 mm × 7.8 mm, Phenomenex, Macclesfield, UK) and UV-975 detector (Jasco, Tokyo, Japan). The mobile phase was 0.005 M sulfuric acid solution with a linear gradient of 0.6 mL/min, and elution curves were monitored at 210 nm. Calibration curves were established for SCFAs with concentrations ranging from 1 to 100 mM.
5
Analyzing Short-Chain Fatty Acids in Gut Microbiota
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The supernatant from the colon microbiota culture was filtered and 0.2 μL were injected into an HPLC system (Jasco, Tokyo, Japan) equipped with a UV975 detector and automatic injector37 . SCFA were separated using a Rezex ROA Organic Acids column (300 × 7.8 mm) (Phenomenex, Macclesfield, UK) thermostated at 50 °C following the method described by Sanz et al.43 (link). The mobile phase had a linear gradient of 0.005 M sulphuric acid in HPLC grade water, and the flow rate was 0.6 mL/min. The elution profile was monitored at 210 nm and peak identification was carried out by comparison between retention times and standards. ChromNAV data system software (Jasco) was used for data acquisition and processing. Calibration curves of acetic, butyric, formic, lactic and succinic acid were built up in the range concentration of 1 to 100 mM.
6
SCFA Analysis of Bioreactor Samples
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SCFA in the sample supernatants from the R1, R2 and R3 reactors were analysed by HLPC as described earlier (Barroso et al., 2015) (link). SCFA were separated in a Rezex ROA Organic Acids column (Phenomenex, Torrance, CA, USA) by using a linear gradient of 0.005 mM sulphuric acid at 0.6 mL/min. The ammonium content was determined using the Nessler's reagent (Sigma) as described by Doo et al. (Doo, Chassard, Schwab, & Lacroix, 2017) (link).
7
HPLC-based SCFA Quantification
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Supernatants from the faecal homogenates were filtered and 0.2 µL were injected on a HPLC system (Jasco, Tokyo, Japan) equipped with a UV-975 detector. SCFA were separated using a Rezex ROA Organic Acids column (Phenomenex, Macclesfield, UK) following the method described by Sanz et al. 24 The mobile phase was a linear gradient of 0.005 M sulphuric acid in HPLC grade water, and flow rate was 0.6 mL/min. The elution profile was monitored at 210 nm and peak identification was carried out by comparing the retention times of target peaks with those of standards. Calibration curves of acetic, propionic, butyric, formic, succinic and lactic acids were built up in the concentration range of 1 to 100 mM.
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