SCFA identification and quantification was performed as in Panzella et al. (2017) (link). The equipment used was an Accela 600 HPLC (Thermo Scientific) with a quaternary pump, an autosampler and a UV-VIS PDA detector set at 210 nm. We used 0.1 M phosphate buffer (pH 2.8)/acetonitrile 99:1 v/v with a flow rate of 1.25 mL/min as mobile phase. An Aquasil C18 reverse phase (Thermo Scientific) (150 × 4.6 mm, 5 μm) was used with a run time of 30 min. The samples did not require any pretreatment before injecting except for centrifugation at 13000 rpm and further filtration through 0.22 μm nylon filter. One mL of fermentation supernatant was centrifuged and filtered prior injection of 20 μL. The SCFA standards (acetate, propionate and butyrate) were prepared in the mobile phase at concentrations ranging from 5 to 10000 ppm. Results were expressed as mM.
Aquasil c18 reverse phase
Manufactured by Thermo Fisher Scientific
Aquasil C18 is a reverse phase chromatography packing material. It is composed of silica particles coated with a C18 hydrocarbon chain. Aquasil C18 is used for the separation and purification of a wide range of organic compounds in liquid chromatography applications.
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2 protocols using aquasil c18 reverse phase
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SCFA Analysis via HPLC
2
HPLC Analysis of Short Chain Fatty Acids
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Short chain fatty acids were analyzed as in Pérez-Burillo et al. 18 In vitro fermentation supernatant was filtered through a 0.22 μm nylon filter and injected into the HPLC system (Accela 600). The HPLC system was composed of a PDA detector, a multisampler and a quaternary pump. The detector was set at 210 nm; the mobile phase used was Milli-Q water with 1% of formic acid/acetonitrile with 1% of formic acid at a rate of 99 : 1 v v -1 delivered at a 1.25 mL min -1 flow rate; the column used was an Aquasil C 18 reverse phase (Thermo Scientific) (150 × 4.6 mm, 5 µm), with a total run time of 30 min. Calibration curves with standard acetate, propionate and butyrate were made at a concentration range of 10-0.01 mM. The results were expressed in mM.
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