Fusion rp 80a column

The quantitative and qualitative analysis of polyphenolic compounds was performed using the high-performance liquid chromatography (HPLC) method described earlier by Król et al. [47 (link)]. Briefly, a 3 mL sample of coffee brew was combined with 5 mL of 80% methanol. The mixture was then mechanically shaken and subjected to extraction in an ultrasonic bath (30 °C, 10 min). Following extraction, the sample was centrifuged (5 °C, 3780× g, 10 min) and was subsequently subjected to chromatographic analysis (HPLC-DAD using two LC-20AD pumps, a CBM-20A controller, an SIL-20AC column oven, and UV/Vis SPD-20 AV, and SPD-M20A spectrometers; Shimadzu, Kyoto, Japan).
Chromatographic separation was carried out on a Phenomenex Fusion-RP 80A column (Torrance, CA, USA, 250 mm × 4.60 mm). The mobile phase consisted of two components: (A) 90% water and 10% acetonitrile, and (B) 45% water and 55% acetonitrile, both with a pH of 3.0. The applied gradient program is detailed in Table 3. The injection volume was set at 100 μL, with the sample temperature maintained at 30 °C, and the column temperature also held at 30 °C. UV detection was performed at wavelengths: λ = 250 nm for flavonoids (quercetin-3-O-rutinoside, kaempferol-3-O-glucoside, quercetin, quercetin-3-O-glucoside, kaempferol, epigallocatechin) and λ = 370 nm for phenolic acids (gallic, chlorogenic, caffeic, salicylic).

Individual phenolics were extracted and measured by HPLC [38 (link)]. One hundred milligrams of powdered plant material was extracted with 80% methanol by sonication (10 min., 6000 Hz, temp. 30 °C). After extraction, the samples were centrifuged (10 min, 6000 rpm, temp. 0 °C). Fifty microliters of supernatant was injected into a Fusion RP-80 A column (250 × 4.6 mm, Phenomenex, Warsaw, Poland). A flow rate of 1 mL min⁻¹ of gradient phase prepared from acetonitrile and water with phosphoric acid (pH 3.0) was used. Pump pressure was in the range of 13.00–14.50 mPa. Time phases are as follows: 1.00–22.99 min, phase A 95% and phase B 5%; 23.00–27.99 min, phase A 50% and phase B 50%; 28.00–28.99 min, phase A 80% and phase B 20%; and 29.00–38.00 min, phase A 95% and phase B 5%. The analysis time was 42 min, the detection wavelength for flavonoids was 360 nm, and the detection wavelength for phenolic acids was 250 nm. Polyphenols were identified based on retention time and external standards (gallic acid, p-hydrobenzoic acid, caffeic acid, p-coumaric acid, benzoic acid, kaempferol-3-O-glucoside, myricetin, luteolin, quercetin, quercetin-3-O-glucoside, and kaempferol). Chromatograms from phenolics analysis are presented in Figures S2 and S3 (Supplementary Materials). Standard curves with their equations and R² are presented in Figures S4–S6 (Supplementary Materials).