HPLC analyses were performed on instrument Agilent 1200 RR HPLC (Agilent, Waldbronn, Germany), equipped with a DAD detector, using reverse-phase analytical column Zorbax SB-C18 (Agilent) as we previously described in detail [45 (link)]. Mobile phase A was the solution of orthophosphoric acid in water (1% v/v) whereas mobile phase B was acetonitrile. The flow rate was 1 mL/min. Gradient elution was employed according to the following scheme: 98–90% A (0–5 min); 90% A (5–15 min); 90–85% A (15–20 min); 85–70% A (20–25 min); 70–40% A (25–30 min); 40–0% A (30–34 min). Detection was done at 260 and 320 nm. The quantity of investigated compounds was calculated using calibration curves of authentic standards (punicalagin, punicalin, gallic acid, and ellagic acid). The results are expressed as mg per gram of dry weight. Experiments were repeated three times.
Agilent 1200 rr hplc
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The Agilent 1200 RR HPLC is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It is capable of separating and analyzing a wide range of compounds with high resolution and accuracy.
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4 protocols using agilent 1200 rr hplc
1
HPLC Analysis of Phenolic Compounds
2
Quantitative Analysis of Phytochemicals
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HPLC analyses were performed on the apparatus Agilent 1200 RR HPLC (Agilent, Waldbronn, Germany), equipped with a DAD detector, using reverse-phase analytical column Zorbax SB-C18 (Agilent), as previously described in detail [73 (link)]. Orthophosphoric acid in water (1% v/v) and acetonitrile were mobile phases A and B, respectively. The parameters were as follows. Flow rate: 1 mL/min; gradient elution: 98–90% A (0–5 min); 90% A (5–15 min); 90–85% A (15–20 min); 85–70% A (20–25 min); 70–40% A (25–30 min); 40–0% A (30–34 min); detection—260 and 320 nm. The quantity of investigated compounds in the PEx (PG, PN, and EA) and GA was determined using calibration curves of their standards, and the results were expressed as mg/gram of dry weight. As shown in our previous paper [24 (link)] and the Supplementary Figure S1 of this paper, the main compounds of PEx were PG (67.26 ± 0.81 mg/g), PN (31.91 ± 0.22 mg/g), EA (25.11 ± 0.06 mg/g), and GA (9.75 ± 0.05 mg/g).
3
Quantification of Polyphenols in Extracts
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Total phenolic content was analyzed spectrophotometrically using the slightly modified Folin-Ciocalteu method. The absorbance was measured at 765 nm by UV/VIS spectrophotometer (HP 8453, Agilent Technologies, USA). The calibration curve was prepared using gallic acid (GA) (0–100 mg/L). The results were expressed as milligrams of GA equivalents per gram of dry weight of the extract (mg GAE/g DW) or mg GAE/100 mL juice. The tannin content analyses were performed according to the European Pharmacopeia 7.0 using phosphomolybdotungstic reagent [11 ]. The absorbance of the final mixture was measured at 760 nm by UV/VIS spectrophotometer (HP 8453, Agilent Technologies, USA). The results were calculated and expressed as a percent of pyrogallol.
HPLC analysis was carried out on instrument Agilent 1200 RR HPLC (Waldbronn, Germany), equipped with DAD detector, using reverse-phase analytical column Zorbax SB-C18 (Agilent), (5 μm particle size; 150 m × 4.6 mm i.d.). Mobile phase A was the solution of orthophosphoric acid in water (1%, v/v), and mobile phase B was acetonitrile; flow rate was 1 mL/min. The injection volume was 5 μL, and the column temperature was maintained at 25 °C. Detection was done at 260 and 320 nm. The amounts of investigating compounds were calculated using calibration curves of standards (punicalagin, punicalin, gallic acid, and ellagic acid).
4
Analytical Characterization of Pomegranate Peel Extract
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The detailed procedure for preparing and analyzing PoPEx was published in our previous paper [27 (link)]. The powdered pomegranate peel was prepared from pomegranate fruits collected at a natural locality in southern Bosnia and Herzegovina. The peel was extracted with 50% ethanol, using 1:10 as a solid-to-solvent ratio. After filtration and evaporation, the extract was analyzed spectrophotometrically by the Folin–Ciocalteu method, where gallic acid was used to prepare the calibration curve. The results were expressed as mg of gallic acid equivalents per gram of dry weight. The pomegranate peel was deposited in the Botanical Garden “Jevremovac” University of Belgrade (voucher specimen No. BEOU 17742). HPLC analysis was performed on Agilent 1200 RR HPLC (Agilent, Waldbronn, Germany), equipped with a DAD detector, using reverse-phase analytical column Zorbax SB-C18 (Agilent, Waldbronn, Germany). Detection was performed at 260 and 320 nm. The quantity of analyzed compounds (punicalagin, punicalin, gallic acid, and ellagic acid) was calculated using calibration curves of authentic standards (Supplementary Figure S4 ). The results are expressed as mg per gram of dry weight. Experiments were repeated three times. Their mean content was as follows: punicalagin 67.26 ± 0.81 mg/g, punicalin 31.91 ± 0.22 mg/g, ellagic acid 25.11 ± 0.06 mg/g, and gallic acid 9.75 ± 0.05 mg/g.
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