Waters e2695

Manufactured by Waters Corporation

Sourced in United States, China

The Waters e2695 is a high-performance liquid chromatography (HPLC) system designed for a variety of analytical applications. It features automated sample handling, precise solvent delivery, and reliable performance for consistent results.

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53 protocols using waters e2695

Quantification of Catechins and Theaflavins in Tea Extracts

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Catechin levels were measured with an HPLC-photodiode array detector (Waters 2996 PDA Detector; Waters, USA) and a liquid chromatography system (Waters e2695; Waters). The extract sample (10 µL) was injected into the HPLC column (5 µm, 250 × 4.6 mm, Shiseido CAPCELL PAK C18 UG 120; Shiseido, Japan). Mobile phase A was composed of 0.1% acetic acid and mobile phase B was composed of 100% acetonitrile. The catechins were eluted with 95% mobile phase A at 0 min, 75% mobile phase A at 20 min, 100% mobile phase B at 21 min, and 95% mobile phase A at 36 min. The flow rate was 1.0 mL/min at 40℃. Peaks were monitored at 280 nm and UV spectra were recorded.
Theaflavins were quantified with an HPLC-photodiode array detector (Waters 2998 PDA Detector; Waters) and a liquid chromatography system (Waters e2695; Waters). The extract sample (10 µL) was injected into the HPLC column as described above. Mobile phase A was composed of 0.1% acetic acid:acetonitrile:tetrahydrofuran (96:2:2), and mobile phase B was 100% acetonitrile. The theaflavins were eluted with 100% mobile phase A at 0 min, 40% mobile phase A at 45 min, and 100% mobile phase A at 47 min. The flow rate was 1.0 mL/min at 40℃. Peaks were monitored at 310 nm and UV spectra were recorded. The individual catechins and theaflavins were identified by comparing the retention times of the analytes with those of reference standards.

Kim Y.C., Choi S.Y, & Park E.Y. (2015). Anti-melanogenic effects of black, green, and white tea extracts on immortalized melanocytes. Journal of Veterinary Science, 16(2), 135-143.

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HPLC-based ACE Inhibitory Assay

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The ACE inhibitory activity of the samples was determined using a high-performance liquid chromatograph (HPLC) (model Waters e2695, Alliance, San Diego, CA, USA) equipped with a SunFire C18 column (4.6 mm × 150 mm; Merck, Darmstadt, Germany), as previously described by Su et al. (2023) [17 (link)].

Wang Y., Chen S., Shi W., Liu S., Chen X., Pan N., Wang X., Su Y, & Liu Z. (2024). Targeted Affinity Purification and Mechanism of Action of Angiotensin-Converting Enzyme (ACE) Inhibitory Peptides from Sea Cucumber Gonads. Marine Drugs, 22(2), 90.

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Flavanone Composition Analysis in Juice

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The flavanone composition was measured according to Huang et al. [29 (link)] with some modifications. Briefly, juice was diluted 2.33 times with methanol and flavonoid standards (eriocitrin, neoeriocitrin, narirutin, naringin, neohesperidin from Sigma-Aldrich (St. Louis, MO, USA)) were dissolved in methanol and sufficiently mixed. After filtration through a PTFE membrane with φ = 13 mm and pore size of 0.45 mm, 10 μL of samples (or standard solutions) were injected into the HPLC system (Waters e2695, Waters, Milford, MA, USA) coupled with a UV/vis detector (Waters 2489, Waters, Milford, MA, USA). The XDB-C18 column (250 × 4.6 mm, Agilent, Santa Clara, CA, USA) was kept at 25 °C with a flow rate of 0.7 mL/min. Solution A (0.1% formic acid) and solution B (methanol) were used as the mobile phase with the gradient elution as follows: 0–20 min, 63–50% A; 20–25 min, 50–20% A; 25–30 min, 20–0% A; 30–35 min, 0% A; 35–40 min, 0–63% A; 40–42 min, 63% A. The detection was performed at 283 nm. The flavonoids were identified and quantified according to the standard curves (Figure S1).

Zhou X., Wang W., Ma X., Xu E, & Liu D. (2021). Ultrasonication of Thawed Huyou Juice: Effects on Cloud Stability, Physicochemical Properties and Bioactive Compounds. Foods, 10(8), 1695.

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Quantifying Piceid and Resveratrol in Rice

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To determine the piceid and resveratrol content in rice grains, a fine powder of each sample was mixed with 80% methanol (300 mg fine powder: 900 µL of 80% methanol). The mixture was sonicated for 30 min at room temperature. The tube was centrifuged at 10,000× g at 4 °C for 5 min. After centrifugation, the supernatant was collected and filtered through a 0.2 m nylon membrane filter. The filtered supernatant (1 µL) was used for HPLC analysis of piceid and resveratrol amounts on a Waters e2695 (Waters, Milford, MA, USA). HPLC was performed as previously described [53 (link)]. Piceid and resveratrol contents were quantified by comparing them to the calibration standard curve (Figure 6).

Monmai C., Kim J.S, & Baek S.H. (2023). Use of Germination to Enhance Resveratrol Content and Its Anti-Inflammatory Activity in Lipopolysaccharide-Stimulated RAW264.7 Cells. Molecules, 28(13), 4898.

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OTA Analysis by HPLC with Fluorescence Detection

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The measurement of OTA was carried out by HPLC (Waters E2695; Waters Corporation, USA). The detection of OTA was done using 333 and 477 nm as wavelengths of excitation and emission, respectively. The separation was performed on Chromolith^® Performance RP‐18e (200 mm × 4.6 mm, i.d., 3 μm) with fluorescence detector and a particle size of 3 µ. The mobile phase delivered at a flow rate of 1 ml/min consisted of water: acetonitrile: methanol: acetic acid (30:39:30:1, v/v/v/v). HPLC measurements were carried out in triplicates.

Mozaffary P., Milani J.M, & Heshmati A. (2019). The influence of yeast level and fermentation temperature on Ochratoxin A decrement during bread making. Food Science & Nutrition, 7(6), 2144-2150.

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Phenolic Compound Extraction and Identification

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The extraction and identification of phenolic compounds were conducted according to our previous method (Xi, Lu, Qun, & Jiao, 2017). Fifteen milliliters of 80% methanol were added to one gram of frozen powder. After mixing by vortexing, the phenolic compounds were extracted ultrasonically at 60°C for 60 min and then centrifuged at 5,000 r/min for 15 min, after which the supernatant was collected. The extraction was repeated three times, and the supernatants were combined, increased to 50 ml with 80% methanol, and stored for phenolic compound determination.
The phenolics were detected by an HPLC system (Waters e2695, USA) equipped with a Sunfire C18 (4.6 mm × 250 mm, 5 μm, Waters) and were detected at wavelengths of 260, 280, and 320 nm with a 2,998 PDA detector. The phenolic compounds were identified by comparing their retention times with the spectral characteristics and peak times of the standard. Their contents were quantified using the standard curve method and expressed in mg/g FW. The three biological replicates were used for each sample.

Yang C., Chen T., Shen B., Sun S., Song H., Chen D, & Xi W. (2019). Citric acid treatment reduces decay and maintains the postharvest quality of peach (Prunus persica L.) fruit. Food Science & Nutrition, 7(11), 3635-3643.

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HPLC Analysis of Compound Purity

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Purity of all compounds tested in biological assays was determined to be >95% by HPLC analysis. The following methods were used: HPLC-Agilent 1260 (Agilent Technologies Inc., Palo Alto, CA, USA), Agilent zorbax eclipse ZOEBAX SB-C18 (150 mm × 4.6 mm, I.D. 5 μm), DAD (254 or 260 nm) detector, water (mobile phase A), methanol (mobile phase B), 0 min 20% B, 8 min 55% B, 20 min 80% B; or Waters e2695 (Waters, Milford, MA, USA), shim-pack VP-ODS (150 mm × 4.6 mm, I.D. 5 μm), DAD (254 or 260 nm) detector, water (mobile phase A), methanol (mobile phase B), 0–8 min 20% B; 9 min 55% B; 20 min 80% B.

Zhang S., Zhao Y., Wang S., Li M., Xu Y., Ran J., Geng X., He J., Meng J., Shao G., Zhou H., Ge Z., Chen G., Li R, & Yang B. (2020). Discovery of novel diarylamides as orally active diuretics targeting urea transporters. Acta Pharmaceutica Sinica. B, 11(1), 181-202.

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HPLC Identification of Antioxidant Compounds

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To confirm the identity of the antioxidant compounds corresponding to the structure identified by the NMR measurement, HPLC was performed using Lee et al.’s method [36 (link)]. Samples and standards were added to the methanol, followed by filtration through a 0.45-μm membrane filter (Millipore Co., Burlington, MA, USA) and analysis by HPLC (Waters e2695, Waters Co., Milford, MA, USA). The column comprised Capcell pak C₁₈ UG120, S5 (4.6 × 250 mm) (Shiseido co., LTD, Tokyo, Japan). The eluents were 0.1% formic acid in water (A) and 0.1% formic acid in 90% acetonitrile with water (B) and the flow rate used was 0.1 mL/min. The gradient was as follows: 0–4 min, 10% B; 4–20 min, 60% B; 20–23 min, 10% B; 23–30 min, 10% B. The injection volume was 10 µL and the detection wavelength was 310 nm (Waters 2489 UV/visible detector, Waters Co., Milford, MA, USA).

Kim S.H, & Huh C.K. (2022). Isolation and Identification of Fisetin: An Antioxidative Compound Obtained from Rhus verniciflua Seeds. Molecules, 27(14), 4510.

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Synthesis and Characterization of Catalpol

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Catalpol was extracted from Rehmannia glutinosa in laboratory, purity is 98%. All the chemicals and organic solvents used in the synthetic process are AR grade. Propionic anhydride, pyridine, sodium bicarbonate and anhydrous sodium sulfate were purchased from Shanghai Alpha Chemical Co., Ltd., China. Methylsilicone oil was purchased from Changzhou Longcheng Organosilicon Co., Ltd., China. Chromatographic grade methanol and acetonitrile were purchased from Tianjin Siyou Fine Chemicals Co., Ltd., China. HPLC were performed using Waters-E2695 (Waters Inc., USA). High resolution mass spectra (HRMS) were recorded by Thermo Fisher-Exactive Obitrap Mass Spectrometer (Thermo Fisher Inc., USA). NMR spectra were recorded on Agilent 400 MHz NMR spectrometer (Agilent Inc., USA). FT-IR spectra were measured on Nicolet iS5 (Thermo Fisher Inc., USA). Solvent CDCl₃ used in NMR were obtained from Aldrich Chemical Co., Inc. RE-2000B rotary evaporator is purchased from Zhengzhou Kaipeng Experimental Instrument Co., Ltd., China. Ultrapure water was prepared using Milli-Q Century (Millipore Company, USA).

Dong C., Liu S., Cheng X., Wang Q., Jiang S, & Wang G. (2019). Design, synthesis, and preliminary biological evaluation of catalpol propionates as antiaging drugs. BMC Chemistry, 13(1), 109.

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Determination of Encapsulation Efficiency of Salicylate-Loaded Lipid Nanoparticles

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To determine EE, 1 mL Sal-LPNPs solution was centrifuged for 70 min (1.20 × 10⁴ g/min) at 4 °C. The precipitate was dissolved in DMSO. The amount of Sal in precipitate was defined as the capsulated part and determined by high-performance liquid chromatography (HPLC) assay (Waters e2695, Waters Co., Shanghai, China). A reverse-phase Cosmosil C18 column (150 × 4.6 mm, pore size 5 μm, Global chromatography) was used for the chromatographic separation with an injection volume of 20 μL. The mobile phase was a mixture of methanol/water (75/25, v/v) at a rate of 1.0 mL/min. Sal was detected at 278 nm [45 (link)].

Fang D.L., Chen Y., Xu B., Ren K., He Z.Y., He L.L., Lei Y., Fan C.M, & Song X.R. (2014). Development of Lipid-Shell and Polymer Core Nanoparticles with Water-Soluble Salidroside for Anti-Cancer Therapy. International Journal of Molecular Sciences, 15(3), 3373-3388.

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