Acquity arc

Manufactured by Waters Corporation

Sourced in United States

The Acquity Arc is a high-performance liquid chromatography (HPLC) system designed for a wide range of analytical applications. It features a modular design and advanced technology to provide reliable and consistent performance in the laboratory.

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16 protocols using acquity arc

Molecular Weight Estimation by SEC

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Molecular
weights (MWs) were estimated by size exclusion chromatography (SEC)
on an Acquity Arc equipped with a 2998PDA Detector, a Sample Manager
FTN-R, and a Quaternary Solvent Manager-R (Acquity Arc, Waters Corporation,
Milford, MA, USA) with a XBridge TM Protein BEH SEC 200 Å 2.5
μm 4.6 mm × 150 mm column. The standard proteins (Waters
Corporation, Milford, MA, USA) were used to calibrate the column:
uracil (0.112 kDa), ribonuclease A (13.7 kDa), albumin chicken egg
white (44.2 kDa), and thyroglobulin bovine (669 kDa).^{3 (link)} A volume of 10 μL dissolved in 100 mM sodium phosphate
buffer and 0.02% (w/v) sodium azide adjusted at pH 6.8 at a concentration
of 1 mg/mL was injected. Protein elution was recorded by measuring
its absorbance at 280 nm and analyzed with Empower 3 Personal GPC/SEC
software (Waters Corporation, Milford, MA, USA).

Villanueva A., Rivero-Pino F., Martin M.E., Gonzalez-de la Rosa T., Montserrat-de la Paz S, & Millan-Linares M.C. (2024). Identification of the Bioavailable Peptidome of Chia Protein Hydrolysate and the In Silico Evaluation of Its Antioxidant and ACE Inhibitory Potential. Journal of Agricultural and Food Chemistry, 72(6), 3189-3199.

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Synthesis and Characterization of Rupestonic Acid Derivatives

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All chemicals were purchased from commercial suppliers and used without further purification. The purity of rupestonic acid and M5-0 were determined by high performance liquid chromatography (Waters Acquity Arc), and purity of other synthesized compounds were confirmed by thin-layer chromatography (TLC) using several solvent systems of different polarities. ¹H NMR, ¹³C NMR were measured on a Bruker AVANCE II 500 MHZ. Digital NMR Spectrometer with tetramethyl silane (MS) as internal standard and DMSO-d₆ as diluent. Mass spectra were measured on a Waters UPLC-MS. UV-vis spectra were recorded on a UV-2600i (A12595931269) at 25 °C. Melting points of compounds were measured by METTLER TOLEDO MP70 Melting Point System. Fluorescence measurements were carried out using an Edinburgh Instruments FLS1000 fluorescence spectrophotometer. Confocal fluorescence images were recorded using a Thermo Fisher Invitrogen™ EVOS™ FL Auto 2.

Cui Z., Zhang Y., Zhang Z., Abudurexiti A, & Yusuf A. (2023). Synthesis of an aggregation-induced emission-based fluorescent probe based on rupestonic acid. RSC Advances, 13(36), 25369-25378.

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HPLC Quantification of BCL-Loaded PLGA Microspheres

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The EE% and DL% of BCL-loaded PLGA microspheres were analyzed by high-performance liquid chromatography (HPLC, Acquity Arc, Waters Co., Ltd., Milford, MA). Briefly, a known mass of BCL-loaded PLGA microspheres was dissolved in THF. The sample was filtered through a 0.22 μm filter (Jinteng Laboratory Equipment Co., Ltd., Tianjin, China) after ultrasonic breakdown and dissolution and then injected into an HPLC system to determine the concentration of BCL. The determination was performed on a Kromasil C18 column (250 mm × 4.60 mm, Nouryon, Amsterdam, Netherlands) with the column temperature maintained at 30 °C. The mobile phase was 0.1% potassium dihydrogen phosphate aqueous solution/acetonitrile (47:53, v/v) and was delivered at a flow rate of 1 mL/min. The detection wavelength was 270 nm and the injection volume was 10 μL. All measurements were conducted in triplicate and the results were expressed as mean ± SD. Finally, the DL and EE were calculated by the following equations:

DL (%) = \frac{w_{1}}{w_{2}} \times 100 % (2)

EE (%) = \frac{actual DL}{theoretical DL} \times 100 % (3)

where W₁ and W₂ represent the weight of the drug in microspheres and the weight of the whole microsphere, respectively. Results are expressed as mean ± SD (n = 3).

Su Y., Liu J., Tan S., Liu W., Wang R, & Chen C. (2022). PLGA sustained-release microspheres loaded with an insoluble small-molecule drug: microfluidic-based preparation, optimization, characterization, and evaluation in vitro and in vivo. Drug Delivery, 29(1), 1437-1446.

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Amino Acid Composition Analysis

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Amino acid composition was determined according to the method of Alaiz et al. [33 (link)] with slight modifications. In this sense, samples were hydrolyzed by incubation in 6 N HCl at 110 °C for 24 h in tubes sealed under nitrogen. Amino acids were determined in the acid hydrolysate by ultra-high-performance liquid chromatography (Acquity Arc, Waters, USA), after derivatization with diethyl ethoxymethylenemalonate, using D,L-α-aminobutyric acid as internal standard, and a 3 mm × 150 mm reversed-phase column (XSelect HSS T3 XP, 2.5 μm; Waters). A binary gradient system with the solvents (A) 25 mM sodium acetate 0.02% sodium azide (pH 6.0), and (B) acetonitrile was used. Calibration curves for each amino acid were developed using a mix of amino acid standard at the same hydrolysis conditions of the samples (Merck, Madrid, Spain) and the resultant peaks were analyzed with EMPOWER software (Waters, Santa Clara, CA, USA). Besides, tryptophan content was assessed according to the Yust et al. method [34 (link)].

la Paz S.M., Martinez-Lopez A., Villanueva-Lazo A., Pedroche J., Millan F, & Millan-Linares M.C. (2021). Identification and Characterization of Novel Antioxidant Protein Hydrolysates from Kiwicha (Amaranthus caudatus L.). Antioxidants, 10(5), 645.

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HPLC Analysis of Phenolic and Anthocyanin Compounds

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The methodology was carried out as reported by Alcázar-Valle et al. [8 (link)] with minor modifications. Extracts and fractions were diluted into ethanol (1% trifluoroacetic acid (TFA)) and refrigerated at 4 °C until analysis. PDA Detector (Acquity Arc, Waters, Milford, MA, USA) was used for HPLC analysis. Starting 90% A from 0–10, 82% A from 10–18 min, 72% from 18–19 min, 60% A from 19,239 min, and 90% A from 23–25 min were gradient conditions. Acidified water (0.1% TFA) was used as solvent A and acetonitrile (0.1% TFA) was used as solvent B. Flow rate was 0.7 mL/min. C18, 2.7 μm, 4.6 × 150 mm (Cortects, Waters, Milford, MA, USA) column was used. The oven was set at 30 °C, and 20 μL of the sample was injected. Phenolic compounds were quantified and analyzed at 280 nm, and Anthocyanin compounds at 520 nm. Standard curves were based on a range from 1 to 40 mg/L and prepared from pure standards. Results were expressed as mg/L of each phenolic compound or anthocyanin.

Contreras J., Alcázar-Valle M., Lugo-Cervantes E., Luna-Vital D.A, & Mojica L. (2023). Mexican Native Black Bean Anthocyanin-Rich Extracts Modulate Biological Markers Associated with Inflammation. Pharmaceuticals, 16(6), 874.

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HPLC Detection of Ochratoxin A

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An HPLC system (ACQUITY, Arc, Waters, Milford, MA, USA) with a fluorescent detector and C₁₈ column (150 mm × 4.6 mm; Particle size: 5 μm) was used for OTA detection. The column temperature was 35 °C. The mobile phase consisted of acetonitrile–water–glacial acetic acid (96:102:2, v/v/v). The flow rate was 1.0 mL min⁻¹. The injection volume was 50 μL. The fluorescence excitation wavelength (λem) was 333 nm, and emission wavelength (λex) was 460 nm. OTA standard solutions of 0, 0.2, 1, 2, 5, 10, and 20 ng mL⁻¹ were used to establish the standard curve, and the results were analyzed by the external standard method.

Ren Y., Tian R., Wang T., Cao J., Li J, & Deng A. (2023). An Extremely Highly Sensitive ELISA in pg mL−1 Level Based on a Newly Produced Monoclonal Antibody for the Detection of Ochratoxin A in Food Samples. Molecules, 28(15), 5743.

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Quantification of Soy-Deprestatin in Enzymatic Digest

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SPD (soy protein enzymatic digest) was prepared as follows; soy protein isolate (SPI, SUPRO 661, Koyo Mercantile) was digested with thermolysin (THERMOASE PC10F, Amano Enzyme) at 60 °C, and then dried to powder. SPD was also available for purchase from UHA Mikakuto as SOYLAX®.
The amount of soy-deprestatin in SPD was measured as follows. SPD powder dissolved in 0.1% polypeptone was analyzed by injecting 10 μL into LC/MS/MS. Analyses were performed using Acquity Arc (Waters) for liquid chromatography (LC), 3200 Q Trap (ABsciex) for mass spectrometry (MS), and CAPCELL PAK C18 UG80 (φ2.0 × 150 mm, 5 μm; Osaka soda) for columns. Separation was performed using 0.1% formic acid - 0.1% formic acid/acetonitrile at a flow rate of 0.2 mL/min, under 0–30% (0–15 min), 30% (15–20 min), and 30–100% (20–25 min) gradient conditions. MS was performed by using the positive ion mode of electrospray ionization. The monitored precursor/fragment ions were m/z 1113.4/101.2 for soy-deprestatin. MS conditions were as follows: curtain gas, 20 psi; collision gas, 3; ion spray voltage, 5000 V, temperature, 600 °C; ion source gas 1, 30 psi; ion source gas 2, 30 psi. Quantitative measurements of soy-deprestatin were performed by creating a calibration curve using synthetic peptides (LSSTQAQQSY; Genscript).

Yasuda T, & Kashima Y. (2024). A soy protein enzymatic digest mitigates Nrf2-related oxidative stress and attenuates depression-like behavior in a mouse model of sub-chronic restraint stress. Heliyon, 10(6), e27826.

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Phytochemical Profiling of Bean Flour

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Bean flour was weighed at 0.5 g and extracted into 10 mL of ethanol with 1% trifluoroacetic acid (TFA) and refrigerated at 4 °C for 24 h. Then, the samples were centrifuged (3,000 rpm) for 5 min, and the supernatant was collected. Finally, the residue was washed once and centrifuged under the same conditions; both supernatants were mixed for analysis by liquid chromatography (UHPLC).
UHPLC analysis was performed with a PDA Detector (Acquity Arc, Waters, Milford, MA, USA), and the gradient conditions started with 90% solvent A, followed by 82% A at 10 min, 72% A at 18 min, 60% A at 19 min, and 90% A at 23 min. Solvent A was water with 0.1% TFA, and solvent B was acetonitrile with 0.1% TFA, with a flow rate of 0.7 mL/min. The column used was a C18, 2.7 μm, 4.6 × 150 mm (Cortects, Waters, Milford, MA, USA), the oven was set at 30 °C, and the volume injected was 20 μL. The compounds quantified in this study were detected at different wavelengths; ferulic acid was analyzed at 250 nm, kaempferol 3-glucoside, quercetin 3-glucoside, and genistein were analyzed at 300 nm, and cyanidin 3-glucoside was analyzed at 550 nm. All samples were injected in duplicate [24 (link)].

Alcázar-Valle M., Lugo-Cervantes E., Mojica L., Morales-Hernández N., Reyes-Ramírez H., Enríquez-Vara J.N, & García-Morales S. (2020). Bioactive Compounds, Antioxidant Activity, and Antinutritional Content of Legumes: A Comparison between Four Phaseolus Species. Molecules, 25(15), 3528.

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Phlorotannin Analysis of I. okamurae Extract

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Chromatographic analyses were conducted on an Acquity™ Arc equipped with a 2998 PDA detector and an Acquity™ QDa detector system (Waters Corporation, Beverly, MA, USA). The phlorotannin compounds in I. okamurae extract were analyzed using a Poroshell 120 EC-C18 column (4 µm, 4.6 mm × 100 mm; Agilent(Santa Clara, CA, USA)). The mobile phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile containing 0.1% formic acid. The HPLC elution condition consisted of an isocratic system of 32% B at a flow rate of 0.4 mL/min and an injected volume of 10 µL.

Kim M., Cho C., Lee C., Ryu B., Kim S., Hur J, & Lee S.H. (2021). Ishige okamurae Ameliorates Methylglyoxal-Induced Nephrotoxicity via Reducing Oxidative Stress, RAGE Protein Expression, and Modulating MAPK, Nrf2/ARE Signaling Pathway in Mouse Glomerular Mesangial Cells. Foods, 10(9), 2000.

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Enzymatic Hydrolysis of Cellulose: Optimizing pH and Additives

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Whatman filter paper was used as the representative of pure cellulose. The conditions of enzymatic hydrolysis of GL-pretreated substrate and pure cellulose were 2% (w/v) solid content in acetate buffer (0.05 M, pH 4.8), and hydrolyzed for 72 h under 50 °C at 150 rpm/min. In the experiments to explore the pH influence to the enzymatic hydrolysis in the presence of PSS, the buffer with the pH range of 4.0 to 7.0 was prepared by disodium hydrogen phosphate and citric acid. The cellulase dosage (CTec2) was 6.3 FPU/g-glucan for Whatman paper and 13.3 FPU/g-glucan for GL-P. LS and PSS were added to the enzymatic hydrolysis system at the designed amount. After enzymatic hydrolysis for 3, 6, 9, 12, 24, 36, 48, 72 h, enzymatic hydrolysate (1 mL) was extracted and boiled for 10 min, then the supernatant was collected and diluted 5 times to analyze the substrate enzymatic digestibility (SED) or the glucose yield using high-performance liquid chromatography (HPLC, ACQUITY Arc, Waters, USA) equipped with the analytical column BIO-RAD Aminex HPX-87H Column (300 × 7.8 mm) and the guard column CationH Refill Cartridges (30 × 4.6 mm). The test was operated at 55 °C with 5 mM H₂SO₄ as mobile phase at the flow rate of 0.6 mL/min.

Liu T., Wang P., Tian J., Guo J., Zhu W., Jin Y., Xiao H, & Song J. (2022). Polystyrene sulfonate is effective for enhancing biomass enzymatic saccharification under green liquor pretreatment in bioenergy poplar. Biotechnology for Biofuels and Bioproducts, 15, 10.

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