Five‐millimeter‐sized devices containing .1% wt/wt PPS (∼.017 mg) were prepared and release study was conducted similar to aforementioned protein release study procedure. The concentration of PPS in the samples was evaluated using liquid chromatography–mass spectrometry (LC–MS). For chromatographic separation, Waters alliance 2695 separation module (Waters Corporation, Milford, MA, USA) was used that was attached to Thermo biobasic 18; 150 × 2.1 mm; 5 u pore size column. Mobile phase used for separation was water (A) and methanol (B) containing .1% vol/vol formic acid. The samples were separated using a gradient elution of 5–98% B in the first 15 min, that was kept constant for the next 10 min and brought back to 5% B for a total run period of 30 min. Flow rate used in the study was .2 ml/min and 25 μl was injected into LC using an autosampler. For the MS analysis, Waters Micromass QTOF 2 (Waters Incorporation, Milford, MA, USA) using electrospray ionization (3.5 kV ionization) and Masslinx software was used. PPS concentration was determined from the area under the curve obtained from LC separation and corresponding ion current intensity obtained from MS analysis and based on this information, the release profile of PPS was determined and plotted as percent cumulative PPS release over time.
Waters alliance 2695 separation module
Manufactured by Waters Corporation
Sourced in United States, Japan
The Waters Alliance 2695 separation module is a high-performance liquid chromatography (HPLC) system designed for efficient separation and analysis of a wide range of samples. It features a modular design with independent components for sample management, solvent delivery, and data management, allowing for customized configuration to meet specific analytical needs.
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Lab products found in correlation
Waters 474 fluorescence detector, by Waters Corporation (1 mentions)
Waters 2414, by Waters Corporation (1 mentions)
Waters 1525 binary hplc pump, by Waters Corporation (1 mentions)
Jupiter c18 column, by Phenomenex (1 mentions)
μbondapack c18 column, by Waters Corporation (1 mentions)
Bds c18 column, by Thermo Fisher Scientific (1 mentions)
Potato dextrose agar (pda), by Waters Corporation (1 mentions)
Genesys 20, by Thermo Fisher Scientific (1 mentions)
2410 refractive index detector, by Waters Corporation (1 mentions)
2996 uv detector, by Waters Corporation (1 mentions)
Waters 2996 photodiode array detector, by Waters Corporation (1 mentions)
Tskgel super ods, by Tosoh (1 mentions)
Whatman, by Cytiva (1 mentions)
13 protocols using waters alliance 2695 separation module
1
PPS Release Evaluation from Micron-Sized Devices
2
Quantification of Total Homocysteine in Whole Blood
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Total homocysteine in whole blood sample was determined using an HPLC method as described previously with minor modifications [29 (link), 30 (link)]. Briefly, 1 ml EDTA blood samples were lysed by vigorously shaking for at least 10 s after adding 10 μl Nonidet P40 (pure) and 10 μl citric acid monohydrate (2.5 M). The lysates were then centrifuged at 10,000g for 3 minutes at room temperature. Following reduction of the sample with tri-n-butylphosphine, precipitation of protein with perchloric acid and derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, the samples were then analyzed using reversed-phase high-performance liquid chromatography (Waters Alliance 2695 separation module) followed by fluorescence detection (Waters 474 fluorescence detector) (Waters Corporation, Milford, MA).
3
HPLC Analysis of Sap and Juice Sugars
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Sugar profiling was performed using a high‐performance liquid chromatography (HPLC) comprised of Waters Alliance 2695 separation module (Waters Corporation) equipped with a refractive index detector (RID; Waters 2414 Corporation). Coconut sap, sugar palm, and sugarcane juices were diluted 10 times with deionised water and then filtered using a 0.45 µm nylon filter (Labserve). Twenty microliters of sample were injected into a LiChroCART® Single bond NH2 column (Merck) with dimensions of 250 mm × 4.6 mm, particle size of 5 µm. The temperature of the column was set at 40°C. The mobile phase consisted of HPLC‐grade acetonitrile and double‐distilled water (80:20, v/v ratio; Chang, Karim, Mohammed, & Ghazali, 2018 ). The sugars were separated isocratically at a flow rate of 1.5 ml/min. Standard curves were constructed based on sugar reference standards (fructose, glucose, and sucrose) by plotting peak area against various concentrations of each sugar (0%–5% w/v; Chang et al., 2018 ; Hunt, Jackson, Mortlock, & Kirk, 1977 ).
4
Quantification of Milk Macromolecules
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The oligosaccharide content of the BCF was estimated using High pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) as per Morrin et al. (2019a (link)). Samples were separated and quantified for lactose using an Aminex HPX 87C Carbohydrate column (30,067.8 mm) (Bio-Rad, UK) fixed ion resin column as per Morrin et al. (2019a (link)). Levels of β-lactoglobulin (β-lg) and α-lactalbumin (α-LA) were determined using a TSK G2000SW (300 × 7.5 mm) and a TSK G2000swxl column (300 × 7.8 mm, from Tosu Hass, Japan) linked in series and fitted to a Waters Alliance 2695 separation module (Waters Corporation, Milford, Mass, USA) as per Morrin et al. (2019a (link)). Levels of LF, immunoglobulin G (IgG) and A (IgA), were determined using ELISA quantification kits (Bethyl Laboratories, Inc. Cambridge Bioscience, Cambridge, UK) as per Morrin et al. (2019a (link)).
5
Peptide Purification and Quantification
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Synthesized peptides were purified with Semi-preparative HPLC (Waters 1525 Binary HPLC Pump with a 998 detector, Waters). The column used was a Jupiter C18 column (250 mm × 10 mm, 300 Å, Phenomenex, Torrance, CA, USA). Eluent A was H2O + 0.1% of TFA, eluent B was CH3CN + 0.1% TFA. The UV absorption spectrum was set at 214 nm, flow rate at 4 mL/min. Eluents gradients were optimised for each peptide. Fractions, that were manually collected, were dried and analysed with UPLC-MS analysis (as described in Section 3.5 ) to verify peptides purity. The quantification was performed with HPLC-UV analysis (Waters Alliance 2695 separation module equipped with a dual λ absorbance detector 2487, Waters) using a Jupiter 5 μm C18 (250 × 2.0 mm, Phenomenex). Eluent A was water with 0.1% TFA, eluent B was acetonitrile with 0.1%. For gradient elution, the following steps were applied: isocratic 90% A for 5 min, from 90% A to 40% A by linear gradient in 50 min plus washing step at 100% B and reconditioning. Flow rate was set at 0.20 mL/min, injection volume 10 μL, column temperature 35 °C. Detection was performed at 214 nm. Quantification was obtained applying the Lambert-Beer equation and calculating the ε as reported in literature [33 (link)].
6
HPLC Analysis of Seasoning Compounds
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The BA profile of seasonings was determined by HPLC analysis using a Waters Alliance 2695 Separation Module (Waters, Milford, MA, USA) with a photodiode array detector, a binary pump, and a vacuum degasser, based on the method published in our earlier paper [21 (link)]. Twenty microliters of sample solution was injected onto a C18 Supelco column (4.6 mm × 250 mm, i.d., 5 μm; Shiseido, Kyoto, Japan) thermostated at 35 °C. The mobile phase was 0.1 M ammonium acetate (solvent A) and acetonitrile (solvent B) with a flow rate of 0.5 mL/min, and the detection wavelength was 210 nm. The gradient elution procedure was as follows: 0 min, 35% A, 65% B; 5 min, 30% A, 70% B; 10 min, 19% A, 81% B; 15 min, 17.5% A, 82.5% B; 20 min, 100% B; 25 min, 35% A, 65% B; 35 min, 35% A, 65% B. Fig. 1 demonstrates the chromatograms of the standard and sample. The method validation was conducted as described previously [21 (link)], and the results are presented in Table S1 .Fig. 1 ![]()
HPLC chromatograms of standard (
7
Reversed-Phase HPLC Analysis Protocol
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The experiments of reverse-phase HPLC in this study were done on a Waters Alliance 2695 separation module (Waters) with a Hypersil base-deactivated silica (BDS) C18 column (Thermo Fisher Scientific) and μBondapack C18 column (Waters). Samples were injected into the column with an isocratic flow of 50 mM ammonium formate, pH 4.3, at 1 mL min−1. Quantification of the peak area was analyzed with Empower 3 data chromatography software (Waters).
8
Separation Module Development and Specificity
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The Waters Alliance 2695 Separation Module equipped with PDA (Waters Corporation, Milford, MA, USA) was used for development studies and specificity.
9
Quantifying Bacterial Growth Metrics
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Cell density was determined by plating onto MRS agar at 37°C for 48 h. Cell density was also monitored using either optical density at 620 nm (OD 620 ) with a Genesys 20 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA), or cell dry weight (CDW). For CDW determination, a known volume of fermentation broth was centrifuged for 10 min at 4°C and 3,000 ×g in pre-weighed tubes, washed once with water, and dried into a microwave oven at 600 W until stable weight was reached. CDW was routinely determined using a standard curve relating OD 620 values to CDW; the correlation factor was CDW (g/l) = 0.43 × OD 620 .
Concentrations of glucose, acetic and lactic acids, glycerol, 1,3-PDO, and ethanol were determined by HPLC analysis using a Waters Alliance 2695 separation module (Waters, Milford, MA, USA) equipped with a Rezex ROA-Organic Acid H+ (8%) 300 mm × 7.8 mm column (Phenomenex Inc., USA), coupled to a Waters 2410 refractive index detector and a Waters 2996 UV detector. Separation was carried out at 65°C with 0.005 M H 2 SO 4 as the mobile phase at a flow rate of 0.6 ml/min.
Concentrations of glucose, acetic and lactic acids, glycerol, 1,3-PDO, and ethanol were determined by HPLC analysis using a Waters Alliance 2695 separation module (Waters, Milford, MA, USA) equipped with a Rezex ROA-Organic Acid H+ (8%) 300 mm × 7.8 mm column (Phenomenex Inc., USA), coupled to a Waters 2410 refractive index detector and a Waters 2996 UV detector. Separation was carried out at 65°C with 0.005 M H 2 SO 4 as the mobile phase at a flow rate of 0.6 ml/min.
10
Quantification of Mepenzolate in Blood and Lung
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After administration of mepenzolate, blood samples (800 μl) were taken periodically into centrifuge tubes containing heparin (50 μl) and centrifuged immediately (1000 × g, 10 min) to obtain the sample. Whole lungs were taken from mepenzolate-treated mice, homogenised in sterile PBS containing 50 U/ml heparin, and centrifuged (14, 000 × g, 1 min) to obtain the sample. An aliquot (300 μl) of each sample was ultrafiltered with an Amicon utra-0.5 centrifugal filter to extract the mepenzolate. The filtrate was analysed by analytical HPLC with a reverse-phase column (TSKgel Super-ODS, 150 × 4.6 mm, 2 μm, Tosoh Co., Tokyo, Japan), Waters 2695 Alliance separation module, and a Waters 2996 photodiode array detector (Waters, Milford, MA). Solution containing 30% (v/v) acetonitrile and 14 mM potassium dihydrogen phosphate/sodium 1-propanesulfonate buffer was used at a flow rate of 0.3 ml/min. Detection was performed at an optical density of 220 nm.
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