Catechins, theanine and caffeine were analyzed on a Waters High Performance Liquid Chromatography (HPLC) system equipped with Waters 600 controller and Waters 2489 UV Detector (280 nm). The Empower TM 3 software was used for instrument operation control and data collection. Chromatographic separation was performed on a Gemini 5u C18 110A column (250×4.60 mm, 5 μm, Phenomenex Inc., Torrance, CA), with a solvent system consisting of 0.17% (v/v) acetic acid (A) and 100% acetonitrile (B); a linear gradient at a flow rate of 1.0 mL/min was set as follows: B from 8 to 28% (v/v) in 30 min was initiated, followed by B from 28 to 100% (v/v) between 30 and 37 min, and B from 100 to 8% (v/v) between 37 and 46 min. Peaks were identified by comparison of retention times with those of standards49 (link). Total sugar content of tea infusion was determined by the anthrone-sulfuric acid method50 (link). Polysaccharides were isolated from tea infusion using an Amicon Ultra-0.5 Centrifugal Filter Device (UFS 503024, 3,000 Dalton cutoff).
600 controller
Manufactured by Waters Corporation
Sourced in United States
The 600 controller is a device used to control and monitor various laboratory equipment. It provides a user interface to configure and manage the operations of the connected equipment.
Automatically generated - may contain errors
Lab products found in correlation
717 plus autosampler, by Waters Corporation (4 mentions)
2996 photodiode array detector, by Waters Corporation (3 mentions)
717 autosampler, by Waters Corporation (3 mentions)
High performance liquid chromatography system, by Waters Corporation (2 mentions)
717 plus, by Waters Corporation (2 mentions)
2489 uv detector, by Waters Corporation (1 mentions)
Gemini 5u c18 110a column, by Phenomenex (1 mentions)
α tocopherol, by Merck Group (1 mentions)
Hplc system, by Waters Corporation (1 mentions)
486 tunable absorbance detector, by Waters Corporation (1 mentions)
Delta 600 pump, by Waters Corporation (1 mentions)
Symmetry c18 column, by Waters Corporation (1 mentions)
Flexanalysis, by Bruker (1 mentions)
Flexcontrol software, by Bruker (1 mentions)
Microflex, by Bruker (1 mentions)
Hplc grade solvent, by Merck Group (1 mentions)
Liquid chromatograph, by Waters Corporation (1 mentions)
20 l loop, by Waters Corporation (1 mentions)
Empowered 2, by Waters Corporation (1 mentions)
J sphere ods h80 column, by YMC (1 mentions)
26 protocols using 600 controller
1
HPLC Analysis of Tea Bioactive Compounds
2
HPLC Analysis of α-Tocopherol in Oils
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Samples of 0.1 g oil were dissolved in mL acetone, shaken, and filtered through –4 m syringe cellulose filters. An aliquot of 20 L of this solution was injected onto the HPLC system (Waters, Milford, MA, USA), which was equipped with a pump unit (600 Controller; Waters) and an auto-sampler (717 plus; Waters). The chromatography column (Spherisorb ODS2; 250 × × 5 m) was kept at 25 °C, with a pre-column used (Phenomenex cartridge, Torrance, CA, USA, C18 AJO-4287). The mobile phase was acetonitrile and methanol (1:1; v/v) at a flow rate of 1 mL/min. -Tocopherol was detected using a photodiode array detector (996; Waters) at a wavelength of 295 nm, using a run time of 18 min. An Enpower 2 Work Station was used for the data processing. The -tocopherol concentrations were initially in mg/L based on the calibration curve, with -tocopherol (Sigma-Aldrich) as the external standard. From the oil weight in the 2 mL sample (see above), the -tocopherol was finally expressed as mg per kg oil (mg/kg).
3
HPLC Quantification of Serum Betaine
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Serum betaine (trimethylglycine) levels were measured using high-performance liquid chromatography (HPLC) as described42 (link). Serum samples (50 μL) were mixed with an equal volume of 100 mM KH2PO4 in screw-top microcentrifuge tubes, then derivatization solution (900 μL; 12.5 mM 18-crown-6, 50 mM 4-bromophenacyl bromide in acetonitrile) was added. The tubes were vacuum-packed in plastic bags and placed in a water bath at 80 °C for 1 h. After cooling to room temperature, the samples were centrifuged at 1000×g. Supernatants (15 μL) were directly injected into the HPLC comprising a 600 Controller (Waters Corp., Milford, MA, USA), a 486 Tunable Absorbance Detector (Waters Corp.), Supelcosil™ LC-SCX HPLC column, particle size 5 μm, 25 cm × 4.6 mm) (Sigma-Aldrich, St. Louis, MO, US), mobile phase, acetonitrile:ultra-pure-water (9:1) containing 22 mM choline, at a flow rate of 1.5 mL/min.
4
HPLC analysis of botanical extracts
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HPLC analysis was carried out on a Waters 600 Controller equipped with Waters 996 photodiode array (PDA) detector using a Novapak C18 column (4.6 × 250 mm, 5 µm) included with 717-Autosampler & Inline degasser. The mobile phase consisted of solvent A, 0.1 % trifluoroacetic acid in acetonitrile and B, 0.1 % trifluoroacetic acid in water and performed gradient elution. The flow rate was 1 mL/min and chromatogram extracted at 290 nm. The mobile phase consists of solvent A, acetonitrile (ACN) and B, 0.4 % formic acid in water. Gradient elution was carried out starting from 95 to 90 % of B in 5 min, from 90 to 85 % of B in 3 min, from 85 to 80 % of A in 2 min, from 80 to 78 % of B in 2 min, from 78 to 76 % of B in 1 min, from 76 to 74 % of B in 1 min, from 74 to 73 % of B in 2 min, from 73 to 72 % of B in 2 min, from 72 to 65 % of B in 1 min, from 65 to 50 % of B in 3 min and finally from 50 to 40 % of B in 2 min. Fully dried MPE (5 mg/mL w/v) and 1, 2, 3, 9 and 10 (1 mg/mL w/v) were dissolved in HPLC grade MeOH and then filtered through 0.22 µm membrane filter.
5
Preparative Purification of Peptides
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Purifications
were performed on a system composed of an automatic gradient controller
(Waters 600 Controller), a quaternary pump (Waters Delta 600 Pump),
a UV detector (Waters 2487 Dual λ Absorbance), a manual sample
injector (Rheodyne 3725i-119), and a register (Kipp & Zonen recorder
124 SE) connected to a preparative C18 column (10 μm,
300 Å, 2.2 × 25.0 cm2, Vydac). The conditions
employed are listed inTable S1 . The percentages
of ACN in solvents B and the linear gradients chosen are related to
the nature of the peptides analyzed.
were performed on a system composed of an automatic gradient controller
(Waters 600 Controller), a quaternary pump (Waters Delta 600 Pump),
a UV detector (Waters 2487 Dual λ Absorbance), a manual sample
injector (Rheodyne 3725i-119), and a register (Kipp & Zonen recorder
124 SE) connected to a preparative C18 column (10 μm,
300 Å, 2.2 × 25.0 cm2, Vydac). The conditions
employed are listed in
of ACN in solvents B and the linear gradients chosen are related to
the nature of the peptides analyzed.
6
Peptide Purity Determination by RP-HPLC and MALDI-TOF-MS
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Peptide purity was determined as described previously [29 (link)] by analytical RP-HPLC using a Waters 717 plus Autosampler, In-line Degasser AF, 600 Controller, and 2996 Photodiode Array Detector; the column used was a Waters Symmetry C18 column (5 μm, 4.6 mm × 250 mm, Milford, MA, USA). An aqueous acetonitrile (ACN) gradient with 0.1% TFA added (mixing eluent A: H2O + 0.1% TFA and eluent B: 90:10 ACN-H2O + 0.1% TFA) was employed using milli-Q water. Preparative RP-HPLC was performed by using a Waters XSelect Peptide CSH C18 OBDTM column (5 μm, 19 mm × 250 mm, Milford, MA, USA) with the same eluents as for analytical HPLC.
MicroflexTM (Bruker Corporation, Bremen, Germany) equipped with FlexControl software (Bruker Daltonik GmbH, Bremen, Germany) was used to obtain the MALDI-TOF-MS spectra, and the data were processed using flexAnalysis (Bruker Daltonik GmbH). All of the reagents and solvents were used without further purification.
MicroflexTM (Bruker Corporation, Bremen, Germany) equipped with FlexControl software (Bruker Daltonik GmbH, Bremen, Germany) was used to obtain the MALDI-TOF-MS spectra, and the data were processed using flexAnalysis (Bruker Daltonik GmbH). All of the reagents and solvents were used without further purification.
7
Measuring Chlorophyll-a in Seawater
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For measuring the photosynthetic pigment Chl-a one liter of seawater samples were taken from Niskin bottles, immediately filtered on GF/F filters, frozen in liquid nitrogen, and stored at -80°C until further analyses by high-performance liquid chromatography (HPLC) at the home laboratory of the Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research after the cruise. The samples were measured using a Waters 600 controller equipped with an auto sampler (717 plus), a photodiode array detector (2996) and the EMPOWER software. Chl-a was analyzed by reverse-phase HPLC using a VARIAN Microsorb-MV3 C8 column (4.6 3 100 mm) and HPLC-grade solvents (Merck). The solvents gradient and routine of analysis are fully described in Taylor et al. [54 ]. Chl-a concentrations were quantified based on peak area of the external standard, which was spectrophotometrically calibrated using extinction coefficients published in Jeffrey et al. [55 ].
8
Estimation of Withanolide Metabolites in Leaf Tissues
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The estimation of the three metabolites were performed on a Waters liquid chromatograph equipped with a Waters 600 controller, a Waters Delta 600 solvent delivery system, a Rheodyne 7125 sample injector fitted with a 20 µL loop, and a Waters 2996 Photodiode Array Detector, with Waters Empowered 2.154 software. A Supelco 516 C18 (4.6 mm×25 cm) reverse phase analytical column equipped with a Waters µBondapak C18 10 µm precolumn was used for estimation. The wavelength scan range of the PDA was set to 190–350 nm and the presence of withanosides-V, withaferin-A and withanolide-A was detected at 227 nm. A. The isocratic mobile phase consisted of 60% acetonitrile containing 0.1% acetic acid (solvent A) and 40% water containing 0.1% acetic acid (solvent-B) at a flow rate of 1.0 mL min−1. The metabolites were estimated in comparison to the external standards and the results were presented as µg mg−1 of dry weight of leaf tissue.
9
Quantitative Analysis of Retinal Lipids
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An aliquot of total lipids from neural retina was saponified (0.4 N KOH in 80% methanol, 50°C for 1 hour). Saponified fatty acids were acidified and extracted with diethyl ether according to Wang et al.17 (link) and stored in methanol containing 1 mM butylated hydroxytoluene. Saponified free fatty acids were fractionated and quantitated by RP-HPLC using a YMC J-Sphere (ODS-H80) column (Allentown, PA, USA) and a sigmoidal gradient starting at 86.5% acetonitrile + acetic acid (0.1%) (Sigma-Aldrich Corp.) and ending at 100% acetonitrile + acetic acid (0.1%) over 50 minutes with a flow rate of 1.0 mL/min using a Waters Corporation 600 controller (Milford, MA, USA). Fatty acids were introduced to the HPLC by injection in methanol and detected using ultraviolet absorbance and evaporative light scatter as previously described.17 (link) Authentic fatty acid standards (Nu-Chek Prep, NIC, Elysian, MN, USA) were used to generate calibration curves for verification and quantification of fatty acids.
10
Amino Acid Analysis by RP-HPLC
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The concentration of amino acids was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) following the methodology of Enrione et al. (2020) [20 (link)] with modifications. The amino acids were separated and quantified using a chromatograph (Waters 600 controller, Milford, MA, USA) with a diode array detector (Waters 996) using an RP18 column (150 mm × 4.6 mm, particle size 5 µm) (Luna, Phenomenex, Los Angeles, CA, USA). The elution of compounds was achieved using a gradient elution method with two mobile phases. Mobile phase A consisted of an acetate buffer adjusted to pH 5.8, while mobile phase B comprised acetonitrile: water (60:40). The oven temperature was maintained at 40 °C, and the flow rate was set at 1.5 mL/min. A sample volume of 20 µL was injected for analysis. The amino acid content was reported as g/100 g of protein.
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