An overnight culture was divided into three aliquots, mixed thoroughly, and 10 µL was used to inoculate MM containing 10 μg/mL ZEN toxin as the experimental group. Pure MM without the culture broth (containing ZEN) was used as the control. After inoculation, the samples were incubated in a shaking incubator at 37 °C and 200 rpm for 12 h. At 4 h intervals, 0.5 mL of the culture solution was taken and combined with 0.5 mL of cold methanol. All samples were filtered through an organic filter membrane with a pore size of 0.22 µm. ZEN standard was dissolved in methanol, and the concentrations of ZEN standards were 0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL, respectively. Then, the concentration of ZEN was analyzed using HPLC (Agilent 1200, UV detector). As described in previous study [52 (link)], The HPLC consisted of an Agilent 1200 autosampler, an Agilent 1200 UV detector and an Agilent 1200 pump. An Agilent Eclipse XDB-C18 column (4.6 mm × 150 mm, 5 m) was used for chromatographic separation and the detection UV wavelength was 254 nm. The mobile phase was acetonitrile–water 60:40 (v/v) with a flow rate of 1 mL/min. the column was kept at 30 °C, and the injection volume was 20 μL.
Agilent 1200
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The Agilent 1200 is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It features a modular design, enabling customization to specific laboratory needs. The Agilent 1200 provides reliable and efficient separation of complex mixtures, with precise control over flow rates, temperatures, and other parameters.
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Lab products found in correlation
Eclipse xdb c18 column, by Agilent Technologies (13 mentions)
Chemstation software, by Agilent Technologies (11 mentions)
Agilent 1100, by Agilent Technologies (10 mentions)
Zorbax sb c18, by Agilent Technologies (7 mentions)
Zorbax eclipse xdb c18, by Agilent Technologies (7 mentions)
Zorbax eclipse xdb c18 column, by Agilent Technologies (6 mentions)
Capcell pak mg 2 c18 column, by Shiseido (5 mentions)
Double sector jms ax505ha mass spectrometer, by JEOL (5 mentions)
Eclipse xdb c18, by Agilent Technologies (5 mentions)
Zorbax sb c18 column, by Agilent Technologies (4 mentions)
Symmetry shield rp18 column, by Waters Corporation (4 mentions)
300 instrument, by Bruker (4 mentions)
Heleos 2 with qels capability, by Wyatt Technology (4 mentions)
C18 column, by Agilent Technologies (4 mentions)
Pack pro c18, by YMC (3 mentions)
Proteinase k, by New England Biolabs (3 mentions)
Prism software, by GraphPad (3 mentions)
Agilent 6410, by Agilent Technologies (3 mentions)
Kinetex evo c18 column, by Phenomenex (3 mentions)
Chemidoc recorder, by Bio-Rad (3 mentions)
622 protocols using agilent 1200
1
Analyzing Zearalenone Toxin Levels
2
Quantification of Caffeine and Labeled Compounds in Acceptor Phases
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The concentration of caffeine (0.2 kDa) in the samples of the acceptor phase was assessed using HPLC (Agilent 1200 equipped with UV/VIS and FLD detectors, Agilent, USA). The amount of caffeine was determined using a Zorbax Eclipse plus C18 column (250 x 4.6 mm, 5 μm) and methanol:0.2% aqueous solution of formic acid 1:3 (v/v) as the mobile phase with the flow rate 1.5 mL/min. The UV/VIS detector wavelength was set at 272 nm. The retention time of caffeine peak was 5.75 min. Spectrofluorimetric quantification of fluorescently labelled dextrans (FD4, FD20, FD40 and FD70) in samples of the acceptor phase was carried out on Aminco Bowman Series 2 (ThermoFisher, USA). The excitation wavelength was set at 419 nm and emission wavelength at 529 nm. The system was calibrated using the individual dextran standards dissolved in acceptor buffer pH 7.4. Spectrofluorometric quantification of fluorescently labelled bovine serine albumin FITC-BSA (66 kDa) in samples of the acceptor phase was carried out on Agilent 1200 HPLC with FLD detector using a column bypass (Agilent Technologies, USA). The excitation wavelength was set at 495 nm and emission wavelength at 523 nm. The system was calibrated using standard FITC-BSA solutions in acceptor buffer pH 7.4.
All HPLC data were analysed using Agilent ChemStation software Rev.C.01.06 (Agilent Technologies, USA).
All HPLC data were analysed using Agilent ChemStation software Rev.C.01.06 (Agilent Technologies, USA).
3
Measuring Serum Vitamin D, Retinol, and Tocopherol
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Serum 25-hydroxyvitamin D (25(OH)D) levels were determined using a 1470 Wizard gamma counter (Perkin Elmer, Turku, Finland) and a radioimmunoassay (RIA) (DiaSorin, Stillwater, MN, USA). Vitamin D deficiency was defined as a serum 25 (OH)D concentration of <50 nmol/L. Serum retinol (mg/L) and serum α-tocopherol (mg/L) levels were measured with an Agilent1200 (Agilent1200; Agilent, Santa Clara, CA, USA) using Chromsystems (Chromsystems; Chromsystems Instruments & Chemicals, Munich, Germany) reagents. The reference normal ranges for serum retinol and α-tocopherol in adults are 0.30 to 0.70 mg/L and 5.00 to 20.00 mg/L, respectively [20 (link)].
4
Enantioselective HPLC Analysis of Chiral Compounds
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High performance liquid chromatography (HPLC) experiments were carried out on an Agilent 1200 high-performance liquid chromatograph (Agilent Technology, Santa Clara, CA, USA) equipped with a G1322A degasser, a G1310A isocratic pump, a G1314B UV detector set at 225 nm, a G1328B manual injector of 20 μL and an Agilent 1200 chemical analytical workstation. Chromatographic separation was conducted at 25 °C, and the flow rate was maintained at 1.0 mL/min. Chiral analysis was conducted according to the enantioselective HPLC method CIPAC/4907 provided from Bayer, using a Chiralcel OD-H column (Daicel Chemical Industries Ltd. Japan, 250 × 4.6 mm i.d., 5 μm particle size), protected with a guard column of the same phase. A mixture of n-hexane/isopropanol (95:5 v/v) was used as eluent, whereas the samples were dissolved in n-hexane [39 (link)].
5
Amino Acid Digestibility Determination
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Feed samples and terminal ileal digesta samples (0.5 g) were accurately weighed and put into an ampere tube, 10 ml of 6 M hydrochloric acid was added. The tube was sealed with an alcohol torch, hydrolyzed at 110 ± 2°C for 24 h, and then transferred to a 100 ml volumetric flask after cooling. Took a constant volume of 1–25 ml from the above solution. Then filtered into the injection flask with a 0.22 μm membrane. The AAs content was determined by high-performance liquid chromatography (Agilent 1200, Agilent Technologies, USA).
The feed samples and ileal digesta after freeze-drying were weighed in parallel samples for analysis and determination. The AA profiles were detected by high-performance liquid chromatography (Agilent 1200, Agilent Technologies, USA). Lysine and threonine were detected after hydrolyzing with 6 mol/L HCl at 105°C for 24 h. Methionine was analyzed as methionine sulfone after cold performic acid oxidation overnight before hydrolysis. Tryptophan was determined after hydrolyzing with 4 mol/L LiOH at 110°C for 20 h. The apparent ileal digestibility (AID) of AAs was calculated using the following formula:
Where (Diet component/Chromium) d = ratio of diet component to Chromium in the diet and (Diet component/ Chromium) i = ratio of diet component to Chromium in the ileal digesta (20 (link), 21 (link)).
The feed samples and ileal digesta after freeze-drying were weighed in parallel samples for analysis and determination. The AA profiles were detected by high-performance liquid chromatography (Agilent 1200, Agilent Technologies, USA). Lysine and threonine were detected after hydrolyzing with 6 mol/L HCl at 105°C for 24 h. Methionine was analyzed as methionine sulfone after cold performic acid oxidation overnight before hydrolysis. Tryptophan was determined after hydrolyzing with 4 mol/L LiOH at 110°C for 20 h. The apparent ileal digestibility (AID) of AAs was calculated using the following formula:
Where (Diet component/Chromium) d = ratio of diet component to Chromium in the diet and (Diet component/ Chromium) i = ratio of diet component to Chromium in the ileal digesta (20 (link), 21 (link)).
6
Comparative Venom Profiling by HPLC
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The analysis was conducted using 200 μg of the adult pooled venoms of MCa-PV and FCp-PV and the individual hybrid samples MH1, MH4, MH6, MH7, and FH3 using a C18 column (150 × 4.6 mm; particle size: 3.5 μm) equilibrated with solution A (0.1% trifluoroacetic acid TFA in H2O) using an Agilent 1200 chromatograph (Agilent 1200, Agilent Technologies, CA, USA). Elution was performed at 1 mL/min using a gradient toward solution B (acetonitrile ACN and 0.1% of TFA) as follows: 0% B by 5 min, 0–60% B over 60 min. Absorbance was monitored at 280 nm.
7
Quantification of Organic Acids and Catechins in Miang Samples
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Eight major organic acids, including glucuronic acid, tartaric acid, lactic acid, acetic acid, citric acid, succinic acid, gallic acid, and butyric acid, in the Miang sample were determined using the method of Han et al. (2019) (link) with slight modifications. Briefly, the analysis was conducted using an Agilent 1200 reversed-phase HPLC coupled with a UV detector equipped with Luna Omega Polar C18 UHPLC column (150 mm × 3.0 mm, 1.6-μm particle diameters, Phenomenex). The column was operated using 20 mM KH2PO4 as a mobile phase with a flow rate of 0.6 ml/min. The UV detection wavelength was set at 210 and 254 nm and the barbituric acid was used as the internal standard. All samples were analyzed in triplicate. The contents of extracted catechins and related compounds from Miang samples were analyzed by a reversed-phase HPLC using Agilent 1200 equipped with the Symmetry Shield RP18 column (4.6 mm × 250 mm, 5-μm particle diameters, Water Co., Ltd.). The column was operated at a flow rate of 1.0 ml/min using 10% acetonitrile in 0.1% acetic acid and H2O as mobile phase. The peaks were detected using a UV detector at 210 and 270 nm. All samples were measured in triplicate.
8
Quantitative Analysis of Paeoniflorin and Salvianolic Acid B in CALG
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According to the results of a pre-extraction experiment, extraction technology, and preparation study, paeoniflorin, and salvianolic acid B were selected as the detection indexes. The content measurement experiment was performed by weighing 0.3 g of compound, dissolving it in mobile phase (Acetonitrile-0.1% phosphoric acid solution (14:86) for paeoniflorin, Methanol-water (8:2) for salvianolic acid B), filtering, and using the filtrate as the test solution. An appropriate amount of reference substance was dissolved with the mobile phase, shaken, and used as the reference substance solution. According to high-performance liquid chromatography (HPLC) (General rule 0512), 10 µL of each solution was absorbed and injected into the liquid chromatography column for determination. The content in CALG was determined using an Agilent 1200, MWD detector, Agilent 1200 workstation, and chromatographic column (waters T3, 4.6 mm × 250 mm, 5μm). The following parameters were used: filler, octadecyl silane bonded silica gel; mobile phase, 0.1% acetonitrile 1% phosphoric acid solution (14:86); detection wavelength, 230 mm.
9
Measuring Bacterial Growth and Metabolites
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Bacterial cell growth was monitored by measuring the optical density at 600 nm (OD600) with a spectrophotometer (AOE instruments A380, Shanghai, China).
The concentration of GABA in the culture broth was analyzed by high performance liquid chromatography (HPLC) with the o-phthaldialdehyde derivatization method [44 (link)]. Cell-free supernatant was filtered through a 0.45 μm membrane filter (Millipore, USA). GABA was detected by using the pre-column derivatization of Agilent HPLC system (Agilent 1200, USA) equipped with Agilent Zorbax Eclipse AAA column (Agilent, USA) according to the manufacturer’s instructions. GABA concentration was calculated from the integrated peak area comparing with standard curve constructed using GABA standard (Sigma, Aldrich Co., St. Louis, MO, USA).
The concentration of glucose in the culture broth was analyzed by HPLC (Agilent 1200, USA) system equipped with Aminex HPX-87H column (300 × 7.8 mm; BioRad) [45 (link)]. Samples were eluted by 5 mM H2SO4 with a flow of 0.60 mL/min and detected by refractive index detector. The temperature of the column was maintained at 60 °C.
The concentration of GABA in the culture broth was analyzed by high performance liquid chromatography (HPLC) with the o-phthaldialdehyde derivatization method [44 (link)]. Cell-free supernatant was filtered through a 0.45 μm membrane filter (Millipore, USA). GABA was detected by using the pre-column derivatization of Agilent HPLC system (Agilent 1200, USA) equipped with Agilent Zorbax Eclipse AAA column (Agilent, USA) according to the manufacturer’s instructions. GABA concentration was calculated from the integrated peak area comparing with standard curve constructed using GABA standard (Sigma, Aldrich Co., St. Louis, MO, USA).
The concentration of glucose in the culture broth was analyzed by HPLC (Agilent 1200, USA) system equipped with Aminex HPX-87H column (300 × 7.8 mm; BioRad) [45 (link)]. Samples were eluted by 5 mM H2SO4 with a flow of 0.60 mL/min and detected by refractive index detector. The temperature of the column was maintained at 60 °C.
10
Chromatographic Profiling of Herbal Extracts
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Valerian rhizome with root (FS.2.5.0009.15), pe ppermint leaves (FS.2.5.0029.15), hops cones (FS.2.5. 0046.15), buckbean leaves (State Pharmacopoeia XI), ethanol 40.70, 95% (State Pharmacopoeia X, GOST), and pure water (FS 42-0324-09) were used in the study. The test object was separated by reverse phase HPLC.
Chromatographic studies were carried out on a device (Agilent Technologies 1200 Infinity) with an automated sample selector Agilent 1200, vacuum microdegasifier, gradient pump, and a thermostat of the same series, and diode matrix spectrophotometric detector Agilent 1200 (λ=190-195 nm) with a 2-nm scanning step. Spectral data and chromatograms were recorded and processed using Agilent Chem Station software.
Chromatographic column Supelco Ascentis express C 18 2.7 µ×100 mm×4.6 mm was used; mobile phase velocity 0.5 ml/min, temperature 35 o C, sample volume 1 µl. Mobile phase: 1.0% water solution of formic acid (A) -95% ethanol (B) in the gradient elution mode. The mobile phase composition varied from 90 to 10% phase A over 40 min [6] . Flavonoids were detected at λ=350 nm, hydroxycinnamic acids and flavones at λ=325 nm, and chalcones at λ=370 nm.
Chromatographic studies were carried out on a device (Agilent Technologies 1200 Infinity) with an automated sample selector Agilent 1200, vacuum microdegasifier, gradient pump, and a thermostat of the same series, and diode matrix spectrophotometric detector Agilent 1200 (λ=190-195 nm) with a 2-nm scanning step. Spectral data and chromatograms were recorded and processed using Agilent Chem Station software.
Chromatographic column Supelco Ascentis express C 18 2.7 µ×100 mm×4.6 mm was used; mobile phase velocity 0.5 ml/min, temperature 35 o C, sample volume 1 µl. Mobile phase: 1.0% water solution of formic acid (A) -95% ethanol (B) in the gradient elution mode. The mobile phase composition varied from 90 to 10% phase A over 40 min [6] . Flavonoids were detected at λ=350 nm, hydroxycinnamic acids and flavones at λ=325 nm, and chalcones at λ=370 nm.
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