Complete rat brain was dissected, washed with cold saline and stored at -70°C until estimation of neurotransmitters, i.e., dopamine and serotonin. For neurotransmitter analysis, tissues were homogenized in phosphate buffer and centrifuged at 14000 rpm for 10 min at 4°C. After centrifugation, the supernatant was filtered by a syringe filter (0.2 μm), and 20 μl of the sample was injected into the Agilent HPLC-1200. Isocratic mobile phase of 50 mM potassium phosphate buffer in 3% methanol was used for analysis. The flow rate was adjusted to 1.5 ml/min with total run time of 25 min. HPLC separation was carried out using a C18 (5 μm × 250 mm × 4.6 mm) column and detected at 270nm by a UV detector. Experiments were performed in triplicate. Neurotransmitter levels were quantified using standard linear regression equations [18 (link), 24 (link)].
1200 hplc
Manufactured by Agilent Technologies
Sourced in United States, Germany
The 1200 HPLC is a high-performance liquid chromatography (HPLC) system designed for analytical and preparative applications. It is a modular system that can be configured with various components, including pumps, autosamplers, detectors, and column compartments, to meet the specific needs of the user's application.
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Lab products found in correlation
Ctc pal chilled autosampler, by Agilent Technologies (4 mentions)
Masshunter software, by Agilent Technologies (4 mentions)
Zorbax sb c18 column, by Agilent Technologies (3 mentions)
Chemstation software, by Agilent Technologies (3 mentions)
6350 tofms, by Agilent Technologies (3 mentions)
Ft ir 4100, by Jasco (3 mentions)
P 2000, by Jasco (3 mentions)
Spectrum 100, by PerkinElmer (2 mentions)
Xbridge c18, by Waters Corporation (2 mentions)
Lcms 1200 6120, by Waters Corporation (2 mentions)
Zorbax sb c18, by Agilent Technologies (2 mentions)
Eclipse plus c18 column, by Agilent Technologies (2 mentions)
Astra 5, by Wyatt Technology (2 mentions)
C18 reverse phase hplc column, by Agilent Technologies (2 mentions)
6410b triple quadrupole mass spectrometer, by Agilent Technologies (2 mentions)
Amazon sl system, by Bruker (2 mentions)
6210a lc tof instrument, by Agilent Technologies (2 mentions)
971 fp flash purification system, by Silicycle (2 mentions)
1260 infinity hplc system, by Agilent Technologies (2 mentions)
Xdb c18, by Agilent Technologies (2 mentions)
113 protocols using 1200 hplc
1
Quantification of Brain Neurotransmitters
2
HPLC-FLD Analysis Protocol
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The analyses were performed with a high performance liquid chromatography system (HPLC, 1200) with a fluorescence detector (FLD, 1290) from Agilent Technologies (Palo Alto, CA, USA).
3
HPLC Analysis of PTX, ALG, and ALG-PTX
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HPLC analysis was carried out using Agilent HPLC 1200 (Santa Clara, CA) system consisting of degasser, quaternary pump, and a fluorescence detector. HPLC chemstation software (Agilent) was used for instrument control, data acquisition, and data analysis. PTX, ALG, and ALG-PTX nanoparticles were eluted using a Zorbax Extend C18 column (150 × 4.6 mm ID × 5 µm particle size) and detected at 232 nm excitation and 353 nm emission with a flow rate of 1.5 mL min−1. Column temperature was maintained at 25°C. The mobile phase consisted of 40% of water, 35% acetonitrile (ACN), and 25% methanol. pH was adjusted using a Jenway 3505 pH meter (Burlington, NJ).
4
Isolation and Characterization of Capsular Polysaccharide
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The slime layer and cell pellet from 3 L of RJX1120 growth in defined medium was resuspended in lysis buffer (50 mM Tris [pH 7.5], 50 mM MgSO4, 20% sucrose). Cells were lysed with 100 units of mutanolysin (M0991) at 37 °C overnight. To complete lysis, sodium dodecylsulfate was added to 0.5% and stirred at room temperature for 1 h and then at 55 °C for 1 h. Cell debris was removed by centrifugation, and the supernatant, containing liberated polysaccharides, was treated with 1 mg proteinase K and incubated at 37 °C for 3 h to remove protein contaminants. The supernatant was concentrated with a 3-kDa molecular-weight cutoff filter and dialyzed against water extensively. Dialysate was applied to 5 mL HiTrap Q HP pre-equilibrated with 10 mM Tris, pH 8. Flow-through and fractions were collected with increasing concentrations of NaCl. Capsular material was found in the flow-through via H2SO4-phenol staining. Molecular weight of capsular polysaccharide was determined on Agilent HPLC1200 equipped with refractive index detector with a Superdex 200 Increase column and 50 mM NH4OAc, pH 6, as mobile phase. Dextran blue and polyethylene glycols were used as molecular-weight standards.
5
Chemotaxonomic Analysis of Bacterial Strains
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Biomass of strains AD7-25T and AB-11 for chemotaxonomic analysis was harvested from cultures after incubation on improved Gibbson medium at 33 °C for 18 h. The analysis of the cell-wall peptidoglycan was carried out with O. neutriphilus CGMCC 1.7693T as a reference according to the method described by Schleifer and Kandler (1972 (link)) and Schleifer (1985 (link)). Cell-wall hydrolysates were separated by one-dimensional chromatography on micro-cellulose thin layers. Menaquinones were analyzed as described previously (Collins 1985 ) using reverse-phase HPLC (Agilent HPLC-1200). Extraction and analysis of polar lipids by two-dimensional TLC was performed according to Ventosa et al. (1993 (link)). Cellular fatty acids were extracted and methylated according to the standard protocol of Sherlock Microbial Identification System version 6.0 (MIDI), analysed by GC (model 6890; Agilent) and identified using the TSBA6 database of the Microbial Identification System (Sasser 1990). The physiological age at the point of harvest of the three bacterial strains tested was the logarithmic growth phase. O. oncorhynchi subsp. oncorhynchi JCM 12661T was used a reference in chemotaxonomic analysis tests.
6
HPLC-DAD Analysis of Total Phenolic Compounds in MOMAST Products
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HPLC-DAD analysis. For all MOMAST® (Plus30 , HY100 , and PW25 ), the analysis of total phenolic compounds was performed according to the IOC official method [25 ]. HPLC-DAD analysis was carried out with an HPLC 1200 (Agilent Technologies, Palo Alto, CA, USA) equipped with a degasser, quaternary pump solvent delivery, thermo-stated column compartment, and a diode array detector. Chromatographic separation was performed by using SphereClone ODS (2), 5 μm, 250 × 4.6 mm; the elution solvents were acidified H2O by phosphoric acid (pH 3.2), CH3CN, and MeOH. The flow rate was 1 mL min−1, with an injection volume of 20 µL; the applied gradient was in accordance with the IOC method. The area values were registered at 280 nm with syringic acid as the internal standard. The results were expressed as mg of tyrosol per g of sample.
7
Chitin Deacetylase Kinetics Quantified by HPLC-MS
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Chitin deacetylase reactions were performed at final concentrations of 0.2 mM GlcNAc5 (A5), GlcNAc4 (A4) or GlcNAc3 (A3) substrates, 3.2 nM protein, in 50 mM K2HPO4, 300 mM NaCl, buffer at pH 8 in a total volume of 200 µL at 37 °C. At different time intervals, 10 μL aliquots were withdrawn and mixed with 90 μL of H20:propanol (1:1) in HPLC vials to stop the reaction. Samples were analysed by HPLC-MS (HPLC 1200, ESI-MS 6100 series SQ, Agilent Technologies) using a XBridge BEH Amide 2.5 μm 3.0 × 100 mm Column XP, (Waters) in combination with a XBridge Amide Guard Cartridge (2PK) pre-column (2.5 µm 4.6 × 20 mm; Waters), 5 μL injection, and isocratic elution at 60 °C with acetonitrile/water 65:35 v/v, 0.1% formic acid, at a flow rate of 0.4 ml/min. MS detection monitored (SIM mode) the following [M+H]+ ion masses: m/z 628 (A3), 586 (A2D1), 544 (A1D2), 831 (A4), 789 (A3D1), 747 (A2D2), 1034 (A5), 992 (A4D1) and 950 (A3D2). Data was analysed with the ChemStation Software (Agilent Technologies).
8
Spearmint Ethanolic Extract Analysis
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HPLC-DAD-electrospray ionization (ESI)/MS was used to analyze the ethanolic extracts of spearmint using an Agilent HPLC 1200 equipped with a photodiode array detector and a mass detector in series (Agilent Technologies, Waldbronn, Germany). An ion trap spectrometer (Model G2445A), which included an electrospray ionization interface and was controlled by LCMS software (Agilent, version 4.1), was used as a mass detector. The column was a Phenomenex Luna C18 250 × 4.6 mm with a 5 µm particle size. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), and the linear gradient used was as follows: 0 min, 10% B; 1–24 min, 24% B; 24–28 min, 32% B; 28–36 min, 40% B; 36–40 min, 60% B; 40–47 min, 95% B; 47–50 min 10% B; and 50–55 min, 10% B. The injection volume was 20 μL. The ionization conditions were a capillary temperature of 350 °C and a voltage of 4 kV. The nitrogen flow rate was 11 L/min, and the nebulizer pressure was 65.0 psi. The full-scan masses covered the range from m/z 100 to m/z 1200. Helium was used as a colliding gas in the ion trap, with voltage ramping cycles from 0.3 to 2.0 V.
9
Quantification of Brain Neurotransmitters
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Complete rat brain was dissected, washed with cold saline and stored at -70°C until estimation of neurotransmitters, i.e., dopamine and serotonin. For neurotransmitter analysis, tissues were homogenized in phosphate buffer and centrifuged at 14000 rpm for 10 min at 4°C. After centrifugation, the supernatant was filtered by a syringe filter (0.2 μm), and 20 μl of the sample was injected into the Agilent HPLC-1200. Isocratic mobile phase of 50 mM potassium phosphate buffer in 3% methanol was used for analysis. The flow rate was adjusted to 1.5 ml/min with total run time of 25 min. HPLC separation was carried out using a C18 (5 μm × 250 mm × 4.6 mm) column and detected at 270nm by a UV detector. Experiments were performed in triplicate. Neurotransmitter levels were quantified using standard linear regression equations [18 (link), 24 (link)].
10
Spectroscopic Analysis of Organic Compound
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IR spectra were recorded on a PerkinElmer Spectrum 100 with KBr disks. 1 H NMR and 13 C NMR spectra 1 H and 13 C NMR spectra were recorded at 25 C with an Avance 500 MHz spectrometer(Bruker). The LR-ESI-MS analysis was performed using a Bruker amaZon SL. HPLC analysis was carried out using Agilent HPLC1200, with main chromatograph conditions, including chromatograph column CNW Athena C18-WP (4.6 mm × 250 mm, 5 µm); mobile phase 10:80:10 (v/v) ethanol/ratio of acetonitrile/KH2PO4 (ф = 0.3%) with flow rate 1.0 mL·min−1; detection wavelength 210 nm.
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