LC-MS analysis was performed on a system equipped with a Waters 2795 separation Module (Alliance HT), Waters 2996 Photodiode Array Detector (190-700 nm) and a Micromass LCT-TOF Premier mass spectrometer. Samples were run over an XBridge BEH300 C18 column (5 μm, 4.6 x 100 mm, T = 40°C) using a gradient of 30 − 60% B (over 3.5 min or 6 min). Samples were run at 0.8 mL/min using a gradient of two mobile phases: A = 1% acetonitrile and 0.1% formic acid in water; B = 1% water and 0.1% formic acid in acetonitrile. Preparative HPLC was performed on a Waters XBridge™ Prep C18 column (30 x 250 mm, 5μm OBD™). Samples were run at 25 ml/min using a gradient of two mobile phases: A = 5% acetonitrile and 0.05% trifluoroacetic acid in water; B = 5% water and 0.05% trifluoroacetic acid in acetonitrile. Gradient: 0 – 6 min: 5 – 10% B; 6 – 10 min: 10 – 30% B; 10 – 26 min: 30 – 50% B; 26 – 27 min: 50 – 95% B. Data processing was performed using Waters MassLynx 4.1 software.
Waters 2795 separation module
Manufactured by Waters Corporation
Sourced in United States
The Waters 2795 Separation Module is a high-performance liquid chromatography (HPLC) system designed for a wide range of analytical applications. It features precise solvent delivery, sample handling, and column temperature control capabilities to enable efficient separation and analysis of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Masslynx 4, by Waters Corporation (3 mentions)
Xbridge prep c18 column, by Waters Corporation (3 mentions)
2996 photodiode array detector, by Phenomenex (1 mentions)
Avance 300 spectrometer, by Bruker (1 mentions)
Micromass zq, by Waters Corporation (1 mentions)
Waters 2996 photodiode array detector, by Waters Corporation (1 mentions)
Avance 3 hd 400 spectrometer, by Bruker (1 mentions)
5 protocols using waters 2795 separation module
1
LC-MS Analysis of Organic Compounds
2
Purification and Analysis of Peptides
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All commercial materials (Aldrich, Fluka, Novabiochem) were used without further purification. All solvents were reagent grade or HPLC grade. LC-MS analysis was performed on a system containing a Waters 2795 separation module (Alliance HT), Waters 2996 Photodiode Array Detector (190–750 nm), Phenomenex Kinetex XB-C18 (2.1 × 50 mm) reversed-phase column, and a Micromass LCT-TOF mass spectrometer. Samples were run at 0.80 ml/min with the use of a gradient of two mobile phases: A) aq. formic acid (0.1%), and B) formic acid in CH3CN (0.1%). Data processing was performed using Waters MassLynx 4.1 software. Preparative HPLC was performed on a Waters XBridge™ Prep C18 Column (30 × 150 mm, 5 μm OBD™) at a flow rate of 37.5 ml/min using aq. 0.05% TFA (Solvent A) and acetonitrile containing 0.05% TFA (Solvent B) as eluents. For purification of peptides, a gradient from 25% B to 95% B over 18 min was used. All samples containing pure peptides were pooled and lyophilized.
3
Photoleucine-Containing Peptide Synthesis
4
Synthesis and Characterization of Substituted Pyrazoles
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Unless otherwise noted, reactions were conducted using commercially available reagents and solvents, which were used as received. 1H and 13C NMR spectra were recorded on Bruker Avance III HD 400 spectrometers. Chemical shifts are reported in δ units (ppm). NMR spectra were referenced relative to residual NMR solvent peaks. Coupling constants (J) are reported in hertz (Hz). Solvent removal was accomplished with a rotary evaporator at ca. 10–50 Torr. Column chromatography was carried out using a Biotage Isolera flash chromatography system and silica gel cartridges from Silicycle. Mass analyses and compound purity were determined using Waters Micromass ZQ, equipped with Waters 2795 Separation Module, Waters 2996 Photodiode Array Detector, and Waters 2424 ELS detector Separations were carried out with an XTerra® MS C18, 5µm, 4.6 × 50 mm column, at ambient temperature (unregulated) using a mobile phase of water-methanol containing a constant 0.1 % formic acid.
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5
Quantification of Delta Toxin by HPLC-MS
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Delta toxin was quantified as described previously.20 (link) Briefly, 5 μL of culture supernatant was infused into a C8 column (ZORBAX SB-C8, 2.1 × 30 mm, 3.5 μm, Agilent, Santa Clara, CA, USA) connected to Waters 2795 separation module (Waters, Milford, MA, USA). Delta toxin was eluted by 0.05% trifluoroacetic acid (TFA) in double distilled water (eluent A) and 0.05% TFA in acetonitrile (eluent B) at a flow rate of 0.3 mL min−1 for 10 min. The gradient program for elution is as follows: 0% eluent B for 1.5 min; 0% to 50% of eluent B for 1 min; 50% to 100% of eluent B for 4 min; 100% eluent B for 2.5 min; 0% eluent B for 1 min. The separated delta toxin was ionized and analyzed by the conjunct Waters ZQ 2000 MS system (Waters, Milford, MA, USA) equipped with an electrospray ionization (ESI) source. Ion source parameters are as follows: capillary voltage, 3.3 kV; cone voltage, 50 V; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow, 900 L h−1; cone gas flow, 50 L h−1. Ions were analyzed in the positive ion full scan mode for the range of m/z 600 to 1600. Scan time is 1 s with an inter-scan delay of 0.1 s following parameters. The production of delta toxin was measured by integrating the extracted ion chromatograms based on the m/z 1504 and 1003 of doubly- and triply-charged ions, respectively.
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