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Column: Symmetry® C18 (150 mm × 4.6 mm i.d., 5 μm particle size), Waters, Ireland.
Flow rate was 1.2 mL/min, column used was Synergi-4µ-hydro-RP-80A (250 × 4.6 mm) with a guard column Symmetry Shield RP-18 (15 mm × 4.5 mm, 5 µm) with an ultraviolet Detector set at 254 nm, column temperature was set to 30 °C, injection volume was 10 µL, run time for the run was 15 min.
Peptides were desalted onto a trap column (Symmetry® C18, Waters).
Each sample was loaded onto a C18 trap column, (Symmetry 180 μm × 20 mm 5 μm; Waters, Milford, MA) and washed with 2% acetonitrile, 0.1% formic acid at 15 μL/min for 2 min.
10, 15, 30 FITC-BSA was quantified using a gradient HPLC method (Agilent Technologies 1200 Series, Stockport, UK) in which the separation was performed on a C4 (4.6 mm × 50 mm, 5 μm) analytical column (Symmetry 300, Waters Ireland, Dublin).
For RP-nano-UPLC-ESI-MS/MS, a sample (2 μL) of the desired peptide digest was loaded into the reverse phase column (Symmetry C18, 5 μm, 180 μm × 20 mm) by an autosampler.
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Samples were dissolved in 0.1% TFA and, on the nanoAcquity system, peptides were trapped using a pre-column (Symmetry® C18, 5 µm, 180 µm ×20 mm, Waters) which was then switched in-line to an analytical column (BEH C18,1.7 µm, 75 µm ×250 mm, Waters).
Peptides were trapped using a pre-column (Symmetry C18, 5 µm, 180 µm × 20 mm, Waters), which was switched in-line to an analytical column (BEH C18, 1.7 µm, 75 µm × 250 mm, Waters) for separation.
The columns Symmetry and Mid show the result of each transformation from image into waveform.
Separations were performed with a reverse phase column (Waters Symmetry C18 column, 5 μm, 4.6 × 150 mm).
Please note that in the first column, the symmetry is for a single harmonophore.
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