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From 0 to 10 minutes isocratic flow of 100% solvent A was performed, from 10 to 15 minutes a linear gradient up to 26% solvent B, from 15 to 40 minutes a linear gradient to 35% solvent B from 40 to 45 minutes a linear gradient to 60% solvent reaching 100% solvent B at 49 minutes.
For the first two minutes, isocratic elution was carried out using 10% A in B. From 2 to 25 minutes, a linear gradient was applied using 10 to 30% A in B. From 25 to 27 minutes, a linear gradient was applied using 30 to 70% A in B. The volume of the sample injected was 20 μl.
For the first 2 minutes, isocratic elution was carried out using 100% of A. From 2 to 5 minutes, a linear gradient was applied using 100 to 30% A in B. From 5 to 5.5 minutes, a linear gradient was applied using 30 to 0% A in B. In the final minute concentration of A was returned to 100%.
The pump was programmed to generate the following gradient: 6% A and 94% B (0 to 3 minutes), a linear increase of A to 25% and a linear decrease of B to 75% (3 to 16 minutes), 25% A and 75% B (16 to 22 minutes), a linear decrease of A to 6% and a linear increase of B to 94% (22 to 27 minutes), and 6% A and 94% B (27 to 35 minutes).
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The gradient system was 90% (B) for 3 minutes → to 60% (B) in 7 minutes in a linear slope → to 0% (B) in 25 minutes in a linear slope and held at 0% (B) for 5 minutes.
The initial mobile phase composition was 100% A for 3.5 minutes then a linear gradient was applied for 5.5 minutes to 90% B. This was maintained for 1.0 minute and a quick linear gradient back to 100% A for 0.5 minute was followed by a 3.5 minutes equilibrium period.
A well defined peak separation was obtained when applying the following elution gradient: 0.1 M NaOH for 5 minutes, then a linear increase from 0.1 M NaOH to 0.1 M NaOH with 0.3NaOAc in 35 minutes, then to 0.1 M NaOH/1 M NaOAc in 5 minutes.
The gradient was as follows: 10% was also maintained at the first 3 minutes, then a linear gradient of 10 20% B for 3 10 min, 20 35% B for 10 35 min, and 35 60% B for 35 50 min; this composition was maintained from 50 to 55 min and then returned to the initial condition in 5 minutes.
Stimulation was applied for a period of 5 minutes, with a linear fade in/fade out of 10 seconds and was congruent with the duration of the three encoding trials (see VLMT test).
To compare fidelities for the five trainers with respect to mental strain, experience of flow and number of interventions per minutes a mixed linear model involving fidelities as the within-subject variable was used.
To estimate the "initial rate" (v 0 ) of the GSs reaction, the production of hydroxamate was measured at several time points during 15 minutes and exhibited a linear increase during the first 3 minutes for all four isoenzymes (data not shown).
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