Waters 1500-Series, Руководство оператора

Страница: 16 / 188

1-2 Introduction
HPLC pump operating principles
This section describes these topics:
• Isocratic and gradient LC system operation
• Effects of dissolved oxygen in the mobile phase
• Removing gases from eluents using in-line degassing
Isocratic and gradient LC system operation
Two basic elution modes are used in HPLC: isocratic elution and gradient
elution.
In isocratic elution, the mobile phase, either a pure solvent or a mixture,
remains the same throughout the run. For LC system operation, a single
pump system, such as the 1515 isocratic pump, delivers a controlled amount of
solvent into the column to maintain consistent and reproducible mobile phase
composition.
In gradient elution, the mobile phase composition changes during the
separation. This mode is useful for samples that contain compounds that span
a wide range of chromatographic polarity. As the separation proceeds, the
elution strength of the mobile phase is increased to elute the more strongly
retained sample components.
In the simplest configuration, there are two bottles of solvent and two pumps,
the case when using a 1525 binary pump. The speed of each pump is managed
by the gradient controller to deliver more or less of each solvent over the
course of the separation. The two streams are combined using a mixer to
create the actual mobile phase composition that is delivered to the column
over time. At the beginning, the mobile phase contains a higher proportion of
the weaker solvent (solvent A). Over time, the proportion of the stronger
solvent (solvent B) is increased according to a predetermined timetable.
Because the mixer is downstream of the pumps, the gradient is created under
high pressure.
Other HPLC systems, such as Waters Alliance systems, are designed to mix
multiple streams of solvents under low pressure, ahead of a single pump. A
gradient proportioning valve selects from the multiple solvent bottles,
changing the strength of the mobile phase over time.