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Optimization of the Agilent 1260 Infinity Binary LC
How to Configure the Optimum Delay Volume
How to Configure the Optimum Delay Volume
Delay Volume and Extra-Column Volume
The delay volume is defined as the system volume between the point of
mixing in the pump and the top of the column.
The extra- column volume is defined as the volume between the injection
point and the detection point, excluding the volume in the column.
Delay Volume
In gradient separations, this volume causes a delay between the mixture
changing in the pump and that change reaching the column. The delay
depends on the flow rate and the delay volume of the system. In effect, this
means that in every HPLC system there is an additional isocratic segment
in the gradient profile at the start of every run. Usually the gradient profile
is reported in terms of the mixture settings at the pump and the delay
volume is not quoted even though this will have an effect on the
chromatography. This effect becomes more significant at low flow rates and
small column volumes and can have a large impact on the transferability of
gradient methods. It is important, therefore, for fast gradient separations to
have small delay volumes, especially with narrow bore columns (that is,
2.1 mm i.d.) as often used with mass spectrometric detection.
As an example, in HPLC methods using 5 µm packing material flow rates
of 1 ml/min are typically used in a 4.6 mm i.d. column and about
0.2 ml/min in a 2.1 mm i.d column (same linear velocity in the column).
On a system with a typical delay volume of 1000 µl and using a 2.1 mm
column, there would be an initial "hidden" isocratic segment of 5 min
whereas on a system with 600 µl delay volume the delay would be 3 min.
These delay volumes would be too high for run times of one or two
minutes. With sub- two µm packings, the optimum flow rate (from the Van
Deemter curve) is a little higher and so fast chromatography can use three
to five times these flow rates yielding delay times of about one minute.
However, the delay volume must be reduced further to achieve delay times
which are a fraction of the intended run time. This is achieved with the
Agilent 1260 Infinity Binary LC due to the low delay volume of the pump
flow path and low- volume of the flow path through the autosampler.
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1260 Infinity Binary LC - System User Guide
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