Delay Volume And Extra-Column Volume; Delay Volume - Agilent Technologies 1290 Infinity LC System System Manual And Quick Reference

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Optimization of the Agilent 1290 Infinity LC System

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.

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 (e.g., 2.1 mm i.d.) as often used

with mass spectrometric detection.

The delay volume in a system includes the volume in the pump from the point

of mixing, connections between pump and autosampler, volume of the flow

path through the autosampler and connections between autosampler and

column.

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

Agilent 1290 Infinity LC System Manual and Quick Reference

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