03/08/2023, 18:53 Understanding Dead Volume, Dwell Volume, & Extra Column Volume

Understanding Your HPLC System: Dead Volume, Dwell

Volume, and Extra Column Volume
By Alec Valenta, (https://www.thermofisher.com/blog/analyteguru/tf-author/alec-valenta/)
02.28.2022

If you’ve ever had to troubleshoot an HPLC method, you already know there are a TON of factors that
impact the quality of a separation. To confuse things further, we chromatographers love our jargon a lot
more than we love being consistent.

Understanding the different volumes associated with an HPLC system can be crucial for things like method
development (https://www.thermofisher.com/us/en/home/industrial/chromatography/liquid-
chromatography-lc/hplc-uhplc-systems/vanquish-method-development-hplc-and-uhplc-systems.html?
ce=E.22CMD.PM109.05966.01&cid=E.22CMD.PM109.05966.01), troubleshooting poor peak shape, and
method transfer (https://www.thermofisher.com/us/en/home/products-and-
services/promotions/industrial/simplified-hplc-method-transfer.html?
ce=E.22CMD.PM109.05966.01&cid=E.22CMD.PM109.05966.01). So, I thought I would take some time to
clarify some terms that seem to cause a great deal of confusion: HPLC dead volume, dwell volume, and
extra column volume.

Before we talk about when each of these terms becomes important, let’s get some definitions out of the
way.

Dead volume – most often used (incorrectly) as a substitute for the term extra column volume. Dead
volume actually refers to “volumes within the chromatographic system which are not swept by the mobile
phase.” 1

Extra column volume – all volume with an HPLC system from the sample loop to the detector, excluding
the column. Dead volume contributes a portion of this volume.

Dwell volume – a.k.a gradient delay volume, is the volume from the point of mobile phase mixing to the
inlet of the column.

HPLC method transfer

Now we can take these definitions and start to think about when each term is important.

HPLC Dead Volume

You can think of dead volume as all the nooks and crannies in your LC system – excluding the column – the
sample comes in to contact with that aren’t actively flushed by mobile phase.

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03/08/2023, 18:53 Understanding Dead Volume, Dwell Volume, & Extra Column Volume

Why is this important?

Dead volume is intrinsically tied to separation efficiency and has a huge impact on peak width and
peak shape.

You can start by imagining an analyte peak, or band, flowing between two pieces of tubing joined by a
union (1). As the band enters the union, the analyte in solution spreads out to fill the now larger available
volume (2). Putting aside the complexities of fluid dynamics, you can imagine the corners of the union will
be flushed by the mobile phase at a slower rate than the rest of the union – think little

whirlpools in each corner.

Then, as the band moves into the next piece of tubing, it will take
longer for the portion of the band that reached the corners by
diffusion or active flow to exit the union (3). This delay ultimately
leads to a type of peak asymmetry known as peak tailing in which
the front side of a band is more concentrated than the back (4).

More, the addition of any volume to the flow path will increase
band broadening due to diffusion – this will come up again in the
extra column volume section.

The best way to avoid peak tailing is to ensure all connections in

the sample flow path have minimal dead volume. To this end,
Thermo ScientificTM ViperTM and Thermo ScientificTM nanoViperTM
Fingertight Fittings

(https://www.thermofisher.com/us/en/home/industrial/chromatography/liquid-chromatography-lc/hplc-
uhplc-related-products/fittings.html?
cid=E.22CMD.DL104.05704.01&ef_id=Cj0KCQiAr5iQBhCsARIsAPcwRONgQ9JuUyNnPg_2m9A3HvTjoRaxDqNKYziEqhxmWWZOmfkIbSXupm
nearly eliminate dead volume – avoiding peak distortion and minimizing band broadening.

HPLC Extra Column Volume

Extra column volume is divided into two categories: pre-column volume and post-column volume.

Pre-column volume is all the volume in the sample flow path from the point of injection to the inlet of the
column. A larger pre-column volume equals more time for an analyte band to diffuse outward,
resulting in band broadening.

While it is always important to minimize pre-column volume, the band broadening effects are most keenly
felt in isocratic separations. This fact is because isocratic separations do not benefit from the band focusing
effect that occurs at head of the column during gradient separations, which mitigates some of the pre-
column band broadening.

Understanding the different volumes associated with your HPLC

system is crucial for successful method development and transfer.

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03/08/2023, 18:53 Understanding Dead Volume, Dwell Volume, & Extra Column Volume

This guide will teach you everything you need to know about HPLC

dead volume, dwell volume, and extra column volume.

If you’re getting the hang of this, you’ve probably already reasoned that post-column volume is everything
after the column outlet. Regardless of separation type (isocratic or gradient), post-column volume can be a
real efficiency killer. Simply put: the more post-column volume, the more time your analyte bands will
have to diffuse prior to detection and the more resolution you will lose. As I mentioned, since this
volume is post-separation, you won’t get any help from gradient focusing effects.

Both pre- and post-column volume become increasingly detrimental to separations as flow rate and column
inner diameter (ID) decrease. Fortunately, the solution is simple – minimize tubing length, eliminate dead
volume due to poor connections, and choose an appropriate ID tubing for the column dimensions.
Choosing the shortest, narrowest ID tubing possible for a given HPLC system limits extra column band
broadening, preserving all that hard-earned peak resolution.

A word of expert advice: keep in mind that back pressure is proportional to 1/tubing radius2. So, if you cut
the tubing ID in half, the pressure required to deliver the same flow rate is multiplied by a factor of 4!

HPLC Dwell Volume/Gradient Delay Volume

Gradient delay volume (GDV) is probably the most straightforward of the three concepts. It includes the
total volume from the point of mobile phase mixing to the inlet of the column including, but not limited to,
pump heads, tubing, mixer(s), the sample loop, and any valves in the flow path. For a given flow rate, GDV
translates into the time it takes for a change in mobile phase in the pump to reach column inlet.

If you are concerned only with isocratic separations, you can skip this section. Because mobile phase
composition is constant, there is no “delay” between the pump and the column.

The pump type is a major factor in determining a system’s GDV. Pumps fall into two categories based on
gradient formation: low-pressure and high-pressure gradient pumps. Low-pressure pumps contribute more
to GDV.

In low-pressure gradient pumps, mobile phase mixing occurs prior to reaching the pump head at low
pressure. The volume of the proportioning valve, pump inlet tubing, and pump head contributes to
the GDV. In low-pressure gradient pumps, the pump itself is often the major contributor to GDV. Most
quaternary pumps perform low-pressure gradient formation.

High-pressure gradient pumps have multiple pump heads – one for each solvent – and the mixing
occurs after the pump head. Pump heads and inlet tubing do not contribute to the GDV. High-
pressure gradient pumps generally contribute a minor amount of the total GDV. Binary pumps tend to
fall in this category.

While the GDV is known to impact analyte selectivity, the two main effects are on throughput and method
transfer.2 First, it causes an initial isocratic before the gradient reaches the column head. The isocratic hold
is one of the major limitations on method throughput.

When transferring methods between HPLC systems, understanding each system’s GDV is crucial for
obtaining comparable results and avoiding unnecessary method validation. As mentioned previously,
significant differences in GDV can have effects on peak shape, selectivity, and retention time.

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03/08/2023, 18:53 Understanding Dead Volume, Dwell Volume, & Extra Column Volume

Not only is absolute retention time dictated by the GDV, but it also influences the gradient profile. This
gradient profile smoothing (the same process as analyte band broadening) shown below can be a major
factor when attempting to match peak shape and elution order. Complex gradients – think multistep – are
even more challenging to recreate on systems with mismatched GDVs.

Fortunately, a number of resources exist to simplify method transfer onto Thermo ScientificTM VanquishTM
HPLC and UHPLC systems (https://www.thermofisher.com/us/en/home/products-and-
services/promotions/industrial/vanquish-hplc-uhplc-systems.html?
ce=E.22CMD.PM109.05966.01&cid=E.22CMD.PM109.05966.01) such as a method transfer kit to allow a
tunable GDV (https://assets.thermofisher.com/TFS-Assets/CMD/Technical-Notes/tn-73371-lc-gradient-
delay-volume-tn73371-en.pdf).

Helpful Links
Simplified Method Transfer (https://www.thermofisher.com/us/en/home/products-and-
services/promotions/industrial/simplified-hplc-method-transfer.html?
E 22CMD PM109 05966 01& id E 22CMD PM109 05966 01)

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03/08/2023, 18:53 Understanding Dead Volume, Dwell Volume, & Extra Column Volume

ce=E.22CMD.PM109.05966.01&cid=E.22CMD.PM109.05966.01)

White Paper: An Instrument Parameter Guide for Successful (U)HPLC Method Transfer
(https://assets.thermofisher.com/TFS-Assets/CMD/Reference-Materials/wp-72711-lc-method-transfer-
guide-wp72711-en.pdf)

Sources
1. IUPAC. Compendium of Chemical Terminology, 2nd ed. (the “Gold Book”). Compiled by A. D. McNaught
and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). Online version (2019-)

2. Schellinger, AP; Carr, PW. A Practical Approach to Transferring Linear Gradient Elution Methods. J.
Chromatogr. A, 2005, 1077(2), 110-119

Alec Valenta

 HPLC Troubleshooting: Why Signal to Noise Ratio Determines Limit of Detection

(https://www.thermofisher.com/blog/analyteguru/hplc-troubleshooting-tips-why-signal-to-noise-ratio-determines/)

HPLC Troubleshooting: Precipitation of Buffers in Gradient Elution Mode (https://www.thermofisher.com/blog/analyteguru/hplc-

troubleshooting-precipitation-of-buffers-in-gradient/) 

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