In chromatography, resolution refers to the ability to separate two peaks. When components in a sample are poorly resolved, peaks will subsequently be close together – and may even overlap.
In contrast, when columns separate samples with a high level of resolution, peaks representing different components will be further apart. Here, Izon’s qEV columns consistently enable high-resolution separation, which is enhanced with Gen 2 columns (compared to Legacy columns).
The improved resolution of Gen 2 columns is an advantage for the separation of soluble, contaminating protein from extracellular vesicles (EVs) and similarly sized particles; depending on the downstream analysis of choice, researchers may wish to alter the collected volumes in a way that changes EV-protein proportions and subsequently prioritises EV concentration, EV recovery, or EV purity.
As illustrated in the figure below, the enhanced resolution of Gen 2 columns provides the opportunity to customise your collected volumes accordingly. The blue shaded area is known as the Purified Collection Volume (PCV), which corresponds to EV-containing volumes that are most of interest, depending on your priority.
As a result, the volume eluting before the purified collected volumes of interest may change. This is referred to as the ‘Adjusted Buffer Volume’ in the user manuals, and is represented by the pink shaded area in the figures below.
To go through this optimisation process, an elution profile will need to be established, as this will differ slightly by column/sample type. However, elution profiles of human plasma samples – provided in the user manuals – can be used as a guide. User manuals provides guidance on when and how the AFC settings need to be adjusted accordingly.
Generic elution profile of a human plasma sample separated using a Gen 2 column and Automatic Fraction Collector (AFC). Particle concentration (blue bars) was measured on the Exoid, and protein (blue dotted line) was measured via bicinchoninic acid assay. Faded bars represent estimated particle concentration based on calculations and comparisons from various pooled volumes.
By programming the AFC appropriately (see AFC and qEV user manuals), the Purified Collection Volume (PCV) containing your particles of interest can be altered to prioritise: A) EV concentration B) EV recovery C) EV purity. D) The default setting on the AFC prioritises a balance of EV recovery and purity.