Calibrating the Automatic Fraction Collector

The field of extracellular vesicles (EVs) is continuously expanding with applications in biomarker discovery, rapid diagnostics, potential vaccine candidates and as vehicles of drug delivery. One of the major challenges for their application in a clinical setting is finding a scalable EV isolation method that yields functional EVs.

Isolation of EVs using the combination of Izon's qEV size-exclusion columns and the Automatic Fraction Collector (AFC) increases uniformity of sample collection volumes containing EVs, decreases experimental runtime allowing for higher sample throughput, and eliminates many of the inaccuracies that are inherent in the manual collection of EVs. The AFC offers users the ability to scale up EV isolation through automation of several steps, including the equilibration, EV elution, washout, and regeneration of the columns.

The AFC manages the collection of the void volume (total volume of mobile phase in the column) by automatically differentiating between the void volume and the sample collection volumes containing EVs. The void volume does not contain any EVs and collects in the central well of the carousel and will depend on the type of qEV column used in each run. Once the void volume has been collected, the instrument will begin collecting a number of sample collection volumes of a given volume predetermined by the user through the intuitive AFC software. The initial collection volumes correspond to the EV Zone (blue zone in Fig. 1), while later fractions correspond to the Protein Zone (purple zone in Fig. 1) that can also be collected if required.

We have received feedback from our users stating the continuous dripping or over/under filling of the collection tubes while running samples in different instances. This issue relates back to how the AFC has been calibrated.
Correct calibration of the AFC plays is vital in the accurate collection of void volumes and subsequent EV collection volumes. We have now included an accurate 10 g weight with the AFC to ensure correct calibration of the load cell. For a detailed protocol on calibration please click here. Inaccurate calibration leads to inconsistent filling of post qEV sample collection tubes.