Figure 8. Peak
overlays of all blue dextran elutions. Variance in peak height is due to
variations in blue dextran spiking concentration and is irrelevant to
the results.
As can be seen from the results, breakthrough volume was independent of
flow rate, with a standard deviation of only 0.004 column volumes. This
can be explained by the Van Deemter Equation:
\begin{equation}
HETP=A+\frac{B}{u}+(C_{s}+C_{m})\bullet u\nonumber \\
\end{equation}Where A is the constant eddy diffusion parameter, B is the diffusion
coefficient in the longitudinal direction, and Cs and Cm are the
resistance to mass transfer in the stationary and mobile phases,
respectively. Because there is no mass transfer of blue dextran from the
liquid phase to the solid phase or vice versa, this portion of the
equation can be ignored. The flow regime is also operated at a
relatively large u, making the longitudinal diffusion part of the
equation negligible as well. This eliminates any dependence of velocity
on peak broadening. These results are significant because they
demonstrate a consistent breakthrough profile regardless of flow rate,
which may need to be adjusted throughout the course of a continuous
process. Therefore, breakthrough time across the column can be easily
calculated from the current flow rate, column volume, and void volume
coefficient. This is not necessarily true of other methods of flow
through virus inactivation that rely on Dean vortices for axial mixing.
In order to demonstrate that live virus behaves similar to blue dextran,
the column was spiked with inactivated xMuLV virus while running at a
residence time of 30 minutes. Three 6 mL fractions of eluate were
collected corresponding to before, during, and after the expected
breakthrough of virus. Results can be seen in Figure 9.