Using Volumetric Flow to Scaleup Chromatographic Processes - - BioPharm International

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Using Volumetric Flow to Scaleup Chromatographic Processes


BioPharm International
Volume 19, Issue 3

Next, by measuring the gradient response curves with columns off line in lab and pilot the total delay before matching the nominal gradient could be determined. Figure 4 shows the gradient response curve for pilot with the resulting delay volume of 2.6 CV. The corresponding result for lab scale was 0.3 CV. The difference in delay volumes from pilot and lab (Δ delay volume) could now be determined as 2.3 CV as shown in Figure 5, which accounts for the deviation.


Figure 5. Example of measurement of extra column effects (Δ delay volume = 2.3CV).





The general relationship between delay volume and gradient slope (G) is shown in Figure 6. In the extreme situation of zero slope (isocratic elution) the delay volume will in theory be infinite as the response curves will never reach the nominal gradient, but in a practical approach the delay may be chosen as the difference at 95% match.


Figure 6. Delay volume vs. gradient slope (G)





For very steep gradients, on the other hand, the delay volume will approach the dead volume. Therefore, in most cases it will be necessary to apply corrections for the actual delay volume differences in order to obtain matching retention volumes.


Figure 7. Profiles from Fig. 2 corrected for the Δ delay volume shown in Fig. 5. Note that the profiles do not match completely due to the better performance (slightly higher and narrower main peak) in pilot scale as predicted by the increased number of plates. The differences in the tailing edge are caused by different regeneration protocols (for column clean in place) used in lab and pilot.





In Figure 7 the elution profiles from Figure 2 are corrected for the 2.3 CV difference in delay volume. The residual difference in retention volume may be explained from the different gradient responses in lab and pilot runs (Figure 5).


Figure 8. Scaleup example with a different set of materials: Lab: 1 x 25 cm = 20 mL, 6 CV/h (150 cm/h). Pilot: 20 x 35 cm = 11,003 mL, 6 CV/h (210 cm/h). The profiles would be the same height but the detector in the lab saturated.






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