Polishing chromatography processes require highly efficient chromatographic columns. Smaller, mono-sized resins can deliver
high column efficiencies, but only when they are packed in their optimal configuration. This requires highly dense and homogeneous
packing of the resin bed.1,2 Additionally, protocols employed for the packing of reversed-phase silica do not provide satisfactory results when used
with reversed-phase polymeric resins. Therefore, a factorial design study was conducted with a new mono-sized, polymeric reversed-phase
resin, Amberchrom HPR10, to determine what conditions provided the highest efficiencies.
Packing Conditions
Table 1: Column packing factorial design.
The efficiency of the chromatographic column used for purification has a profound influence on the success of the separation.
Therefore, column packing is a critically important task in the preparation for a separation. In particular, packing of polymeric
resins is influenced by a number of different factors, including:
Particle size
Particle density
Composition of the particle
Slurry concentration
Fluid composition
– Viscosity/Lubrication – Solvent Polarity – Fluid density
Surface properties of the sphere
Column packing mode: dynamic axial compression vs flow packing
Column distribution system
Column size
Additives.
Figure 1.
For purposes of this study, we examined four factors in a factorially designed experiment using a flow packing technique.
These four factors were: viscosity of the packing buffer, packing pressure, resin slurry concentration, and packing ramp rate.
The factorial design is shown in Table 1. To simplify the number of experiments, we used only one column size — 10 mm i.d.
× 250 mm L. The stainless steel columns were supplied by Alltech Associates (Deerfield, Illinois, USA), and the packing pump
was a Series 1500 HPLC pump supplied by Lab Alliance (State College, Pennsylvania, USA). Additionally, software from Lab Alliance
was used to control the HPLC pump during column packing. This software control provided a more reproducible packing ramp rate
and better pressure control than manual operation. After packing, the columns were evaluated for efficiency and asymmetry
using a 2% acetone injection in a 100% acetonitrile on an Agilent 1100 HPLC system (Palo Alto, California, USA).
Results and Discussion
Figure 2.
The results of the packing study are shown in Figures 1 to 5. Based on these results, it is evident that the resin slurry
concentration had a profound impact on the column efficiencies. As shown in Figures 1, 2 and 5, the higher slurry concentration
of 65% resulted in column efficiencies that were much lower than efficiencies obtained using a slurry concentration of 35%.
The reason for this difference is most likely because of the fact that the resin bed forms too quickly during packing and
this increased the likelihood of aggregation and bridging of resin beads in the packed bed.
