True moving bed electrophoresis using stepped electric field gradients.

True moving bed electrophoresis has been shown to be an effective technique for the bench-scale separation of enantiomers, and it is desired to increase the maximum possible throughput attainable with the process by using electric field gradients. Homatropine enantiomer separations were performed and results using a stepped electric field gradient were compared to those using a traditional non-gradient separation. In order to accomplish this, a newly designed stator was constructed for use with the Vortex-Stabilized Electrophoresis Apparatus that has three sets of electrode housings, one set at both ends and one in the middle of the chamber. There were several problems related to the membranes used at the middle electrode. The dialysis membranes were permeable to the homatropine enantiomers, and while a switch to anion exchange membranes prevented the permeation of the homatropine, this caused a pH shift that interrupted binding to the hydroxypropyl-ss-cyclodextrin chiral selector. These problems prevented any meaningful data from being collected using homatropine enantiomers, and due to this, a proof of concept study was conducted using two bovine proteins. The separations using fluorescein-labeled BSA and bovine hemoglobin showed that a 63% increase in the maximum processing rate was attainable. The maximum throughput using the non-gradient process was 30.6 mg/h and the maximum was 50.0 mg/h using an electric field gradient that was 10% lower than the non-gradient field in section II and 10% higher in section III.

Increasing the scale of true moving bed electrophoretic separations using filtration to reduce solvent volumetric flows between sections II and III.

Over the past decade the moving bed process has become a commonly used tool for the continuous separation of chiral compounds, and its recent application to electrophoretic separations allows the technique to be used as a model system for moving bed method improvements. Much of the recent research on moving bed separations has focused on improving the technique's efficiency and increasing the maximum attainable throughput. This paper presents a novel method for reducing or reversing the increases in tailing that stem from the addition of the feed stream in a moving bed process by adding a filtration unit which retains the products while removing fluid from the boundary between the sections above and below the feed stream. This filtration-enhanced moving bed process was applied to a true moving bed (TMB) electrophoresis separation in the Vortex Stabilized Electrophoresis Apparatus, and its effect on a homatropine enantiomer separation was studied. Experiments showed that there is a 2.4-fold increase in the homatropine processing rate when 0.5 ml/h of water is removed through a reverse osmosis filter at the boundary between the sections above and below the feed stream. In order to further understand the process, filtration-enhanced TMB (FE-TMB) was also analyzed using a linear model of the system which shows that the 99% purity operating region of the separation is greatly increased even with moderate permeate flowrates.

Continuous voltage gradients and their application to true moving bed electrophoresis.

Gradient elution has been practiced in chromatographic separations for many years. The application of discontinuous "step" gradients in simulated moving bed (SMB) chromatography has been very successful in increasing both processing rates and column productivity, resulting in a reduction in the number of SMB columns required. With the advent of the field gradient focusing techniques, electrophoresis has gained the ability to apply a continuous electric field gradient to a true moving bed (TMB) electrophoretic separation. Application of a spatial gradient allows a large degree of control of the product concentrations inside the separation unit as well as a large increase in product throughput. A model of moving bed electrophoretic separations has been developed that demonstrates the potential advantages of applying a continuous gradient to the moving bed process. These advantages include the reduction of detrimental peak tailing and the ability to decrease the concentrations of the compounds being separated in comparison with commonly used step gradient elution.

Development of a segmented model for a continuous electrophoretic moving bed enantiomer separation.

With the recent demonstration of a continuous electrophoretic "moving bed" enantiomer separation at mg/h throughputs, interest has now turned to scaling up the process for use as a benchtop pharmaceutical production tool. To scale the method, a steady-state mathematical model was developed that predicts the process response to changes in input feed rate and counterflow or "moving bed" velocities. The vortex-stabilized apparatus used for the separation was modeled using four regions based on the different hydrodynamic flows in each section. Concentration profiles were then derived on the basis of the properties of the Piperoxan-sulfated beta-cyclodextrin system being studied. The effects of different regional flow rates on the concentration profiles were evaluated and used to predict the maximum processing rate and the hydrodynamic profiles required for a separation. Although the model was able to qualitatively predict the shapes of the concentration profiles and show where the theoretical limits of operation existed, it was not able to quantitatively match the data from actual enantiomer separations to better than 50% accuracy. This is believed to be due to the simplifying assumptions involved, namely, the neglect of electric field variations and the lack of a competitive binding isotherm in the analysis. Although the model cannot accurately predict concentrations from a separation, it provides a good theoretical framework for analyzing how the process responds to changes in counterflow rate, feed rate, and the properties of the molecules being separated.