Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2001-04-19
2003-06-10
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S198200
Reexamination Certificate
active
06576134
ABSTRACT:
This invention is concerned with chromatographic separation, more particularly with a new and improved method of sample displacement chromatography and applications thereof, as well as with apparatus useful in such methods.
During the last 10-15 years displacement chromatography has been suggested as an alternative to liquid chromatography using elution techniques. In elution chromatography, components of a sample are transported along a chromatography bed comprising stationary phase material, e.g. in a column, by a mobile solvent phase. The various components interact at different levels with the stationary phase material and are therefore separated into bands. Displacement chromatography, on the other hands, utilises as mobile phase a displacer solution which has higher affinity for the stationary phase material than do the sample components. In the case of column chromatography the sample components are thereby displaced and driven down the column ahead of the displacer front, competing for adsorption sites and separating into individual component bands as they proceed.
Whereas elution chromatography normally results in substantial dilution of the sample material, displacement chromatography permits recovery of sample components at significantly higher concentrations and generally makes more efficient use of the stationary phase material. However, displacement chromatography does suffer the disadvantage that, to achieve optimum results, operating conditions such as the composition, concentration and flow rate of the displacer solution must be tailored specifically to individual sample types.
Sample displacement chromatography is a self-displacement technique which was first proposed by Hodges et al. [
J. Chromatogr.
444 (1988), pp. 349-362] for preparative purification of peptides by reversed phase HPLC, and which does away with the need for an extraneous displacer solution. The peptide components are applied to the column input and themselves compete for binding sites on the stationary phase as they are carried through the column or series of columns by an appropriate solvent. The more strongly binding components bind first and displace less strongly binding components to further along the column(s). The components are therefore separated according to their different degrees of hydrophobicity/hydrophilicity and thus their affinity for the stationary phase material. In a representative example a short pre-column is used to trap impurities which are more hydrophobic than the desired sample component, this latter being retained in and saturating the main column, while the more hydrophilic impurities are further displaced and so are washed out of the main column. It is suggested that the size of the pre-column may be adjusted to match the amount of hydrophobic impurities present in a particular sample, whilst the size of the main column may be adjusted to ensure maximum product retention and outflow of hydrophilic impurities.
Curiously Hodges et al. subsequently use gradient elution to recover the desired product from the main column, so that the advantageous potential of displacement chromatography for yielding relatively high concentration product solutions is lost. This would suggest that the initial component separation was incomplete.
Veeraragavan et al. [
J. Chromatogr.
541 (1991), pp. 207-220] report application of the Hodges technique to purification of proteins using high performance anion exchange chromatography columns. The apparatus used was a low pressure fast protein liquid chromatographic system and again gradient elution was employed; this was presumably felt to be necessary in light of the observation that peak overlaps were a problem in the primary separation procedure. One and two column systems are specifically described, the former being applicable on what are said to be the rare occasions where the desired product is either the most or least strongly binding component. The possibility of using a multi-column system “in which theoretically every component of the protein sample could be fixed to a column of the proper dimensions” is noted.
Multi-column HPLC systems for sample displacement chromatography have in fact been described by Hodges, inter alia in CA-A-2059114. A representative illustration shows the use of ten reversed phase HPLC columns or column segments connected in series for the purification of peptide samples; after the sample material has been loaded and distributed/separated over the train of columns, individual columns or segments may be eluted separately, without resort to gradient elution, the desired product component being recovered in substantially pure form from at least one such column or segment. Advantages of this process are said to be that (i) it allows ten-fold greater loading than comparable gradient elution separations; (ii) it involves minimal use of costly HPLC solvents; (iii) it requires minimal use of fraction analyses; (iv) it avoids the need to use displacer solutions during the actual separation; and (v) operating costs in terms of solvents, column packings and machine usage are much lower than typical gradient elutions.
The Hodges multi-column procedure does not appear to have been widely adopted and has been found in practice to give products with insufficient purity as a result of inadequate resolution of the product from closely related impurities. Moreover, by virtue of the need to operate HPLC procedures at high pressure, typically 80-200 bar, the apparatus required is necessarily complicated and expensive.
WO98/46623, the contents of which are incorporated herein by reference, describes sample displacement chromatography procedures which typically utilise low operating pressures and recover the desired product in a non-gradient manner. Such use of low operating pressures greatly simplifies apparatus requirements, permitting the use of simpler and less expensive pumps, taps, connectors and the like than are required for HPLC systems; the consequential low mobile phase flow rates also give rise to good separation of the sample components. By avoiding use of gradient elution the separation procedure also reduces solvent requirements and may facilitate recovery of the desired product in an advantageously high concentration.
In existing sample displacement chromatography separations, the sample components are introduced in the form of a homogeneous sample solution, so that the individual components are each delivered at a constant concentration throughout the sample application step. The driving force for separation is that weak binders are displaced from the limited number of binding sites on the stationary phase material by the more strongly binding bulk of the product. This proceeds in a continuous manner until the product and other stronger binders are fully retarded in the earlier part of the chromatography bed, thus permitting the more weakly binding impurities to stay bound to the stationary phase material further along the chromatography bed. Once all sample molecules are bound to the stationary phase, no further movement of these molecules will be observed.
A problem which may occur because of such use of homogeneous sample solutions, however, is that molecules of strongly binding components introduced during an early part of sample application may inadvertently be displaced by weaker binders introduced during a later stage of sample application. Since it is not possible for sample molecules to move against the carrier flow, such displaced strong binders may end up as impurities in the desired product part of the chromatography bed. Moreover, a weakly binding component entering the chromatography bed at a late stage of application may compete for a site at an early position of the bed; with only a small fraction of the total sample remaining, the likelihood of displacement from such an early position on the bed is decreased. Therefore this impurity may remain in an unexpectedly early position after the separation and so may contaminate the desired product. In a difficult separation, wh
The Firm of Hueschen and Sage
Therkorn Ernest G.
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