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Blending of Size Exclusion Media to Tailor Chromatographic Performance

IP.com Disclosure Number: IPCOM000201769D
Publication Date: 2010-Nov-22
Document File: 6 page(s) / 102K

Publishing Venue

The IP.com Prior Art Database

Abstract

The Kav character of size exclusion chromatography (SEC) resin media determine both the relative molecular weight range over which the media offers good selectivity. It also reflects the capacity of the media in regard to the relative column bed volume accessible to a separation target of given molecular weight (MW). For some time it has been appreciated that different SEC columns can be combined in series to generate a SEC train of improved Kav characteristics for a defined application. Use of dual columns is expensive but allows use of SEC media differing in particle size and composition. However custom tailoring of separation properties requires use of different media and columns. In the present work it is shown that it is possible to achieve similar results by combining resin media of similar particle size but different Kav properties in the same column. Such an approach can offer several advantages including increasing the relative column capacity (column volume available to) for a target of specific MW. This may be particularly attractive in regard to preparative SEC separations involving targets and contaminants differing significantly in MW.

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Blending of Size Exclusion Media to Tailor Chromatographic Performance

Abstract

The Kav character of size exclusion chromatography (SEC) resin media determine both the relative molecular weight range over which the media offers good selectivity. It also reflects the capacity of the media in regard to the relative column bed volume accessible to a separation target of given molecular weight (MW). For some time it has been appreciated that different SEC columns can be combined in series to generate a SEC train of improved Kav characteristics for a defined application. Use of dual columns is expensive but allows use of SEC media differing in particle size and composition. However custom tailoring of separation properties requires use of different media and columns. In the present work it is shown that it is possible to achieve similar results by combining resin media of similar particle size but different Kav properties in the same column. Such an approach can offer several advantages including increasing the relative column capacity (column volume available to) for a target of specific MW. This may be particularly attractive in regard to preparative SEC separations involving targets and contaminants differing significantly in MW.

Introduction

The performance of size exclusion chromatographic (SEC) resin media is related to the available volume ratio (Kav) which relates the column bed volume available to molecule of a certain size to the total column bed volume. This can be expressed as:
Kav = (Vc-Vo) / (Vt-Vo) [ 1 ]
where Vc = mobile phase elution volume for substances of specific MW, Vt = total column volume and Vo = void volume (ref. 1, 2). Note that Vt-Vo includes the solid volume of the matrix inaccessible to all mobile phase components. Therefore Kav is not equivalent to Kd which represents the ratio of stationary phase fraction available for diffusion of a given molecular species. However given he low amount of polymeric material in modern SEC media Kav and Kd are similar and related by a constant ratio independent of the MW of a specific target molecule.

SEC media is often characterised by plots of Kav versus logarithm of molecular weight for a set of polymeric or protein standards. Such plots are typically sigmoidal in nature with good separation afforded (Figure 1). In general the MW region over which a SEC media affords good selectivity is related to the linear region of the Kav curve (Figure 1). So too Kav plots can be used to provide insight into the "capacity" or volume of target containing mobile phase which can be added to a column and still effect good SEC separation of target from contaminant(s) of known MW. Ideally target and contaminant(s) should lie as far apart as possible on Kav curve. Obviously this means that in some cases one would wish different Kav slope than in other cases.

In general SEC media has changed little the past few decades when SEC was often used to analytically separate molecules to provide an...