The Effect of SEC Column Arrangement of Different Pore Sizes on Resolution and Molecular Weight Measurements

Aug 01, 2011
Volume 29, Issue 8, pg 668–671

In the practice of size-exclusion chromatography (SEC), the suggested arrangement of columns with different pore sizes appears to be contradictory. Although most column manufacturers recommend that the large-pore-size column be placed first in a set, for example connected to the injector, followed by columns with progressively larger pores, other workers in the field suggest the reverse. In view of these conflicting practices, a study was conducted to determine the influence of column arrangement on molecular size separation and peak broadening using a high-precision SEC chromatographic system and a two-column set with exclusion molecular weight limits of 2 × 10 3 and 2 × 10 6 g/mol. As would be expected, SEC column order was found to have no effect on peak resolution, column efficiency, calibration curve fit, or measured molecular weights on a series of polystyrene standards and poly(tetramethylene ether glycol) samples. These results suggest that any column order can be employed provided that the direction of flow through each column established by manufacturer is used and that the minimum length of low-dead-volume capillary tubing is used for column coupling.

Although size-exclusion chromatography (SEC) is a well-established technique used by most laboratories involved with macromolecule characterization, there is still disagreement about which pore-size column to place first when using a series of SEC columns with different pore sizes. For example, Striegel and colleagues (1) recommend that the smallest-pore-size column should be placed first in a bank of SEC columns, while Mori and Barth (2), as well as most column manufacturers, propose installing columns in order of decreasing pore size. Nevertheless, because any multicolumn or multidimensional separation process, in general, is strictly reversible, the arrangement of individual SEC columns containing packings with different pore sizes should make no difference regarding the final molecular weight (MW) separation.

However, there is an instance whereby SEC column order could influence final results: if a constant-pressure pump with noticeable flow-rate pulsations is used in conjunction with conventional SEC columns packed with soft or semirigid, large-particle-size packings. Because these types of columns help dampen flow-rate pulsations, the first column in a series will take the brunt of the pulsations. Because small-pore-size conventional packings are physically more stable than large-pore-size packings, columns of the former type need to be placed first, followed by the more fragile larger-pore-size packings; in this manner, particle compression or fracture of the latter packing is reduced.

Because of the inherently lower efficiency of conventional columns (for example >50 µm), significant sample dilution would occur when the injected sample reaches the larger-pore-size packing where most of the high-MW separation would occur (1). Consequently, somewhat higher polymer concentrations could be injected without seriously worsening "concentration effects" that would otherwise distort the MW distribution.

By coupling columns with packings of different pore sizes, a desired MW separation range and degree of resolution can be achieved. The downside of this approach is that often it is difficult to arrive at a linear calibration, which inherently has less error than a higher-order polynomial fit. To circumvent this difficulty, individual columns packed with a mixture of two or more different pore sizes (for example, mixed-bed columns) to give a linear-calibration curve are now readily available. Moreover, new packing technology has been introduced in which SEC packings consist of layers of different, tightly controlled pore sizes (3,4). With the use of high-performance mixed-bed and multipore packings, column order is no longer an issue. Nevertheless, the use of columns with individual pore sizes remains popular because of the flexibility they afford.

For this study, we employed an advanced SEC instrument well suited for determining polymer MW with high accuracy and precision (5), especially when used in conjunction with high-resolution SEC columns. Two columns were selected that had substantially different separation ranges and average pore sizes but similar column dimensions to give an overlapping "MW vs. elution volume" calibration curve. Parameters measured and compared for the two column-set arrangements were elution times, peak asymmetry, peak efficiency, resolution, calibration curve polynomial coefficients, and MW measurements and polydispersities. Test polymers were nearly monodisperse polystyrene standards and low-MW poly(tetramethylene ether glycol)s.

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