The State of the Art and Future Trends of Size-Exclusion Chromatography Packings and Columns - - Chromatography Online
The State of the Art and Future Trends of Size-Exclusion Chromatography Packings and Columns

LCGC North America
Volume 7, Issue 30, pp. 544-563

Packing Pore Size

The calculated pore sizes in Table II require some explanation. Usually for rigid packings, like silica, mercury intrusion porosimetry is used to measure the average pore size, as well as the pore size distribution. However, for polymeric packings an erroneous, but historical method is applied based on the extended chain length of polystyrene. For example, all three cross-linked poly(styrene-co-divinylbenzene) packings (SDV, Polyolefin, and MCX) offered by Polymer Standards Service are available with pores as large as 1 107 or 1-mm pore size opening (see Table II). These data suggest that the pore size of a 10-m particle is 1 mm or the pores are 100 larger than the particles themselves! The reason for this obvious impossibility is that most manufacturers still use an early proposed, but incorrect pore-size convention that is based on the extended chain length of the lowest molecular weight, excluded polystyrene standard.

This pore-size designation incorrectly assumes that a polystyrene chain length of 1 corresponds to a molecular weight of 40. For example, a polystyrene extended chain-length designation of 1 106 corresponds to a polystyrene molecular weight of 40 106 . Based on the corresponding hydrodynamic radius of a 40 106 MW excluded polystyrene, the estimated pore size is approximately 3000 , a more realistic value. Unfortunately, most column manufacturers not only use this extended-chain designation, but to complicate matters, fail to inform users. In Table II, we have tried to identify those packings that still rely on this antiquated designation. Regardless of the pore-size designations that are used, these extremely large polymer molecular weight exclusion limits offered by suppliers are beyond the practical range of polymer separations, unless one is using these packings for the separation of nanosize particles or associated molecular structures.

Coupled Columns, Mixed-Bed Columns, and Wide-Pore Packings

Table II: Continued
There are three methods available for extending the molecular weight separation range and improving the linearity of the SEC calibration slope. The first method calls for coupling together columns that have adjacent molecular weight separation ranges but the same calibration slopes, or matched V i /V 0 ratios, where V i is the pore volume and V o is the interstitial volume. The second approach, which has been used quite successfully, is to mix together (in the same column) calculated fractions of two or three single-pore packings of slightly overlapping molecular weight ranges but with the same pore volume or V i /V 0 ratio. These "mixed-bed" or "linear columns" have linear and shallow calibration slopes, as presented in Table III. These columns are very useful for laboratories that receive a variety of polymer samples of unknown and sometimes broad distributions. Nevertheless, small calibration slope discontinuities or deviations from linearity can still occur.

Table III: Characteristics of specialty high-performance SEC packings*
The third type of column packing, which was met with rave reviews, comprises the so-called "multipore" or "wide-pore" packings that are listed in Table III. These packings consist of particles that contain multiple or a broad distribution of pore sizes. Calibration plots of the three molecular weight ranges of multipore columns manufactured by Tosoh Bioscience are shown in Figure 2. TSKgel SuperMultiporeHZ-M (4 m) and SuperMultiporeHZ-H (6 m) give extraordinarily linear calibration plots with no observable calibration slope discontinuities. SuperMultiporeHZ-H has a linear molecular weight range from small molecules (1 102 ) to ultrahigh-molecular-weight polymers (1 107 ) with respect to polystyrene: a five-order molecular weight range for a single 15-cm column with a separation time of less than 6.5 min. The separation range for the -M column is about an order of magnitude lower, but the calibration slope is much lower, which is a desired feature. The molecular weight separation range of the -N column is only three-orders of magnitude, 1 102 to 1 105 , but the slope is remarkably the lowest of the three columns. Unfortunately this column has a second, steeper region from 1 105 to 5 106 that will distort the shape of the high-molecular-weight end of the chromatogram tracing. Please note that Tosoh Bioscience recently has introduced wide-pore-size packings, the TSKgel SuperMultipore PW series, for SEC analysis of water-soluble polymers. Agilent Technologies and BioChrom Labs also market their versions of wide-pore SEC packings, as listed in the third row of Table III.


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