Column selection for SEC is usually a straightforward task. After choosing an appropriate SEC-column vendor, one next picks
a packing that is compatible with the mobile phase required, and a pore size that will cover the anticipated molecular weight
range of samples, using Table II for guidance. If the expected molecular weight range is narrow, that is, less than two orders
of magnitude, it is best to choose a single-pore column; if the molecular weight spread is broad, that is, greater than two
orders of magnitude, a mixed-bed or wide-pore column should be selected.
The best way of choosing packing pore size is by comparing SEC calibration plots that are posted for each column type. The
appropriate column or column set can be chosen from the molecular weight range, the slope linearity in which smooth slopes
without inflections or discontinuities are desired, and the slope steepness, whereby shallow slopes give the best resolution.
Please remember, however, that these calibration plot characteristics supplied by the manufacturer depend on the chemical
composition of the polymer and the nature of the mobile phase used to construct the plot.
If you find that you can tailor-make a column set with superior characteristics compared to purchasing either mixed bed or
wide-pore size columns, the following guidelines should be used:
- The inner diameters of all columns in the set should be the same.
- The type of packing in each column should be matched.
- The slopes of all of the SEC calibration plots should be the same.
- All columns should have the same pore volume or pore volume to interstitial volume ratio.
- SEC calibration plots should be adjacent to one another, but not overlapping (12).
- The pore-size order of the column set is inconsequential (13).
After the correct pore size is selected, a column with the smallest particle size, or highest efficiency should be chosen.
It is curious that many SEC column manufacturers tend to keep an extensive backlog of less-efficient high-performance columns,
even though they have more highly efficient columns listed. If there is a choice, users should always purchase the state-of-the-art,
highest efficiency column that meets their needs. Even a small increase in column efficiency or resolution may uncover hidden
components that may otherwise go undetected, or reveal that product failure was caused by a slight polydispersity increase.
More importantly, however, is the fact that accuracy of molecular weight measurements increases somewhat exponentially with
column efficiency (12).
As we have previously discussed (1), any HPLC column in principle can be used for SEC by adjusting the mobile-phase composition
so that there are no enthalpic interactions between packing and sample. Pore size and pore-size distribution for a given single-pore
column should be uniform and tightly controlled, and available in at least three or four pore sizes to cover a broad molecular
weight range from oligomers to ultrahigh-molecular-weight components of about 106 . Because high-molecular-weight solutes have diffusion coefficients orders of magnitude smaller than those normally encountered
in HPLC, it is critical that the smallest SEC particles be used.
Although column pore-size listings are quite useful for selecting columns of the correct molecular weight range, we feel that
it is more instructive to consult actual SEC calibration plots of a number of test polymers to choose columns that give shallow
and smooth linear slopes. Consequently, we recommend that users study product literature before making a selection of columns.
However, bear in mind that the slope and log MW and elution volume intercepts depend on the molecular conformation and monomer
molecular weight of the polymer under study. When comparing columns, choose the ones that have the highest resolution — that
is, the lowest amount of broadening and the lowest calibration slope according to the following equation (12):
R*sp = 0.58/σ'D
where σ' is the amount of peak broadening, that is, standard deviation in units of volume of a small monodisperse solute;
is the negative slope of the SEC calibration plot (log MW vs. elution volume); and R*sp is the specific resolution normalized with respect to column length L.
Many first-time SEC users assume incorrectly that a single-pore-size packing is only capable of separating or retaining macromolecules
whose molecular size matches the pore openings of a packing. However, size separation does not depend on whether or not a
macromolecule can penetrate or occupy a pore; rather, it depends on the fraction of pore volume a macromolecule can occupy,
provided that it is sufficiently small to penetrate the pores in the first place. This pore-volume fraction is actually defined
by the SEC distribution coefficient K
is the elution volume of a polymer, V
is the interstitial volume of the packed column, and V
is the total pore volume of the packed column. Because a single-pore-size packing can accommodate a wide molecular-size or
molecular-weight range (12), the slope of the SEC calibration plot of log M vs. V
R, is finite, rather than infinite or perpendicular to the abscissa. Except for some tailor-made porous materials, silica and
polymeric SEC packings have finite pore-size and pore-opening distributions, as well as different pore shapes, further decreasing
the SEC calibration curve slope (12).