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

Entropic HPLC (SEC) vs. Enthalpic HPLC

Figure 1: During an SEC analysis, the entire polymer sample is eluted within the pore volume of an SEC column. Provided that the column is calibrated with polymers of known molecular weight, the elution profile of a sample is transformed into its molecular weight distribution, from which all statistical average molecular weights can be readily determined in minutes. Before the age of SEC, these measurements would have taken many weeks to accomplish using a range of sophisticated instrumentation.
Because of the similarities between entropic HPLC (SEC) and enthalpic HPLC, SEC closely follows in the footsteps of enthalpic HPLC in terms of packing and instrument developments. Nevertheless, there are major differences between the two modes of chromatography:
  • The total SEC separation occurs within a relatively small elution volume window (that is, the total pore volume of the column), whereas with HPLC, there is no theoretical upper elution volume limit.
  • Many of the polymer's characteristics (molecular weight distribution [MWD], average molecular weights, molecular size parameters, the nature of the polymerization reaction, and the sample composition) lie within the pore volume of the column (see Figure 1).
  • Extracolumn peak broadening and skewing from the injector, column, detector cells, and interconnecting tubing can severely distort the polymer peak shape and compromise information within the pore volume.
  • Because the diffusion coefficient and relative viscosity of a polymer sample are orders of magnitude greater than those of a small molecule, precautions must be taken to reduce their influence.
  • Modern SEC with light-scattering, viscosity, and spectroscopic detectors can be used for determining molecular weights, long-chain branching, molecular size parameters, and molecular conformation all as a function of molecular weight.
  • To facilitate spectral interpretation of complex spectra, SEC often is used to "clean up" complex, multicomponent samples for on-line or off-line Fourier transform infrared (FT-IR), mass spectrometry (MS), or nuclear magnetic resonance (NMR) measurements.

SEC Packings: State of the Art

Composition of SEC Packings

Table II: Characteristics of high-performance SEC "single-pore-size" packings
Table II lists "single-pore-size" packings, along with their particle and average pore sizes offered by major SEC companies. The polarities of mobile phases that are compatible with specific packings are designated as follows: Aq stands for aqueous (hydrophilic) mobile phases; N stands for nonpolar (hydrophobic) mobile phases; P stands for polar organic mobile phases including aqueous organic solutions; and F stands for fluorinated mobile phases. Example mobile phases for each class are given in the footnote of Table II. (When changing columns over to different mobile phases, be certain to follow manufacturer's instructions to avoid damaging the packed bed.)

There are essentially two types of SEC packing compositions: silica, with or without surface modification, and cross-linked polymeric packings, which are nonpolar (hydrophobic), hydrophilic, or ionic. The most common silica packings consist of chemically bonded 1,2-propanediol functional groups that render the surface hydrophilic. This stationary phase blocks or reacts with many of the acidic silanol groups, neutralizing the surface and making it ideal for SEC separation of biopolymers and synthetic water-soluble polymers, except for polyelectrolytes or charged polymers. Cationic polyelectrolytes or amino-containing polymers will ion exchange onto residual acidic silanol groups on bare or even diol-modified silica. Anionic polyelectrolytes or carboxylic acid–containing polymers will be ion excluded from negatively charged pores unless a significant amount of electrolyte is added to the mobile phase to shield anionic functionalities. When choosing diol-coated silica packings, especially small-pore-size packings, the pore size is reduced because of the pore volume taken up by the diol stationary phase.

Bare silica is a useful packing for the analysis of nonaqueous polar or nonpolar organic mobile phases, especially for high-temperature applications for the analysis of nonionic polymers. However, bare silica is not recommended with aqueous mobile phases because of the presence of active silanol adsorptive sites and the finite solubility of silica in aqueous buffers, especially at elevated temperatures. Furthermore, silica packings will continually degrade (that is, hydrolyze in aqueous mobile phases), exposing more silanol groups with time. A detailed summary of major SEC companies offering silica packings appears in Table II.

The newest type of silica-related packing is an ethylene-bridged hybrid inorganic-organic (BEH) packing offered by Waters Corporation (9). This packing is a mixed composition of silica and organosiloxanes which form poly-ethoxyoligosilane polymers. Compared to silica packings, BEH particles have improved chemical stability, reduced silanol activity, and a larger pore size (9).

The polymeric packing of choice for the separation of nonpolar (hydrophobic) polymers is cross-linked poly(styrene-co-divinylbenzene) (or polydivinyl benzene by Jordi Labs), introduced in 1964 by Moore (6). The popularity of this packing stems from the fact that the degree of cross-linking, which in turn influences the pore size, can be adjusted by carefully controlling polymerization conditions. Secondly, the solubility parameter of poly(styrene-co-divinylbenzene) is within the range of most vinyl types of polymers. If the packing is used with a mobile phase that has a similar solubility parameter, such as tetrahydrofuran or toluene, polymer adsorption onto the packing is prevented.

There have been a number of different hydrophilic cross-linked packings developed for the SEC of biopolymers and synthetic water-soluble polymers. Most of these packings are proprietary hydroxylated derivatives of cross-linked polymethacrylates. Unusual polymeric packings for aqueous SEC include sulfonated cross-linked polystyrene, polydivinylbenzene derivatized with glucose or anion-exchange groups, a polyamide polymer, and Novarose, a high-performance crossed-linked agarose (Table II). However, when using organic or polar SEC packings for aqueous SEC, one must guard against possible adsorption if there are extensive hydrophobic regions on the packing or polymer. Unwanted hydrophobic interactions can also occur with packings if the electrolyte concentration of the mobile phase is too high. This type of adsorption can be eliminated either by lowering the electrolyte concentration of the mobile phase, increasing column temperature, or adding an organic moderator, such as methanol, to the mobile phase (10).


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