Size-exclusion chromatography expressions

Most size exclusion chromatography (SEC) practitioners select their columns primarily to cover the molar mass area of interest and to ensure compatibility with the mobile phase(s) applied. A further parameter to judge is the column efficiency expressed, e.g., by the theoretical plate count or related values, which are measured by appropriate low molar mass probes. It follows the apparent linearity of the calibration dependence and the attainable selectivity of separation the latter parameter is in turn connected with the width of the molar mass range covered by the column and depends on both the pore size distribution and the pore volume of the packing material. Other important column parameters are the column production repeatability, availability, and price. Unfortunately, the interactive properties of SEC columns are often overlooked. 

Figure 2 shows the effect of flow rate on column efficiency using the SW-2000 column with cytochrome C. The column efficiency expressed as the number of theoretical plates (N) was dependent on flow rate, a result typical of size exclusion chromatography. 

EROD activity is measured in the H411E cells as follows. The cells are seeded at 7,000 cells per well in 250 xL of Dulbecco s modified Eagles culture media (Tillitt et al., 1991). After an initial incubation period of 24 hours, the cells are dosed with 5 xL volumes of eiuiched SPMD extracts (cleanup of extracts generally includes dialysis and size exclusion chromatography [SEC]) and incubated for an additional 72 hours. Sample dose is typically expressed as g-equivalents triolein or whole SPMD per mg cellular protein. Multiple exposures are performed at each of six (typically) sample concentrations, using a dilution series. Afterwards, the microtiter plates are washed three times with distilled water to lyse the cells. EROD activity (pmol mg cellular protein per min) in each sample is measured... 

The molecular mass of the expressed PA-PLAi was estimated to be 110 kDa by SDS-PAGE, and 97.6 kDa by matrix-associated laser desorption/ionization [48]. A value of 97.6 kDa was also obtained from the deduced amino acid sequence of the open reading frame (ORF) [48]. The enzyme purified from bovine testes had an apparent molecular mass of 440 kDa, as determined by size exclusion chromatography [48], suggesting that PA-PLAi exists as a homotetramer of 98 kDa subunits in solution. The molecular mass of bovine brain PA-PLAi, estimated by the same method, was 200 kDa, suggesting that it exists as a homodimer in the brain [48]. 

Empirical MMD functions possess two or more adjustable parameters and the MM averages are given by closed expressions. Empirical MMD functions are used to compare MMD data obtained by different methods and especially to compare data from viscosity measurements with data from Size Exclusion Chromatography or Osmometry." They are also used in Dynamic Light Scattering to obtain Mn and Mw (see section on Light Scattering below). 

Often, size exclusion chromatograms (SEC) (compare section 11.7, Size Exclusion Chromatography) of polymers under study are expressed as differential representations of molar mass dispersity. The chromatographic retention volumes are directly transformed into the molar masses. This approach renders useful immediate information about tendencies of molar mass evolution in the course of building or decomposition polyreactions but the absolute values of molar mass can be only rarely extracted from it. As a rale, polystyrene calibrations are applied for molar mass calculation so that one deals with the polystyrene equivalent molar masses, not with the absolute values. The resulting dispersity (distribution) functions may be heavily skewed because the linear part of the calibration dependence for the polymer under study may exhibit well different slope compared with the polystyrene calibration, which was employed for the transformation of retention volumes into molar masses. 

Benoit and coworkers (15) showed that size exclusion chromatography separates polymer molecules by hydrodynamic volume. The hydrodynamic volume can be expressed as the product of intrinsic viscosity and molecular weight ... 

The volume fractions of both micropores (Sp) and voids (inter-microglobule porosity, e ) represent the total porosity (8t). This value indicates a percentage of pores in the monolith. The pore size distribution can be calculated from inverse size exclusion chromatography (ISEC) data or from mercury intrusion. " Together, these two values translate into a total pore volume Vp, expressed in mlg". ... 

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