Size-exclusion chromatography in an analytical perspective

A Method for the Quantitation of Charge by Size Exclusion Chromatography Demonstrated with Components of Ficoll 400

“…It can be seen from Figure 8, as well as from Figure 4, that the pullulans, ficoll F17-17 and myoglobin all fall within this margin and errors are essentially dominated by flow instability. Our performance is quite comparable to other equipment and investigations [39,65,66] and future technology would have to produce at least a fourfold improvement in order that flow rate instability not be the limiting technique factor. As it is unlikely that the mechanics of pump design could be substantially improved, performance will have to be advanced either with a flow feedback system [67] or simply by replacing the fixed time base in current chromatography with accurate monitoring of actual volume flow.…”

Gel Filtration

“…Very small solutes will permeate the entire pore volume, V p , of the matrix and will be eluted in the total liquid volume, V t . The separation of solutes is quantified by the resolution factor R s and for gel filtration is given by eqn [1] (Hagel, 1993):…”

“…However, size differences much smaller than 50% (e.g. 20%) are difficult to resolve by gel filtration (Hagel, 1993). The optimal experimental conditions will depend on the size differences of the solutes and on the processing rate.…”

“…If such calibration standards are not available, calibration may be performed by using a polymer standard (e.g. dextran) and expressing the size as apparent viscosity radius (Hagel, 1993). For globular proteins, the viscosity radius is close to the Stokes radius.…”

Stationary phases for peptide analysis by high performance liquid chromatography: a review

“…The proposed five peptides form a series with repeating tenresidue units (800 to 4000 Da) and with increasing basicity (1 to 5 positively charged residues) ( Table 2) and can be used for column calibration and for monitoring of ionic and hydrophobic interactions. A well calibrated column permits the determination of solute molecular masses with a precision close to that of absolute methods [109]. The peptides remain in a random coil configuration in both denaturing and non-denaturing mobile phases.…”