sec hplc column resistant to sodium hydroxide

), which have anomalous migrations in gel filtration, resulting in a defined overestimation of the molecular weight due to their highly extended conformation.

This is the case for some Ca 2+ sensor proteins, e.g., calmodulins from different organisms ( Sorensen and Shea, 1996

For example, a protein with an elongated shape could elute at a position that does not correspond to its dimension and which is significantly different from the position of a spherical protein having the same molecular weight. However, because the shape of proteins can vary significantly and may be not known for an unknown protein, care must be taken in the determination of molecular size from elution volume. If the protein of interest has the same shape (generally globular) as the standard calibration proteins, the gel filtration experiment provides a good estimate of its molecular weight. The molecular weight of a protein can be determined by comparison of its elution volume parameter K d, which represents the distribution of a given solute between the stationary and mobile phases (see Data analysis below), with those of different known calibration standards. Molecules with intermediate dimensions will be eluted with an elution volume (V e) comprised between the void volume and the total volume available to the mobile phase (the smaller the molecule, the greater its access to the pores of the matrix, and thus the greater is its V e). Molecules smaller than the fractionation range, which are able to diffuse into the pores of the beads, have access to the total volume available to the mobile phase, therefore they move through the bed more slowly and elute last.

Molecules larger than the fractionation range, which are completely excluded from entering the pores, flow quickly through the column and elute first at the void volume (V 0), which is the interstitial volume outside the support particles. Gel filtration separates molecules of different sizes and shapes based on their relative abilities to penetrate a bed of porous beads with well-defined pore sizes, which identifies the fractionation range.

Sec hplc column resistant to sodium hydroxide