Gradient elution in reversed-phase HPLC-separation of macromolecules

Abstract

Wilmingion, Del. 19898 E. I. du Pont de Nemurs 8 CO. Gradient Elutior Separation of Macromolecules have led workers to make new proposah for the basis of such separations. UWd R w h hc, 2281 william e., york. F b r o 1. Fractional migration (XIL) of solute bands along the column and resulting c h r o m a t~ for Isocratlc vs. gradient elution tewn Heights, N.9.10598 ANMYTICAL cHEMismy.

HPLC of Peptides and Proteins

“…According to the Knox equation, h is related to the reduced mobile phase velocity, ν, through the relationship h = A ν 0.33 + B ν −1 + C ν, where ν = ud p / D m , u is the linear velocity, and A , B , and C are constants that describe the packing and solute transport characteristics of a particular sorbent in a column. Observations of reduced plate heights ( h values) in the range 3 < h < 15 for small peptides with reversed‐phase sorbents of average particle size of 3 to 10 µm, and for proteins and other high molecular weight macromolecules where h values are >50, have frequently been made, confirming that the Knox equation can be approximated (Snyder et al, ) for small peptides as h = A ν 0.33 and for proteins as h = C ν, because, in the latter case, only the intraparticle pore transport and surface interaction (term C ), contributes significantly to the band broadening.…”

“…The S values of peptides and proteins can be empirically correlated with their molecular weights (Snyder et al, ), based on a correlation of S = 0.48( MW ) 0.44 . Thus S values, estimated with this equation or, alternatively, values of similar peptides or proteins taken from the literature, can be used to calculate a reasonable starting point for the initial experiments according to the following equation: …”

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

“…There are several beneficial aspects when analyzing large molecules by 2D-LC in comparison to small ones. It has been reported that resolution is independent from column length for RPLC gradient elution of proteins (53,54). Therefore, the gradient slope has a greater contribution than column length on the chromatographic separation, allowing the use of short columns and analyte peak focusing on the 2 D column inlet (54).…”

“…It has been reported that resolution is independent from column length for RPLC gradient elution of proteins (53,54). Therefore, the gradient slope has a greater contribution than column length on the chromatographic separation, allowing the use of short columns and analyte peak focusing on the 2 D column inlet (54). Second, the under sampling issue, which is well known with small molecules (48), is less critical with large ones due to broader peaks caused by slower mass transport due to their larger size (55).…”

Nonlinear Gradient Isotherm Parameter Estimation for Proteins with Consideration of Salt Competition and Multiple Forms

“…+ Current address: Air Products and Chemicals, Inc., Allentown, PA 18195-1501Ghrist and Snyder, 1988a,b;Kang and Mc-Coy, 1989). Snyder and co-workers (Snyder, 1980;Snyder et al, 1983Snyder et al, ,1988Eble et al, 1987;Cox et al, 1988) have put forth a model for multicomponent systems which assumed a linear relation between the log of the capacity factor and the solvent strength (LSS). Snyder measured deviations in peak symmetry in gradient and in isocratic elution to determine generalized correlations for changes in band shape.…”