Chromatography of proteins at high organic solvent concentrations: An inverse-gradient reversed-phase hplc method for preparing samples for microsequence analysis

It is not generally recognized that a skewed U-shaped, or bimodal, dependency exists between retention times and concentration of organic solvent during reversed-phase chromatography (RPC) of low-Mr organic compounds, peptides and small proteins. 1 - 6 Interestingly, those reversed-phase packings which best exhibit this behavior for proteins (Figures 1B, C, and D) (i.e., strong retention at high concentrations of organic solvent) are characterized by large surface areas (200 to 400 m²/g) and high carbon content (7 to 15%). Such packings (e.g., Beckman Ultrasphere-ODS, [Figure 1B] Zorbax-ODS [Figure 1C], ODS-Hypersil [Figure 1D], Brownlee C₈ VeloSep [data not shown]) are characterized by small pore sizes (6 to 12 nm, 60 to 120 Å) and are commonly utilized for RPC of low-Mr substances (e.g., peptides). By contrast, those packings which exhibit only weak retention for proteins at high organic solvent concentrations (e.g., Brownlee RP-300 [Figure 1A]; Beckman RPSC and Toya-Soda TSK-Phenyl- 5PW [data not shown]) are those designed specifically for RPC of proteins (typically, packings with large pore sizes [30 to 100 nm, 300 to 1000 Å] and small surface areas [approx. 60 m²/g]).