Convenient experimental determination of adsorption isotherms in a Hydrophobic Interaction Chromatography system

Summary HPLC was combined with a packable microbore guard column to obtain the adsorption isotherm of lysozyme in a Hydrophobic Interaction Chromatography system. The equipment configuration enabled isotherm determination of the protein on a relatively low pressure chromatographic media (TosoHaas 650M Phenyl).Notation C_m,i is the mobile phase concentration of protein. (M/L³ _(liquid)) - C_m,0 =0 - C_s,i is the stationary phase concentration of protein. It is the concentration of protein on the chromatographic media. (M/L³ _(solid)) - C_s,0 =0 - M,L is the dimensions mass and length - V_r,i is the retention volume of the peak front that corresponds to a mobile phase protein on the concentration C_m,i. (L³ _(liquid)) - i i is a counter that is used to keep track of C_m, C_s, and V_r.For example, i=1 in the term C_m,i denotes the first, and lowest, mobile phase protein concentrations are described by higher values of i. - V_d is the system dead volume. It consists of all of the system volume that the mobile phase "sees" or contacts, includingchromatographic media interparticle and pore volume. (L³ _liquid) - V_s the stationary phase volume. V_s is the nonporous bead volume. For porous beads, V_s is the bead volume - the porevolume. (L³ _(solid)) - V_e is the empty column volume. (L³ _liquid) - V_m is the packed column mobile phase volume and consists of the pore volume and the excluded volume. (L³ _(liquid)) - V_{e system} is the empty column system volume. (L³ _(liquid)) - V_frit the volume of mobile phase that fills the column frits. (L³ _(liquid)) - V_woc the system volume without the column connected. (L³ _(liquid))     

The displacement effect of phenylalanine on lysozyme elution in hydrophobic interaction chromatography

The volume, retention time, and shape of the lysozyme peak eluted from a hydrophobic interaction chromatography column (TosoHaas 650 M Phenyl) was influenced by the presence and concentration of phenylalanine in the elution buffer. Lysozyme peak retention time decreased by a factor of 2.5 with the addition of 86 mM phenylalanine to the elution buffer.