Optimization of preparative hydrophobic interaction chromatographic purification methods

Online Hydrophobic Interaction Chromatography–Mass Spectrometry for Top-Down Proteomics

“…We recently reported that ammonium tartrate provides effective separation of intact proteins when used in HIC and minimizes protein adduction and MS signal suppression, compared to the common sulfate salts. , Nevertheless, ammonium tartrate is not volatile and, thus, a desalting step is necessary prior to MS analysis of the HIC eluates. Ammonium acetate (NH 4 OAc), on the other hand, is volatile and features reduced nonspecific sodium adduction. , Unfortunately, it provides inadequate protein retention when used with conventional HIC materials, such as PolyPROPYL A . Using a HIC column with pentyl ligands, Gooding et al obtained adequate retention of proteins only with 4 M NH 4 OAc .…”

“…Ammonium acetate (NH 4 OAc), on the other hand, is volatile and features reduced nonspecific sodium adduction. , Unfortunately, it provides inadequate protein retention when used with conventional HIC materials, such as PolyPROPYL A . Using a HIC column with pentyl ligands, Gooding et al obtained adequate retention of proteins only with 4 M NH 4 OAc . Hence, we propose to increase the hydrophobic character of the HIC stationary phase to the point that proteins are retained and eluted using NH 4 OAc concentrations of 1 M or less, which is a range that has been previously demonstrated to be compatible with MS analysis .…”

“…Ammonium acetate (NH 4 OAc), on the other hand, is volatile and features reduced nonspecific sodium adduction. 39,40 Unfortunately, it provides inadequate protein retention when used with conventional HIC materials, such as PolyPROPYL A. 38 Using a HIC column with pentyl ligands, Gooding et al obtained adequate retention of proteins only with 4 M NH 4 OAc.…”

Biochemical and Synthetic Polymer Separations

“…The ligands are typically short-chain alkyl or phenyl groups, and retention increases with ligand length ( [70,71]; see the examples of Figure 13 It should be noted that IEC and IMAC columns can each display HIC behavior under high-salt conditions, due to the hydrophobic contributions of spacer and cross-linking groups [68]. The support is typically covered with a polymeric, hydrophilic coating (or linker groups) in order to provide a wettable, noninteractive surface; hydrophobic ligands are then attached to the polymer or linker.…”

The applications of hydrophobic interaction chromatography to the purification of plant proteins

“…The effect of pH on the strength of the hydrophobic interaction must therefore be called secondary and this effect may be unpredictable. Nevertheless, the pH certainly has an effect and this may be used to adjust the retention and selectivity (Engelhardt and Schon, 1986;Gooding et al, 1986;HjertCn et al, 1986;Buckley and Wetlaufer, 1990).…”