New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications (original) (raw)
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Journal of Chromatography A, 2005
This article describes a new complementary peptide separation and purification concept that makes use of a novel mixed-mode reversedphase/weak anion-exchange (RP/WAX) type stationary phase. The RP/WAX is based on N-(10-undecenoyl)-3-aminoquinuclidine selector, which is covalently immobilized on thiol-modified silica particles (5 m, 100Å pore diameter) by radical addition reaction. Remaining thiol groups are capped by radical addition with 1-hexene. This newly developed separation material contains two distinct binding domains in a single chromatographic interactive ligand: a lipophilic alkyl chain for hydrophobic interactions with lipophilic moieties of the solute, such as in the reversed-phase chromatography, and a cationic site for anion-exchange chromatography with oppositely charged solutes, which also enables repulsive ionic interactions with positively charged functional groups, leading to ion-exclusion phenomena. The beneficial effect that may result from the combination of the two chromatographic modes is exemplified by the application of this new separation material for the chromatographic separation of the N-and C-terminally protected tetrapeptide N-acetyl-Ile-Glu-Gly-Arg-p-nitroanilide from its side products. Mobile phase variables have been thoroughly investigated to optimize the separation and to get a deeper insight into the retention and separation mechanism, which turned out to be more complex than any of the individual chromatography modes alone. A significant anion-exchange retention contribution at optimal pH of 4.5 was found only for acetate but not for formate as counter-ion. In loadability studies using acetate, peptide masses up to 200 mg could be injected onto an analytical 250 mm × 4 mm i.d. RP/WAX column (5 m) still without touching bands of major impurity and target peptide peaks. The corresponding loadability tests with formate allowed the injection of only 25% of this amount. The analysis of the purified peptide by capillary high-performance liquid chromatography (HPLC)-UV and HPLC-ESI-MS employing RP-18 columns revealed that the known major impurities have all been removed by a single chromatographic step employing the RP/WAX stationary phase. The better selectivity and enhanced sample loading capacity in comparison to RP-HPLC resulted in an improved productivity of the new purification protocol. For example, the yield of pure peptide per chromatographic run on RP/WAX phase was by a factor of about 15 higher compared to the standard gradient elution RP-purification protocol.
Reverse-Phase Chromatography RP-HPLC for Peptides
Reverse-phase high performance liquid chromatography (RP-HPLC) is an extremely useful tool for analytical biochemists. However, unlike small molecule HPLC, separations of proteins and peptides are nearly always performed under gradient conditions. There are other differences that one needs to be aware of in order to develop RP-HPLC separations of proteins and peptides as efficiently as possible. The general guidelines given in this short article may help reduce your method development time. RP-HPLC of complex peptide or protein mixtures remains a general method of choice because of the resolution it provides. Unlike small organic molecules whose chromatographic behavior is described by a finite partitioning equilibrium between the stationary phase and the mobile phase, proteins and peptides typically do not exhibit such an effect. Instead, they exhibit an adsorption phenomenon in which the polypeptide is adsorbed onto the stationary phase and elutes only when the solvent strength of the mobile phase is sufficient to compete with the hydrophobic forces keeping it there. For this reason, elution of peptides or proteins from reverse-phase supports is by gradients of increasing solvent strength. When run under isocratic conditions, peaks for proteins and peptides are typically much broader than their small molecule counterparts.
Journal of Chromatography A, 2010
Caustic regeneration procedures are often used in chromatographic purification processes of peptides and proteins to remove irreversibly bound impurities from the stationary phase. Silica-based materials are the most commonly used materials in reversed phase chromatography of peptides. Their limited chemical stability at high pH can be, however, problematic when high pH column regeneration (i.e. cleaning in place) is required. The effect of cleaning in place on the surface chemistry of the stationary phase has been investigated using the Tanaka test. It has been shown that the high pH treatment does not significantly affect the hydrophobicity of the material, but it strongly increases its silanol activity. A representative peptide purification process has been used to investigate the impact of cleaning in place on the separation performance. It has been shown that the caustic regeneration increases the peptide retention at high pH (pH 6.5), due to the interactions between the peptide and the negatively charged silanol groups. These unwanted interactions reduce the separation performances by decreasing the selectivity between the late eluting impurities and the main peptide. However, it has been shown that the effect of the silanol groups on the peptide adsorption and on the separation performance can be minimized by carrying out the purification process at low pH (pH ∼ 2). In this case, the silanol groups are protonated and their electrostatic interactions with the positively charged analyte (i.e. peptides) are suppressed. In these conditions, the peptide adsorption and the impurity selectivity is not changing upon high pH column regeneration and the separation performance is not affected.
Column Selection for the Reversed-Phase Separation of Proteins
The ability to analyze amino acid derivatives and small peptides by reversed phase HPLC has led to an active search of column types and mobile phase conditions for the fast separation of larger peptides and proteins. After the earliest publications (in 1977 and 1978) on protein separations using silica gel based alkyl bonded phases, one has seen an exponential growth in the number of scientific papers in this field, but only a modest increase in the number of useful types of stationary phases.
Talanta, 2008
Recent reports from our laboratory presented a comprehensive theory and demonstrated feasibility of reversed-phase liquid chromatography (RP-LC) employing the programmed gradient of pH of the mobile phase. The aim of that work was to explore the usefulness of the pH gradient-based approach in fractionation of peptides. The experiments were performed on a series of peptides separated at various LC conditions.
Journal of Separation Science, 2005
Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions Two-dimensional high performance liquid chromatography is a useful tool for proteome analysis, providing a greater peak capacity than single-dimensional LC. The most popular 2D-HPLC approach used today for proteomic research combines strong cation exchange and reversed-phase HPLC. We have evaluated an alternative mode for 2D-HPLC of peptides, employing reversed-phase columns in both separation dimensions. The orthogonality of 2D separation was investigated for selected types of RP stationary phases, ion-pairing agents and mobile phase pH. The pH appears to have the most significant impact on the RP-LC separation selectivity; the greatest orthogonality was achieved for the system with C18 columns using pH 10 in the first and pH 2.6 in the second LC dimension. Separation was performed in off-line mode with partial fraction evaporation. The achievable peak capacity in RP-RP-HPLC and overall performance compares favorably to SCX-RP-HPLC and holds promise for proteomic analysis.
Separation of Peptides and Proteins by Reversed Phase HPLC
During this presentation we will show the influence of some chromatographic variables on the retention of protein test mixtures during reversed phase liquid chromatography. A discussion on the type of chromatographic interaction is included. Finally we will present data on the quantitative reproducibility of the analysis of proteins by gradient elution RPLC.
Journal of Chromatography A, 1983
Weak anion-exchange and reversed-phase high-performance liquid chromatographic methods for peptide separations were compared using a tryptic digest of "rat small myelin basic protein". In these experiments, a number of tryptic peptides that were-not resolved on the reversed-phase column could be separated on the weak anion-exchange column, and in other instances, as might be expected, reversed-phase chromatography provided better resolution of certain peptides than did the weak anion-exchange method. The results obtained strongly suggest that the combined use of these two methods of separation, which utilize different selectivities, can provide an excellent improvement in resolving power for a number of peptide separations.