Alternative high-performance liquid chromatographic peptide separation and purification concept using a new mixed-mode reversed-phase/weak anion-exchange … (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.
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.
Journal of Pharmaceutical and Biomedical Analysis, 2012
In the pharmaceutical field, there is considerable interest in the use of peptides and proteins for therapeutic purposes. There are various ways to characterize such complex samples, but during the last few years, a significant number of technological developments have been brought to the field of RPLC and RPLC-MS. Thus, the present review focuses first on the basics of RPLC for peptides and proteins, including the inherent problems, some possible solutions and some directions for developing a new RPLC method that is dedicated to biomolecules. Then the latest advances in RPLC, such as wide-pore core-shell particles, fully porous sub-2 m particles, organic monoliths, porous layer open tubular columns and elevated temperature, are described and critically discussed in terms of both kinetic efficiency and selectivity. Numerous applications with real samples are presented that confirm the relevance of these different strategies. Finally, one of the key advantages of RPLC for peptides and proteins over other historical approaches is its inherent compatibility with MS using both MALDI and ESI sources.
Separations
Peptides are a class of biomolecules with a great potential from the therapeutic point of view, because of their unique biological properties. Industrially, the production stategies adopted produce both the target peptide and a series of impurities that must be removed. Preparative chromatography is the technique of choice for the large-scale purification of biomolecules, generally performed in reversed-phase mode, using hydrophobic adsorbents (e.g., C8 stationary phases). A promising and innovative alternative is represented by mixed-mode columns, which bear two different ligands on the particle surface, exploiting two different retention mechanisms to improve the separation. This work represents a proof-of-concept study focused on the comparison of a hydrophobic adsorbent and a mixed-mode one (bearing both hydrophobic groups and charged ones) for the purification of a crude peptide mixture. Thanks to more-favourable thermodynamics, it was found that, when collecting the whole peak...
Analytical and Bioanalytical Chemistry, 2011
We recently introduced a mixed-mode reversedphase/weak anion-exchange type separation material based on silica particles which consisted of a hydrophobic alkyl strand with polar embedded groups (thioether and amide functionalities) and a terminal weak anion-exchange-type quinuclidine moiety. This stationary phase was designed to separate molecules by lipophilicity and charge differences and was mainly devised for peptide separations with hydroorganic reversed-phase type elution conditions. Herein, we demonstrate the extraordinary flexibility of this RP/ WAX phase, in particular for peptide separations, by illustrating its applicability in various chromatographic modes. The column packed with this material can, depending on the solute character and employed elution conditions, exploit attractive or repulsive electrostatic interactions, and/or hydrophobic or hydrophilic interactions as retention and selectivity increments. As a consequence, the column can be operated in a reversed-phase mode (neutral compounds), anion-exchange mode (acidic compounds), ion-exclusion chromatography mode (cationic solutes), hydrophilic interaction chromatography mode (polar compounds), and hydrophobic interaction chromatography mode (e.g., hydrophobic peptides). Mixed-modes of these chromatographic retention principles may be materialized as well. This allows an exceptionally flexible adjustment of retention and selectivity by tuning experimental conditions. The distinct separation mechanisms will be outlined by selected examples of peptide separations in the different modes. Keywords Mixed-mode stationary phase. Reversed-phase/ weak anion-exchange material. Hydrophilic interaction chromatography (HILIC). Hydrophobic interaction chromatography (HIC). Ion-exclusion chromatography Abbreviations AEX Anion-exchange HIC Hydrophobic interaction chromatography HILIC Hydrophilic interaction chromatography IEC Ion-exclusion chromatography IEX Ion-exchange LSS Linear solvent strength (theory) RPLC Reversed-phase liquid chromatography RP/WAX Mixed-mode reversed-phase/weak anionexchange material
Scientific Reports, 2022
Porous silica particles were prepared by sol–gel method with some modification to get wide-pore particles. These particles were derivatized with N-phenylmaleimide-methylvinylisocyanate (PMI) and styrene by reversible addition fragmentation chain transfer (RAFT) polymerization to prepare N-phenylmaleimide embedded polystyrene (PMP) stationary phases. Narrow bore stainless steel column (100 × 1.8 mm i.d) was packed by slurry packing method. The chromatographic performance of PMP column was evaluated for the separation of synthetic peptides mixture composed of five peptides (Gly-Tyr, Gly-Leu-Tyr, Gly-Gly-Tyr-Arg, Tyr-Ile-Gly-Ser-Arg, Leucine enkephalin) and tryptic digest of human serum albumin (HAS) respectively. Number of theoretical plates as high as 280,000 plates/m were obtained for peptides mixture at optimum elution condition. Separation performance of the developed column was compared with commercial Ascentis Express RP-Amide column and it was observed that separation performan...
Stationary phases for peptide analysis by high performance liquid chromatography: a review
Analytica Chimica Acta, 1997
A survey is given of modern stationary phases employed in high performance liquid chromatography (HPLC) analysis of peptides. The physico-chemical properties of peptides and their consequences for the selection and optimization of the separation system are briefly discussed, followed by a summary of the approaches to the selection and characterization of stationary phases. The properties and applicability of various stationary phases are then critically reviewed, including aspects such as size-exclusion, ion-exchange, reversed-phase, hydrophobic-interaction, affinity and chiral systems, as well as some specialized separation techniques. Emphasis is placed on the most recent literature. 0 1997 Elsevier Science B.V.
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.
Use of sodium perchlorate at low pH for peptide separations by reversed-phase liquid chromatography
Journal of Chromatography A, 1997
The reversed-phase liquid chromatography (RPLC) behavior of synthetic model peptides containing positively charged amino acid residues was studied in the presence or absence of 100 mM sodium perchlorate in order to determine the effect on apparent side-chain hydrophilicity of a charged residue at low pH. The peptides used in this study were either non-helical peptides or amphipathic a-helical peptides, where the effect of the negatively charged perchlorate ion on a charged residue in either the hydrophobic face or hydrophilic face of the helix was monitored. We have shown that the addition of 100 mM perchlorate to RPLC separations of positively charged peptides performed in a 20 mM aqueous phosphoric acid-acetonitrile system resulted in an increase in retention time of a peptide relative to the same peptide in the absence of perchlorate. This effect occurred independent of conformation, i.e., whether comparing the effect of positively charged residue substitutions in the hydrophobic or hydrophilic face of an amphipathic a-helix or in a peptide with negligible secondary structure. From these results, suggesting that positively charged side-chain hydrophilicity is decreased by ion-pairing with the perchlorate ion, we have shown practical examples where mixtures of non-helical and amphipathic a-helical peptides showed enhanced resolution in the presence of perchlorate at pH 2, compared to in its absence. In addition, it was shown that an aqueous phosphoric acid-perchlorate-acetonitrile mobile phase may show markedly different selectivity for peptide separations at low pH compared to the more traditional aqueous trifluoroacetic acid-acetonitrile system.
Journal of Chromatography A, 2022
Peptides and proteins have great therapeutic potential, and owing to their unique biological properties, their separation is very challenging. A polar embedded C18 stationary phase was prepared by function- alization of porous silica monolith particles with N-3-(trimethoxysilyl) propyl stearamide. The stationary phase was packed in narrow bore column (100 ×2.1 mm) and evaluated for the separation of peptides and proteins in HPLC. Number of theoretical plates (N) as high as 520,0 0 0 and 340,0 0 0 plates/meter were obtained for peptides and proteins respectively. The separation performance of C18-amide column was compared with four commercial amide columns i.e. Ascentis Express RP-Amide, Accucore 150 Amide, TSKgel Amide-80, and XBridge Amide column. The performance of C18-amide column was much better than these commercial amide columns in terms of efficiency and resolution. Owing to high efficiency and good resolution for peptides and proteins separation, C18-amide column could be used as alternative to expansive commercial columns.