Preparation of mixed-mode stationary phase for separation of peptides and proteins in high performance liquid chromatography (original) (raw)
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Polymers
Separation with high efficiency and good resolution is constantly in demand in the pharmaceutical industry. The fast and efficient separation of complex samples such as peptides and proteins is a challenging task. To achieve high efficiency with good resolution, chromatographers are moving towards small particles packed into narrow-bore columns. Silica monolith particles (sub-2 µm) were derivatized with chlorodimethyl octadecyl silane (C18) and packed into stainless steel columns (100 mm × 1.8 mm i.d) by a slurry-packing method. The developed columns were used for the separation of peptides and proteins. A separation efficiency (N) of 40,000 plates/column (400,000 plates/m) was achieved for the mixture of five peptides. Similarly, the fast separation of the peptides was carried out using a high flow rate, and the separation of the five peptides was achieved in one minute with high efficiency (N ≅ 240,000 plates/m). The limit of detection (DL) and the limit of quantification (QL) for...
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.
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, 2009
This review discusses different liquid chromatographic and capillary electrochromatographic approaches to the separation and quantitation of peptides using silica-based and polymeric-based columns with emphasis on liquid chromatography. Mass spectrometry detection and quantitation of peptides using labeled and label-free procedures, will also be discussed, as well as the effect of amino acids' properties on the solubility of peptides, an important parameter that influences the selection of the mobile phase. A discussion of different column packing materials, reversed-phase, cyclodextrins, macrocyclic antibiotics, porous graphitic carbon, mixed-phases, and normal-phase will be included, as well as a short discussion of multi-dimensional approaches for the separation of complex peptide mixtures.
Journal of Chromatography A, 2007
Chromatographic measurements were made on 17 physicochemically diversified high-performance liquid chromatography (HPLC) columns which were further analyzed in terms of their similarities and dissimilarities for 25 carefully designed, structurally diverse peptides showing distinctly distinguished groups. The goal of the study was to investigate the molecular mechanism of retention and to find an objective manner of quantitative comparison of retention properties and classification of modern stationary phases for reverse phase HPLC (RP-HPLC). We utilized the structural descriptors of peptides obtained from molecular modeling to describe their chromatographic retention behavior under given HPLC conditions. Quantitative structure-retention relationships (QSRR) with the following descriptors were employed: logarithm of the sum of amino acid retention contributions in a given peptide, log Sum AA , logarithm of the Van der Waals volume of the peptide, log VDW vol , and logarithm of its calculated n-octanol/water coefficient, clog P. The best QSRR equations were obtained in the case of monolithic and regular octadecylsilica columns. On the other hand, the combination of QSRR and principal component analysis (PCA) can be considered as the efficient tool allowing column classification and searching for orthogonal HPLC conditions required to separate peptides.
Journal of Pharmaceutical and Biomedical Analysis, 1997
A new method of HPLC using packing materials modified with a temperature responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), was developed. Homogeneous PIPAAm polymer and its copolymer with butyl methacrylate (BMA) were synthesized and grafted to aminopropyl silica by activated ester-amine coupling and they were used as packing materials. The surface properties and functions of the stationary phases are controlled by external temperature. Isocratic elution by aqueous mobile phase alone is the basis for separation of peptides and protein. The separation of the mixture of three peptides, insulin chain A and B and ~-endorphin fragment 1-27 was achieved by changing the column temperature with 0.9% NaC1 aqueous solution as the sole eluent. Retention of peptides and proteins was controlled both by column temperature and by NaC1 concentration in the aqueous mobile phases in this chromatographic system.
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.
Separation of peptides by strong cation-exchange high-performance liquid chromatography
Journal of Chromatography A, 1985
The effects of pH and gradient conditions on the separation of a series of ten peptides (9-36 residues) and carboxamidomethylated troponin I (CM-TnI, 178 residues) on a new commercially available strong cation-exchange silica based 300-A column (Synchropak S300) were examined. The elution times of the peptides were linear with respect to their net charge at pH 3.0 and pH 6.5. The basic protein CM-TnI (pZ % 9.5) and peptides with net charges from + 2 to + 10 were separated with linear AB salt gradients varying from 5 mM to 10 mM B per min (A = 5 mM KH2P04 buffer, pH 6.5 or 3.0; B = 5 mM KH2P04 buffer, pH 6.5 or 3.0, containing 1 M KCl). All peptides and CM-TnI were eluted with KC1 concentrations below cu. 0.6 M. The advantage of performing cation-exchange chromatography over anionexchange chromatography was demonstrated for the separation of peptides which, while acidic or weakly basic at neutral pH, through protonation of the acidic functions results in positively charged peptides at pH 3.0. 0021-9673/85/%03.30 0 1985 Elsevier Science Publishers B.V.