Reversed-phase liquid chromatography of proteins and peptides using multimodal copolymer-encapsulated silica (original) (raw)
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Journal of Chromatography A, 1987
Columns of dimensions 36 x 8 mm I.D., packed with n-octyl-bonded nonporous monodisperse silica particles of diameter 1.5 pm f 2%, were tested for their performance in the reversed-phase gradient elution of proteins, employing methanol, acetonitrile, 2-propanol and 1-propanol as organic solvents and 0.01-0.02 M trifluoroacetic acid or perchloric acid as eluents. The retention of proteins over a wide range of volume fractions of the organic solvent modifier follows the same dependences as are observed on porous reversed-phase packings, with one noticeable exception: the slope of the linear dependence of the logarithmic capacity factors on the composition of the binary eluent yields values between 4 and 7 for the proteins studied, whereas S is usually a function of the molecular weight and approaches values of up to 100. This peculiarity is assumed to be associated with the retention of proteins on a surface with easily accessible n-alkyl ligands and free from any pore diffusion effects. The 1.5-pm particles provide an excellent column performance, enhancing both the resolution and the detection sensitivity. Gradient times of 3 min and flow-rates of 1.5 ml/min allow the rapid separation of proteins. The sample capacity was found to be cu. 1 mg of protein per column volume, without a loss of resolution.
Journal of Chromatography A, 2003
In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic methods. Peak assignment of the various peptides was based on evaluation of the biophysical properties of the individual peptides and via mass spectrometric identification. The performance of several different monolithic sorbents prepared as columns of identical cross-sectional dimensions were investigated as part of these peptide mapping studies and the data evaluated by applying solvent strength theory. These studies revealed curvilinear dependencies in the corresponding relative resolution maps. These findings directly impact on the selection of specific sorbent types or column configurations for peptide separations with silica rod monoliths. Moreover, the influence of variations in the amino acid sequence of the cytochrome cs were evaluated with respect to their effect on intrinsic hydrophobicity, the number of experimental observed tryptic cleavage sites, detection limits of the derived fragments in relation to their molecular size, and the chromatographic selectivity and resolution of the various peptides obtained following enzymatic fragmentation of the parent protein. Finally, the scope of these approaches in method development was examined in terms of robustness and efficiency.
Journal of Chromatography A, 2012
The separation range of superficially porous particles (Fused-core®) has been extended by design of particles with 160 Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of <15,000). The high efficiency and relatively low back pressure of these 2.7 μm fused-core particles has been maintained so that separations can be performed with conventional HPLC instruments. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100 °C. To address the issue of poor peptide peak shape in formic acid-containing mobile phases we show that the addition of 10-20 mM ammonium formate improves peak shape, retention and load tolerance of peptides. Use of the Fused-core particle materials for separations of synthetic peptides and tryptic digests yields peak capacities that are comparable to those obtained using columns packed with sub-2-μm particles, but with less than one-half of the operating back pressure. A peak capacity of 530 was obtained in 150 minutes on coupled columns of HALO Peptide ES-C18 with a combined length of 250 mm.
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.
Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica
Journal of Chromatography A, 1988
A strong cation-exchange material, poly(Zsulfoethy1 aspartamide)-silica (PolySULFOETHYL Aspartamide) was developed for purification and analysis of peptides by high-performance liquid chromatography. All peptides examined were retained at pH 3, even when the amino terminus was the only basic group. Peptides were eluted in order of increasing number of basic residues with a salt gradient. Capacity was high, as was selectivity and column efficiency. This new column material displays modest mixed-mode effects, allowing the resolution of peptides having identical charges at a given pH. The selectivity can be manipulated by the addition of organic solvent to the mobile phases; this increases the retention of some peptides and decreases the retention of others. The retention in any given case may reflect a combination of steric factors and non-electrostatic interactions. Selectivity was complementary to that of reversed-phase chromatography (RPC) materials. Excellent purifications were obtained by sequential use of Poly-SULFOETHYL Aspartamide and RPC columns for purification of peptides from crude tissue extracts. The new cation exchanger is quite promising as a supplement to RPC for general peptide chromatography.
Journal of Chromatography A, 2006
Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 m. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.
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.
Immobilized Stationary Phases for Hydrophobic Interaction Chromatography of Proteins
Chromatographia, 2006
Immobilized stationary phases for hydrophobic interaction chromatography (HIC) of proteins are prepared by coating macroporous silica Daisogel of different porosity with hydrophobized cellulose derivatives. The polymer adsorbed on the silica surface afterwards was cross-linked with bifunctional compounds. A uniform polymer nanocoating was indicated using the nitrogen gas adsorption method BET and scanning electron microscopy. The absence of non-specific protein sorption of the synthesized adsorbents shows that the developed polymeric coating isolates silica surface from contact with the sorbate. The retention data of bovine serum albumin (BSA) in the HIC mode on different synthesized adsorbents were evaluated. It was shown that sorption capacity of such phases may vary over a wide range and depends mainly on the substitution degree of the immobilized polymer. The dynamic sorption capacity of BSA was up to 63 mg mL)1. The results proved that the new stationary phases have significant promise for the separation and purification of proteins in the HIC mode.