High-performance liquid chromatographic molecular weight determination of allergen extracts. Examination of the influence of the column material on allergenic activity and allergen patterns

To investigate whether the column material employed in size-exclusion high-performance liquid chromatography (HPLC) influences the allergenic activity and antigen/allergen patterns of allergen extracts, the molecular weights of a mite and a birch pollen allergen extract were determined using a Bio-Sil TSK 250 column with a guard column. The allergenic activities were measured by RAST inhibition and the antigen/allergen patterns were determined by crossed (radio)immunoelectrophoresis. The original extracts and the corresponding eluates after HPLC runs showed the same allergenic activity and the same number of antigen/allergen precipitation lines. Only slight differences in the peak heights of some precipitates were observed.     

Reversed-phase HPLC of peptides: Assessing column and solvent selectivity on standard, polar-embedded and polar endcapped columns

We desired to evaluate the chromatographic selectivity for peptides of silica-based RP high-performance liquid chromatography stationary phases with various modifications (polar embedding and polar endcapping on C(18) columns; ether-linked phenyl column with polar endcapping) compared with n-alkyl (C(18), C(8)) and aromatic phenylhexyl columns. Thus, we have designed and synthesized two series of synthetic peptide standards with the sequence Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal either contains a free α-amino group (AmC series) or is N(α)-acetylated (AcC series) and where position X is substituted by Gly, Ala, Val, Ile, Phe or Tyr. These represent series of peptides with single substitutions of n-alkyl (Gly相似文献   

Evaluation of New Microparticulate Packings for Aqueous Steric Exclusion Chromatography

Abstract

Two types of high performance aqueous size exclusion columns have recently been developed, one a rigid spherical silica-based packing containing a new hydrophilic bonded phase (MicroPak TSK Gel Type SW) and the other an organic-based, semi-rigid gel (MicroPak TSK Gel Type PW). Characteristics of MicroPak TSK SW and PW columns were compared to other commercially available aqueous SEC columns packed with similar supports. Chromatographic performance of prepacked columns containing microparticulate support materials were compared for exclusion separations of water-soluble organic polymers, biopolymers, and small water-soluble oligomers. Amino acid probes were used to investigate non-exclusion effects of MicroPak TSK SW and PW columns.     

Effect of long-term storage and use on the properties of reversed-phase liquid chromatographic columns

In order to study column deterioration as a result of long-term storage and/or usage in liquid chromatography analyses, 55 pairs (same batch) of different commercial reversed-phase C(18) columns were examined using an already existing column characterisation system. After initial testing, one column was stored and the other was used to analyse different pharmaceuticals. All columns were characterized by four chromatographic parameters reflecting hydrophobicity, silanol activity, metal impurity and steric selectivity at the beginning and at the end of the test. An F-value was calculated to express the change of column properties with one single number. After performing analyses, higher F-values were obtained as compared to the non-used, stored columns. Although the time during which the columns were used to perform analyses was relatively short, an obvious influence was noticed, mainly resulting from small changes in silanol activity and hydrophobicity. Most of the affected columns have no endcapping and/or no base deactivation, making them more vulnerable for degradation, resulting in higher silanol activity and faster ageing. This effect is observed less with columns equipped with polar-embedded groups and/or polar endcapping, protecting the column by blocking the silanol groups and attracting a shielding water layer. Also columns with higher coverages and bulky or long chains show more resistance towards degradation.     

Effect of high pH column regeneration on the separation performances in reversed phase chromatography of peptides

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.     

含酯基包覆聚合物液相色谱柱填料

以丙烯酸甲酯或辛酯和二乙烯苯为原料，在溶液中用游离基聚合法制备了一系列含酯基包覆聚合物反相液相色谱柱填料．用傅立叶红外光谱、电子显微镜和元素分析鉴定了聚合物层，并评价了诸如硅羟基、柱压降、柱效和峰对称性等特性．该类填料适合于含氨基和羟基化合物的分离，作为应用实例，对洛伐他汀（Lovastatin）的分析展示其优良的色谱性能。     

Resolution and quantitation of apolipoproteins A-I and A-II from human high-density lipoprotein by size exclusion high-performance liquid chromatography

Apolipoproteins A-I and A-II, extracted from human high-density lipoprotein (HDL), were resolved and quantified by size exclusion high-performance liquid chromatography on TSK 125 and TSK 250 analytical columns connected in series without the use of chemical denaturants or detergents in the eluent buffer. The columns were pre-equilibrated with a solution containing 0.1 M sodium phosphate, pH 7.2, 0.2 M sodium chloride at a flow-rate of 1 ml/min. Delipidated HDL (1 mg protein per ml) was resolved into two populations of apolipoprotein (apo) A-I: one representing the apo A-I monomer and the other, a self-associated form with a molecular weight of approximately 120,000 daltons. The column eluates were screened for immunoreactivity to apo A peptides, and the identity of each peak was confirmed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis followed by immunoblot analysis. Apo A-I peptides isolated by high-performance liquid chromatography disrupted unilamellar phospholipid vesicles to form smaller phospholipid particles that eluted on gel filtration columns within the size range of HDL. Thus, a rapid method for the isolation and quantitation of non-denatured apolipoproteins from HDL has been developed using size exclusion high-performance liquid chromatography.     

Dry-packed capillary columns for micro HPLC

A dry-packing method has been developed which enables the preparation of packed capillary columns for micro HPLC from 250 μm i.d. tubing and 5 μm packing materials. Pressurized gases, such as hydrogen and argon, were used to transport the packing media, and ethanol or methanol were used as discharge agents. By changing the gas pressure, the packing density could be easily adjusted. It was found that, within experimental limits, the higher the packing density, the greater the column efficiency. Comparison between dry- and slurrypacked columns showed that the former had greatly improved stability; the efficiency of dry-packed columns was about the same as, or even better than, that obtained by slurry-packing.     

Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides

The solution chemistry conditions necessary for optimum analysis of peptides by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry and CZE electrospray ionization tandem mass spectrometry have been studied. To maximize the signal-to-noise ratio of the spectra it was found necessary to use acidic CZE buffers of low ionic strength. This not only increases the total ion current, but it also serves to fully protonate the peptides, minimizing the distribution of ion current across the ensemble of possible charge states. The use of acidic buffers protonates the peptides, which is advantageous for mass spectrometry and tandem mass spectrometry analysis, but is problematic with CZE when bare fused silica CZE columns are used. These conditions produce positively charged peptides, and negatively charged silanol moieties on the column wall, inducing adsorption of the positively charged peptides, thus causing zone broadening and a loss in separation efficiency. This problem was circumvented by the preparation of chemically modified CZE columns, which, when used with acidic CZE buffers, will have a positively charged inner column wall. The electrostatic repulsion between the positively charged peptides and the positively charged CZE column wall minimizes adsorption problems and facilitates high efficiency separations. Full-scan mass spectra were acquired from injections of as little as 160 fmols of test peptides, with CZE separation efficiencies of up to 250,000 theoretical plates.     

Peptide mapping by reversed-phase high-performance liquid chromatography employing silica rod monoliths

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.     

HPLC chiral stationary phases produced with isolated human serum albumin fragments.

The enantioselectivity of HPLC chiral stationary phases produced with human serum albumin (HSA) fragments was investigated. An HSA fragment (HSA-FG75) was isolated by size-exclusion chromatography following peptic digestion of HSA. The isolated HSA-FG75 was mainly an N-terminal half peptide with an average molecular weight of about 35,000 daltons. The HSA and HSA-FG75 proteins were bound to aminopropylsilica gels activated by N,N'-disuccinimidyl carbonate. Though the HSA-FG75 column showed lower enantioselectivities for all of the racemates tested than the intact HSA column, the enantioseparations of the racemates tested were attained with a shorter analysis time on the HSA-FG75 column. These results are ascribable to removal of the non-specific binding sites of HSA, changes in the globular structure of the HSA fragment and/or changes in the local environment around the binding sites. Further, the HSA-FG75 column was as stable as the intact HSA column for repetitive injection of samples.     

Naphthalene sulphonic acids--new test compounds for characterization of the columns for reversed-phase chromatography

Non-substituted naphthalene sulphonic acids are strong acids, which are completely ionised in aqueous and aqueous-organic solutions. Because of repulsive electrostatic interactions, they are more or less excluded from the pores of the column packing materials commonly used in reversed-phase chromatography. The ionic exclusion can be suppressed by increasing the ionic strength of the mobile phase. In aqueous sodium sulphate solutions, very good selectivity was observed for isomeric naphthalene di- and tri-sulphonic acids, allowing reversed-phase separations of these strongly ionic compounds without addition of ion-pairing reagents to the mobile phase. The retention of the isomeric acids increases proportionally to the dipole moment, which can be explained by its effect on increasing exposure of the naphthalene ring to hydrophobic interactions with the non-polar stationary phases. Chromatographic behaviour of isomeric naphthalene di- and trisulphonic acids was investigated on 25 different columns for reversed-phase chromatography. The elution order of the isomers is the same on all the columns, but very strong stationary phase effects were observed on the retention and on the band asymmetry, depending on polar interactions with residual silanol groups and other polar adsorption centres in the stationary phases. These effects are independent of the organic solvents, as the tests are performed in purely aqueous mobile phases and allow classification of the columns into several groups.     

Characterization of the acidity of residual silanol groups in microparticulate and monolithic reversed-phase columns

The residual silanol acidity and activity of several microparticulate and monolithic C18 columns has been measured from the retention of LiNO3 in the columns with a methanol/buffer (1 mM in Na+) (60:40 v/v) mobile phase buffered to different pH values. For Luna C18 (2) and LiChrospher RP-18 columns, at least two different types of silanols with different acidity for each packing, were observed. Purospher RP-18e and Chromolith RP-18e packings present evidence of some active silanols only at pH values close to their basic pH stability limit or higher. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MSC18 and Underivatized XTerra. A modification of an equation previously proposed has been applied to all columns studied and the results obtained have been used to classify the columns according to their silanol acidity and activity. The method allows the prediction of the extent of the silanol activity of the columns studied at a particular mobile phase pH.     

Use of hydrophilic hydroxypropyl methacrylate/ethylene glycol methacrylate packing material in size-exclusion chromatography

Spherical particles of hydroxypropyl methacrylate/ethylene glycol methacrylate copolymer were synthesized in-house for use in size-exclusion chromatography. The porous hydrophilic material was packed in glass and stainless steel columns to evaluate their chromatographic performance. The support particles were small (approximately 20 A), and the average pore size was in the low range of mesopores (approximately 100 A). The packed columns were calibrated by using polysaccharide dextrans, showing a good range of separation for molecular weights between 10000 and 600000 daltons. The packing material appears to separate the large molecules through the size-exclusion mechanism. Polysaccharides and polypeptides dissolved in adequate mobile phases were injected into the packed column. The separation of the macromolecules was consistent with the size-exclusion mechanism. Application of the packing material to the separation of small molecules (alkyl alcohols) was also investigated.     

Reversed-phase liquid chromatographic separation and simultaneous profiling of steroidal glycoalkaloids and their aglycones

Improved and simplified reversed-phase liquid chromatographic conditions for the separation and simultaneous profiling of both steroidal glycoalkaloids and their aglycones, having solanidane- or spirosolane-type structures, are described. The most reproducible retention behavior for these ionizable compounds on C18 columns was achieved under isocratic and gradient elution conditions using acetonitrile in combination with triethylammonium phosphate buffer at pH 3.0, when basic functional groups of solutes and silanol groups on the silica are fully protonated minimizing ionic interactions. Gradient elution was the only feasible approach for the simultaneous separation of steroidal glycoalkaloids and their aglycones. A Zorbax SB C18 column, specially designed for low-pH separations, showed good performance in critical separations. The impurities of the commercial tomatine and tomatidine standards were studied and confirmed using mass spectrometric, liquid chromatographic and thin-layer chromatographic methods.     

High-speed and high-performance size-exclusion chromatography of proteins on a new hydrophilic polystyrene-based resin

High-performance size-exclusion chromatography of some standard proteins, peptides and amino acids on a new hydrophilic packing material obtained by chemical transformation of a cross-linked polystyrene-divinylbenzene copolymer was studied. Columns filled with 4 and 7 micron particles were compared. The influence of the concentration of acetonitrile, isopropanol and trifluoroacetic acid in the mobile phase on the chromatographic performance was investigated. A good linear calibration graph covering the molecular weight range from 200 to 700,000, was obtained under the optimal conditions. The packing material can be used for separations, for molecular weight determinations and for the pre-fractionation of proteins. The high rigidity of the packing material allows relatively high pressures to be used and therefore fast separations to be achieved. The packing material was applied to the chromatography of proteins from beer, bones and milk.     

Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.     

Effect of the surface coverage of endcapped C18-silica on the excess adsorption isotherms of commonly used organic solvents from water in reversed phase liquid chromatography

The excess adsorption isotherms of methanol, ethanol, 2-propanol, acetonitrile, and tetrahydrofuran from water were measured on five different silica-based packing materials by the minor disturbance method. These materials were prepared with the same lot of 5-microm particles (average pore size 90 A), all endcapped with trimethylchlorosilane (TMS), and bonded to octadecyl chains with different surface coverages (0, 0.42, 1.01, 2.03, and 3.15 micromol/m2). The relative adsorption of one eluent by respect to a second one informs on the heterogeneity of the material (alkyl-bonded and bare silica regions) and on the accessibility of the unreacted silanol groups to the mobile phase. It is shown that the total surface area of the adsorbent decreases with increasing degree of surface coverage with octadecyl chains and that the relative surface area of the regions occupied by accessible silanol groups to the regions occupied by alkyl-bonded groups decreases. For the five columns, an average of 10% of the adsorbent surface area is covered of bare silica accessible to the liquid phase, with a minimum of 5% with tetrahydrofuran and a maximum of 12% with ethanol or 2-propanol. Increasing the surface coverage by the C18 chains causes a significant increase of the attraction potential of the hydrophobic surface toward the organic solvent. This result is confirmed by the increase of the number of adsorbate monolayers with increasing bonding density of the octadecyl chains. This number is twice larger for the 315C18 column than for the C1 column.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

Immobilized metal ion affinity chromatography. Effect of solute structure, ligand density and salt concentration on the retention of peptides

The adsorption characteristics of a variety of synthetic peptide hormones and di-, tri- and tetrapeptides on Cu(II) immobilized on two commercially available high-performance chelating gels run under various experimental conditions are described. Methods for determining the concentration of immobilized Cu(II) in situ are also described. The Cu(II)-charged columns exhibit a net negative charge as judged from the significantly higher retention of some basic peptides in the absence of NaCl in the equilibration and elution buffers. At higher NaCl concentrations (2-4 M), aromatic interactions seem to be superimposed on the metal ion affinity characteristics of the peptides. The relationship between resolution of peptides and the concentration of immobilized Cu(II) ions has also been established for the Chelating Superose gel where 40 mumol Cu(II) ml-1 gel apparently gives the optimum resolution. The nature of the gel matrix also plays a role in the resolution of some peptides, the extent of which is difficult to predict. The results obtained also suggest that peptides containing aromatic and hydroxy amino acids are retarded more than those which lack them. Moreover, these same amino acids apparently strengthen the existing strong binding of peptides containing His, Trp or Cys to a Chelating Superose-Cu(II) column. Dipeptides with C-terminal His (i.e., X-His) are neither bound nor retarded on a column of Chelating Superose-Cu(II) whereas those having the structure His-X are strongly bound. Some tri- and tetrapeptides containing His were also found not to bind to the column. The underlying cause of this anomalous adsorption behaviour is discussed and is ascribed to "metal ion transfer" arising from the relatively higher affinity of such peptides towards immobilized Cu(II) ions than the chelator groups (iminodiacetate) which are covalently bound to the gel matrix.