Experimental Studies with a Bonded N-acetylaminopropylsilica Stationary Phase for the Aqueous High Performance Exclusion Chromatogrpahy of Polypeptides and Proteins

Abstract

The potential of the micoparticulate, chemically bonded N-acetylaminopropylsilica stationary phase of nominal pore diameter of 100 angstroms in the high speed gel permeation chromatography of polypeptides and small proteins has been further investigated. The influence of ionic strength on the elution behaviour of a selected group of polypeptides and proteins on this bonded hydrophilic support has been examined. The results obtained with this porous, microparticulate bonded ‘amide’ phase silica support, packed into standard analytical-size stainless steel HPLC columns, indicate that milligram quantities of polypeptides and protein swith molecular mass up to 45,000-50,000 daltons can be efficiently fractionated with excellent recoveries of biological activities. The role of silica-based sorbents in the gel permeation fractionation of polypeptide and protein hormones, including those of pituitary and hypothalamic origin, is discussed.     

Electrokinetically-driven cation-exchange chromatography of proteins and its comparison with pressure-driven high-performance liquid chromatography.

This paper examines protein ion-exchange behavior in electrokinetically-driven open-tubular chromatography with columns produced by immobilization of poly(aspartic acid) on capillary walls. Retention and selectivity are similar in the electrokinetic elution mode to that observed in HPLC. The separation mechanism was found to depend on the relationship of mobile phase pH to that of protein pI and ionic strength. Column efficiency in the electrokinetic elution mode was found to be 10-100-times higher than in HPLC. The best separations were achieved at intermediate ionic strength and high pH. The great advantage of these low-phase-ratio, high-efficiency open tubular columns is that isocratic separations in the electrokinetic elution mode were equivalent to gradient elution in the HPLC mode. Low phase ratio has the net effect of collapsing the chromatogram into a narrow elution window while the very high efficiency produces the requisite resolution.     

Reversed phase high performance liquid chromatography of glycopeptides

A practical procedure for isolating and purifying glycopeptides is described, viz. enzymatic hydrolysis-gel permeation chromatography-ion exchange chromatography-reversed phase HPLC. Using this procedure 28 glycopeptides from hen ovalbumin have been isolated some of which hitherto have not been identified. Water was a suitable mobile phase for preparing pure glycopeptides, and control of column temperature was important for good separations and reproducible retention times. Structural confirmation was by fast atom bombardment mass spectrometry.     

Molecular weight and monosaccharide composition of Astragalus polysaccharides

Two polysaccharides (APS-I and APS-II) were isolated from the water extract of Radix Astragali and purified through ethanol precipitation, deproteination and by ion-exchange and gel-filtration chromatography. Their molecular weight was determined using high performance liquid chromatography and gel permeation chromatography (HPLC-GPC) and their monosaccharide composition was analyzed by TLC and HPLC methods, using a refractive index detector (RID) and an NH(2) column. It was shown that APS-I consisted of arabinose and glucose and APS-II consisted of rhamnose, arabinose and glucose, in a molar ratio of 1:3.45 and 1:6.25:17.86, respectively. The molecular weights (Mw) of APS-I and APS-II were 1,699,100 Da and 1,197,600 Da, respectively.     

Oil-In-Water Microemulsion LC Determination of Pharmaceuticals Using Gradient Elution

Efficient and novel oil-in-water microemulsion HPLC (MELC) separations of a range of solutes have been performed on conventional reversed-phase HPLC columns using gradient elution. This work follows previous successful separations using isocratic oil-in-water MELC [1]. It was found that by changing certain variables, peak-peak resolution, separation selectivity, efficiency and solute retention could be manipulated. The method was compatible with very low UV detection wavelengths. A robust separation method was developed for the quantitative analysis of 2 steroids in a combination-inhaled product for asthma. The method offered similar chromatography and run time when compared with conventional HPLC modes, thus demonstrating its potential for routine use. Stability-indicating methods were developed to separate synthetic and degradative impurities from the main component peaks in 4 pharmaceutical products. The methods offered quicker analysis times and equivalent selectivity to conventional HPLC modes. In developing the separations the effect on the chromatography of varying the operating parameters was studied.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

Analysis of human tear proteins by different high-performance liquid chromatographic techniques

A comparison of the efficiencies of hydrophobic interaction chromatography, ion-exchange chromatography, reversed-phase chromatography and gel permeation chromatography in the separation of tear proteins was made using a variety of different buffers. Separation of immunoglobulins, lactoferrin, albumin, PMFA (protein migrating faster than albumin) and lysozyme was accomplished by gel permeation chromatography in less than 30 min using a TSK-type SW3000 column equilibrated with ammonium acetate buffer (pH 4.1) with a high reproducibility. When gel permeation chromatography was used as a completely automated diagnostic method, only minute volumes (1.0 microliter) of tear samples were necessary for the quantitative analysis of proteins. The other three methods proved to be more suitable for the preparation of individual tear proteins but were less suitable for their quantitation.     

Capillary electrochromatography of peptides in a microfabricated system.

Reversed-phase liquid chromatography of tryptic peptides is shown in the capillary electrochromatography mode using microfabricated columns. Although selectivity is different, a mixture of tryptic peptides from ovalbumin appears to be as easily separated in the CEC as HPLC mode. The major difference between a separation in the macrofabricated CEC column and conventional separations in the HPLC mode is that separations are more readily achieved in the isocratic mode in the lower surface area microfabricated CEC columns.     

Separation of naphthoquinones and lipophilic vitamins by gel and thin-layer chromatography.

A separation of naphthoquinones on silica gel and on silica gel impregnated with polyethylene glycol 200 by thin-layer chromatography was compared with gel permeation chromatography (GPC) on styrene-divinylbenzene copolymer S-832-gel using tetrahydrofuran as mobile phase. Factors affecting the separations attainable are discussed, and it is concluded that GPC is a suitable method for the determination of K vitamins in natural materials.     

Determination of planar chlorobiphenyls in animal fat

The occurrence of the highly toxic non-ortho substituted (planar) chlorobiphenyls (CBs) at low levels in wildlife, (human) fat, and fish generates the need for a routine analytical procedure. The use of a fully automated system for the separation of planar and non-planar CBs, consisting of a gel permeation chromatography system, a sample preparation system employing extraction columns, and a porous graphitic carbon HPLC column is described, and tested with horse fat. Gas chromatography on capillary columns, with electron-capture or high resolution mass spectrometric detection, is used for quantitation of the CBs.     

Industrial application of green chromatography--I. Separation and analysis of niacinamide in skincare creams using pure water as the mobile phase

In this work, chromatographic separation of niacin and niacinamide using pure water as the sole component in the mobile phase has been investigated. The separation and analysis of niacinamide have been optimized using three columns at different temperatures and various flow rates. Our results clearly demonstrate that separation and analysis of niacinamide from skincare products can be achieved using pure water as the eluent at 60 °C on a Waters XTerra MS C18 column, a Waters XBridge C18 column, or at 80 °C on a Hamilton PRP-1 column. The separation efficiency, quantification quality, and analysis time of this new method are at least comparable with those of the traditional HPLC methods. Compared with traditional HPLC, the major advantage of this newly developed green chromatography technique is the elimination of organic solvents required in the HPLC mobile phase. In addition, the pure water chromatography separations described in this work can be directly applied in industrial plant settings without further modification of the existing HPLC equipment.     

Preliminary analysis of the composition of polysaccharide fractions

Conclusions A simple and convenient method for analyzing polysaccharide fractions isolated from natural sources has been proposed.The method is based on ion-exchange chromatography on DEAE-cellulose and gel filtration on Bio-Gels using small columns and automatic monitoring of the separation with the aid of a flow-through ultraviolet densitometer. The method is characterized by rapidity and good reproducibility.Khimiya Prirodnykh Soedinenii, Vol. 5, No. 4, pp. 203–206, 1969     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.     

液体色谱,凝胶色谱分析低聚芳砜及其双烯大分子单体

本文用液相色谱和凝胶色谱对双酚A、双酚S型低聚芳砜及α,ω-双甲基丙烯酸聚芳砜酯大分子单体进行了组分分析,通过改合成条件、测定数均分子量等辅助手段确定液相色谱各峰的归属,计算其分子量及分子量分布指数,并对两种方法测定的结果进行了比较。用液相色谱观察低聚体每个组分的含量及其反应过程中的消长情况比凝胶色谱清晰。     

Polymethacrylate monolithic columns for capillary liquid chromatography

In recent years, continuous separation media have attracted considerable attention because of the advantages they offer over packed columns. This research resulted in two useful monolithic material types, the first based on modified silica gel and the second on organic polymers. This work attempts to review advances in the development, characterization, and applications of monolithic columns based on synthetic polymers in capillary chromatography, with the main focus on monolithic beds prepared from methacrylate-ester based monomers. The polymerization conditions used in the production of polymethacrylate monolithic capillary columns are surveyed, with attention being paid to the concentrations of monomers, porogen solvents, and polymerization initiators as the system variables used to control the porous and hydrodynamic properties of the monolithic media. The simplicity of their preparation as well as the possibilities of controlling of their porous properties and surface chemistries are the main benefits of the polymer monolithic capillary columns in comparison to capillary columns packed with particulate materials. The application areas considered in this review concern mainly separations in reversed-phase chromatography, ion-exchange chromatography, hydrophobic and hydrophilic interaction modes; enzyme immobilization and sample preparation in the capillary chromatography format are also addressed.     

Separation of T-MAZ ethoxylated sorbitan fatty acid esters by reverse phase chromatography

Summary The method for determination of T-MAZ ethoxylated sorbitan fatty acid esters is described. This work demonstrates that with a less retentive C₈ alkyl bonded phase packing, reverse phase chromatography can be used to analyze nonionic polymer mixtures with a molecular weight range of 900 to 1500. Using a gradient elution, a complete separation of T-MAZ oligomers was achieved, comparable to that obtained by Supercritical Fluid Chromatography (SFC). Isocratic elution is used to quantify T-MAZ and the detection limit is 321 ppm, which is acceptable for polymers with high molecular weights and no UV-absorbing chromophores. This work also shows the comparison of the separations of T-MAZ using gel permeation chromatography and reverse phase chromatography.     

Accurate and Reproducible Determination of Molecular Weight Distribution of Sodium Heparin U.S.P. By HPLC

Abstract

Accurate molecular weight averages and molecular weight distributions have been determined for bulk Sodium Heparin, USP. The molecular weight averages were approximately 10,000 daltons (one dalton = one molecular weight unit) and ranged from 3,000 to 17,000 daltons in a given sample. The method uses high pressure liquid chromatography (HPLC) with 5–10 micron particle size controlled porous glass of different pore diameters (40 Å, 100 Å, and 250 Å) and simultaneous detection with a differential refractometer and ultraviolet detector (254 nm). The columns were calibrated with different molecular weight fractions of Sodium Heparin and baseline resolution obtained between 6.5K daltons differences in molecular weight. Analysis time was 25 minutes per sample and the method gave excellent reproducibility for calculated molecular weight averages in a repeated series of analysis. It was determined that the column doing most of the separation in the set was the one packed with 250 Å pore diameter size material.     

Preparation of active ribosomal proteins by HPLC

Summary The proteins of the large ribosomal subunit fromEscherichia coli have been separated by size-exclusion, ion-exchange and reversed-phase high-performance-liquid chromatography (HPLC) using various buffer systems. The biological activity of the isolated proteins was tested via their ability to assemble into active 50S subunits (total reconstitution). The activity of the reconstituted subunits was measured with poly(U)-dependent poly-(Phe) synthesis. Reversed-phase HPLC techniques yielded active proteins (80–100%) by application of 2-propanol or acetonitrile. Proteins prepared by size-exclusion chromatography employing ammonium acetate as buffer also gave highly active proteins (70%). On the other hand, separation of the proteins on ion-exchange columns, using urea containing buffers, resulted in reduced activity (up to 50%).     

Isolation of experimental anti-AIDS glycerophospholipids by micro-preparative reversed-phase high-performance liquid chromatography.

The experimental anti-AIDS glycerophosphatidic acid: nucleoside (sn-1/sn-2 diacylglycerol:dideoxynucleotide) drugs 3'-azido-3'-deoxythymidine monophosphate diglyceride (AZT-MP-DG) and 2',3'-dideoxycytidine monophosphate diglyceride (ddC-MP-DG) were isolated and purified by reversed-phase high-performance liquid chromatography (HPLC). The chromatographic separation was based on the glycerophospholipid moiety of the drugs and detection of the nucleoside component. The separations were optimized on method development columns packed with the stationary phase to be used in the micro-preparative column and monitored by a UV detector. Fractions were collected and analyzed for purity by analytical-scale HPLC and by thin-layer chromatography (TLC). The purity of the recovered drugs based on UV and light-scattering detection and on TLC was greater than 99%. The purified compounds were isolated for studies on structure confirmation, physical, biophysical and formulation properties and anti-HIV efficacy in culture.     

Chromatographic methods for product-profile analysis and isolation of oligosaccharides produced by heparinase-catalyzed depolymerization of heparin.

A two-dimensional chromatography method for monitoring the formation of oligosaccharides produced by heparinase-catalyzed depolymerization of heparin is reported. In the first step of the two-dimensional method, the depolymerized heparin is size-fractionated by high-performance gel permeation chromatography (HPGPC). The size-uniform fractions are then separated on the basis of charge by strong anion-exchange (SAX) HPLC on a high resolution CarboPac PA1 column. To demonstrate application of the two-dimensional product-profile analysis method, data are presented for the heparinase I-catalyzed depolymerization of heparin in the absence and presence of histamine, a ligand that binds site-specifically to heparin. Results from the two-dimensional analysis indicate that histamine alters the extent of depolymerization and the product-profiles for the tetrasaccharide and hexasaccharide fractions. The use of CarboPac PA1 columns for the semi-preparative scale separation of oligosaccharides in size-uniform fractions isolated from depolymerized heparin by low-pressure (gravity flow) GPC is also reported. The semi-preparative scale CarboPac PA1 column gives high resolution and excellent reproducibility after repeated use over an extended period of time, making it possible to reliably combine fractions from multiple separations. The oligosaccharides are eluted from the CarboPac PA1 column with a NaCl gradient at relatively low pH (3 or 7) where they are stable. An efficient two-step procedure is described for desalting oligosaccharides separated