Chromatographic measurement of interactions between unlike proteins

A chromatographic method is used to measure ovalbumin-lysozyme and BSA-lysozyme interactions in aqueous salt solutions as a function of solution conditions (pH, ionic strength, salt type). In this method, one protein is immobilized on the support surface, and the other, dissolved in a buffer/electrolyte solution, flows over that surface. The retention time provides a measure of immobile/mobile protein–protein interactions. Trends in ovalbumin-lysozyme interactions suggest that they are primarily electrostatic. The identity of the electrolyte has a strong influence on the magnitude of the interaction. Assuming a potential of mean force that contains a hard sphere, electrostatic, and square-well potential, experimental results are used to fit the square-well depth. For BSA-lysozyme interactions, the square-well depth depends on which protein is immobilized on the solid phase.     

Characterization of unstable ion-exchange chromatographic separation of proteins.

Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (beta-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within +/-0.1 unit and the ionic strength within +/-0.002 mol/l in order to meet the criteria (+/-5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained.     

流动相组成、浓度和pH对蛋白质在金属螯合柱上的保留特性的影响

 系统研究了流动相中盐的性质和浓度、溶液 pH以及竞争配体对蛋白质在金属螯合色谱中保留值的影响。导出了描述蛋白质在金属螯合色谱中保留特征的数学表达式 ,提出用洗脱强度指数ε表征盐溶液的洗脱能力。根据不同色谱条件下蛋白质的保留特性 ,发现蛋白质在金属螯合色谱中的保留是配位、静电和疏水的协同作用。对与蛋白质强结合的金属螯合柱 ,以配位作用为主 ,静电作用为辅 ;对弱结合的金属柱 ,以静电作用为主 ,配位作用为辅。在流动相中加入高浓度非成络盐 ,可增强蛋白质和固定相间的疏水作用。     

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.     

Analysis of selected ionic liquid cations by ion exchange chromatography and reversed-phase high performance liquid chromatography

The chromatographic behavior of 8 ionic liquids - 7 homologues of 1-alkyl-3-methylimidazolium and 4-methyl-N-butylpyridinium - has been investigated with a strong cation exchange adsorbent. In particular, the dependence of the retention properties of these solutes on mobile phase composition, pH, and buffer concentration was evaluated with the aim of optimizing and improving the selectivity and retention of solute separation. While using the SCX stationary phase, several interactions occurred with varying strengths, depending on the mobile phase composition. Cation exchange, nonspecific hydrophobic interactions, and adsorption chromatography behavior were observed. Reversed phase chromatography occurred at low concentrations of acetonitrile, electrostatic and adsorption interactions at higher organic modifier concentrations. Elevated buffer concentrations lowered the retention factors without affecting the selectivity of ionic liquids. Obtained results were further compared to the chromatographic behaviour of ionic liquids in the reversed phase system. All analyzed ionic liquids follow reversed-phase behavior while being separated. Much lower selectivity in the range of highly hydrophilic compounds is obtained. This suggests preferred use of ion chromatography for separation and analysis of compounds below 4 carbon atoms in the alkyl side chain.     

Effects of electrostatic and hydrophobic interactions on the chromatographic behavior of biopolymers in HPLC columns

The effect of electrostatic and hydrophobic interactions on the chromatographic behavior of biopolymers with the use of chemically bonded silica-based HPLC columns and aqueous buffered mobile phases containing neutral salts in a wide range of concentration is discussed. Two columns packed with stationary phases appositely designed for biopolymer HPLC in size exclusion and anion exchange mode, respectively, are examined. Experimental data are evaluated by plotting the measured isocratic elution volumes of several standard proteins of different isoelectric point against the salt concentration in the mobile phase. Depending on the concentration and nature of salt, both columns exhibit different domains where either sieving effect or electrostatic or hydrophobic interactions are predominant. At sufficiently low salt concentrations electrostatic interactions are predominant leading to either increasing or decreasing elution volumes depending on the sign of the charges on the stationary phase and the protein, respectively. On the other hand, at high salt concentrations of a salt with sufficiently high molal surface tension increment proteins may be retained by hydrophobic interactions.     

High-Performance Aqueous Size-Exclusion Chromatography Using Diol-Bonded Porous Glass Packing Materials. Retention Behavior of Some Proteins

Abstract

Retention volume of proteins increased or decreased with increasing phosphate buffer or neutral electrolyte concentrations in the mobile phase. This variation suppressed or accelerated by changing pH values in the mobile phase. The behavior of proteins can be interpreted by knowing isoelectric points (pI) of proteins and pKa value of the residual silanol groups on the surface of diol-bonded porous glasses. Positively charged surface of proteins below pH 8.0 (cytochrome c, lysozyme) retarded the elution by the ion-adsorption effects and negatively charged proteins around pH 7.0 (egg albumin, bovin serum albumin) eluted earlier than expected by the ion-exclusion effects. These effects suppressed by increasing phosphate buffer and neutral electrolyte concentrations in the mobile phase. Size-exclusion separation was attained in the mobile phase over 0.1 M phosphates and 0.1 M NaCl concentrations at pH 7.0. Mcllvaine buffer and Gomori buffer showed opposite action to proteins for retention comparing with Soerensen phosphate buffer. Potassium thiocyanate showed the different action for retention of proteins comparing with other neutral electrolytes and acted like sodium dodecyl sulphonate.     

High-performance liquid chromatography of some basic drugs on a n-octadecylphosphonic acid modified magnesia-zirconia stationary phase

The high-performance liquid chromatographic behavior of some basic drugs was studied on a n-octadecylphosphonic acid modified magnesia-zirconia (C18PZM) stationary phase. The effect of mobile phase variables such as methanol content, ionic strength, and pH on their chromatographic behavior was investigated. The retention mechanism of basic drugs on the stationary phase was elucidated. The results indicate that both hydrophobic and cation-exchange interactions contribute to solute retention under most chromatographic conditions. The inherent Br?nsted-acid sites and also the adsorbed Lewis base anionic buffer constituents on accessible ZM surface Lewis acid sites play a role in the retention of ionized solutes by cation-exchange interaction. However, especially at high mobile phase pH, the retention of basic drugs depends mainly on hydrophobic interactions between solutes and support. Separations of the basic drugs on the C18PZM phase by a predominantly reversed-phase retention mode were very promising. The mixed-mode retention feature on this phase, as a result of the adsorbed Lewis base anionic buffer constituents acting as sites for cation-exchange, could also be very useful, e.g. for enhancing the chromatographic selectivity of such analytes. The C18PZM seems to be an excellent alternative to silica-based reversed-phase stationary phase for the separation of strongly basic solutes.     

液相色谱法分离纯化烟草疫霉菌激发子

利用阴离子交换色谱和疏水相互作用色谱从烟草疫霉菌培养液中分离了一种新的９０ＫＤ激发子蛋白,他阴离子交换色谱流动相的最佳ｐＨ值,建立了疏水相互作用色谱,硫酸铵与三羟甲基氨基甲烷（Ｔｒｉｓ）缓冲液／水洗脱模式,简化了纯化步骤和活性损失的危险。     

Isolation and purification of chemical constituents from the pericarp of Sophora japonica L. by chromatography on a 12% cross-linked agarose gel

A chromatographic method using 12% cross-linked agarose gel Superose 12 as the separation medium was developed for isolation and purification of the chemical constituents from the pericarp of Sophora japonica L. The mobile phase used for the separation was 2% acetic acid and 7% acetic acid in gradient elution. As a result, eight compounds including four kinds of flavonoids and four kinds of isoflavonoids were obtained in a one-step separation. A straightforward explanation of the separation mechanism of flavonoids and isoflavonoids on Superose 12 is also given. The flavonoids and isoflavonoids are retained on Superose 12 by a combination of hydrogen bonding and hydrophobic interactions between the hydroxyl groups of aglycone and the residues of the cross-linking reagents used in the manufacture of Superose 12.     

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. II. Separation of amino acids and peptides on sulfonated naphthalimido-modified silyl silica gel

The potential of 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP) as a mixed-mode stationary phase for capillary electrochromatography (CEC) was investigated for the separation of charged analytes, taking four amino acids (tyrosine, phenylalanine, tryptophan, histidine) as model analytes. The elution process of these charged analytes in CEC with SNAIP was dominated by a combination of both electrophoretic process and chromatographic process involving hydrophobic as well as electrostatic interactions. In order to study the retention mechanism, the CEC retention factor k* and the velocity factor ke* were measured for the amino acids, which allowed the assessment of the respective contribution from the differential processes underlying the separation. Migration and retention could be mediated by changing various mobile phase compositions, including buffer pH, buffer concentration, and concentration of organic solvent. Based on the results obtained by separation of the amino acids, the separation of eight peptides (Gly-Val, Gly-Phe, Gly-Ile, Gly-His, Gly-Lys, Lys-Lys, Gly-Gly-Gly, Gly-Gly-His) was attempted. A good separation was achieved under an isocratic elution with a mobile phase consisting of 35 mM phosphate buffer (pH 3.8) and 40% methanol.     

Retention behavior of basic compounds on alkylphosphonate-modified magnesia-zirconia composite stationary phase in RPHPLC

Summary The chromatographic properties of an alkylphosphonate-modified magnesia-zirconia composite stationary phase have been investigated by reversed-phase high-performance liquid chromatography with basic compounds as probes. The influence of organic modifier composition and mobile phase pH was studied. The new stationary phase, similar to a silica-based reversed-phase stationary phase, has hydrophobic properties, but greater pH stability. Use of the phase results in more symmetric peaks for basic compounds. A possible mechanism of retention of basic solutes on the new stationary phase is discussed. The chromatographic behavior of the basic solutes depends mainly on hydrophobic interactions between the solutes and the hydrophobic moiety of the stationary phase. Br?nsted acidic and basic sites on the surface of the new stationary phase play an important role in the retention of ionized solutes by ion-exchange interaction. Promising separations of some basic compounds have been achieved by use of methanolic TRIS buffer, pH 10.0, as the mobile phase.     

Effect of the ionic composition of a mobile phase on the chromatographic retention of profen enantiomers on a chiral adsorbent with grafted eremomycin antibiotic

The effect of ionic composition of mobile phase on retention and chromatographic separation of optical isomers of several derivatives of 2-arylpropanoic acid on a chiral stationary phase Diaspher-Chiralsel-E with a grafted eremomycin antibiotic was studied. As was shown, the mechanism of retention differs from that for hydrophobic reverse-phase adsorbents, as it involves a substantial ion-exchange component that determines the nonmonotonic dependence of the retention factor and the enantioselectivity on eluent pH. The effect of the concentration of buffer salt in the eluent on the chromatography of this class of compounds is discussed.     

Chromatographic evaluation of reversed-phase/anion-exchange/cation-exchange trimodal stationary phases prepared by electrostatically driven self-assembly process

This work describes chromatographic properties of reversed-phase/cation-exchange/anion-exchange trimodal stationary phases. These stationary phases were based on high-purity porous spherical silica particles coated with nano-polymer beads using an electrostatically driven self-assembly process. The inner-pore area of the material was modified covalently with an organic layer that provided both reversed-phase and anion-exchange properties while the outer surface was coated with nano-sized polymer beads with strong cation-exchange characteristics. This design ensured spatial separation of the anion-exchange and the cation-exchange regions, and allowed reversed-phase, anion-exchange and cation-exchange retention mechanisms to function simultaneously. Chromatographic evaluation of ions and small molecules suggested that retention of ionic analytes was influenced by the ionic strength, pH, and mobile phase organic solvent content, and governed by both ion-exchange and hydrophobic interactions. Meanwhile, neutral analytes were retained by hydrophobic interaction and was mainly affected by mobile phase organic solvent content. Depending on the specific application, selectivity could be optimized by adjusting the anion-exchange/cation-exchange capacity ratio (selectivity), which was achieved experimentally by using porous silica particles with different surface areas.     

Applicability of alkyl-bonded ultra-pure silica stationary phases for gradient reversed-phase HPLC of folates with conventional and volatile buffers under highly aqueous conditions

Applicability of several alkyl-bonded silica stationary phases was tested for gradient RP-HPLC of folates under highly aqueous conditions. High retention of folates was achieved on alternative phases with enhanced polarity and classical phases with higher carbon content. Phases exhibiting polar secondary interactions were found to provide better selectivity for late-eluting folates, whereas selectivity for early-eluting folates was mostly dependent on hydrophobic interactions. Best selectivity in phosphate buffered mobile phase was achieved on polar-endcapped silica phases (Aquasil C18 and HyPurity Aquastar) followed by alternative Atlantis dC18. Classical phases exhibited poorer separation of 10-formyl-folic acid and 5-formyl-tetrahydrofolate, but it could be considerably improved by increasing the buffer pH. Strong secondary interactions of ion-exchange character on polar-embedded phases resulted in marked peak deterioration, loss of recovery and dramatic changes in retention behaviour for early- and late-eluting folates when changing the mobile phase composition and pH. Therefore, polar-embedded phases such as HyPurity Advance were found to be unsuitable for separating folates. Stationary phases exhibited peak deterioration when using volatile buffer of low ionic strength. Better results were obtained with classical phases, whereas alternative phases showed not only peak deterioration but also a decrease in recovery and poorer selectivity due to increased secondary interactions in volatile buffer.     

Protein separation and enrichment by counter-current chromatography using reverse micelle solvent systems

A protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by high-speed counter-current chromatography using a two-phase aqueous/reverse micelle-containing organic solvent system. About 50% stationary phase retention ratio was obtained in most chromatographic experiments. Separations were manipulated mainly by pH gradients that controlled the electrostatic interactions between the protein molecules and reverse micelles. Separations were further improved by incorporating an ionic strength gradient along with the pH gradient. Control of ionic strength in the aqueous solution helped fine-tune protein partitioning between the stationary and mobile phases. Although non-specific protein interactions affected baseline resolution, recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions.     

Evaluation of multi-modal high salt binding ion exchange materials

The performance and selectivity of novel cation and anion exchange multi-modal chromatographic materials were evaluated. Desorption profiles of 13 proteins possessing a range of properties (e.g. size, charge and hydrophobicity) were determined on the cation exchange materials. Batch experiments were carried out by loading individual proteins on each resin at low salt, and examining the desorption of the proteins during sequential washes with increasing salt concentrations. While all of the resins exhibited some binding of proteins at elevated salt concentrations, this effect was more pronounced on the resins with aromatic ligands as compared to the materials with aliphatic ligands. As expected, materials with higher ionic capacities exhibited higher binding at elevated salts. In addition, some proteins exhibited high binding at elevated salt concentrations to all of the resins. The combined effect of charge and other secondary interactions with these multi-modal chromatographic materials enables high salt binding of a range of proteins as well as unique selectivities for the recovery of certain classes of proteins. Since the anion exchange materials all exhibited high binding at elevated salt concentrations the work with these materials focused on a study of elution strategies to remove proteins from these aromatic based materials. After evaluating various elution protocols, a combined strategy of pH change and chaotropic salt were shown to minimize electrostatic and hydrophobic interactions and was found to be an effective elution strategy for this class of anion exchange materials using peanut lectin as a model protein.     

Use of mixed-mode sorbents for the electrochromatographic separation of thrombin receptor antagonistic peptides

In this study, the thrombin receptor antagonistic peptide TRAP-1 and its alanine-scan analogues, TRAP 2-6, have been employed as probes to characterise the performance of C18/SCX mixed-mode capillary electrochromatographic (CEC) columns. It was found that the resolution of this group of peptides could only be achieved in a narrow pH range with phosphate-based running electrolytes. The influence of the running electrolyte composition, e.g. the buffer choice, the ionic strength, the pH and the organic solvent content, on the electroosmotic flow (EOF) of these mixed-mode CEC columns was investigated. In addition, the retention mechanism for this group of peptide probes in the electrochromatographic process was studied by examining the effect of varying the running electrolyte composition. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving both hydrophobic as well as ion exchange interactions. By modulating the running electrolyte composition, the hydrophobic or ion exchange components of the interaction process could be made to dominate the chromatographic retention of the peptides. Based on this strategy, a high-resolution separation of six closely related synthetic peptides was demonstrated with this mixed-mode CEC system.     

Preparation and characterization of prototypes for multi-modal separation media aimed for capture of negatively charged biomolecules at high salt conditions

Several prototypes of multi-modal ligands suitable for the capture of negatively charged proteins from high conductivity (28 mS/cm) mobile phases were coupled to Sepharose 6 Fast Flow. These new prototypes of multi-modal anion-exchangers were found by screening a diverse library of multi-modal ligands and selecting anion-exchangers resulting in elution of test proteins at high ionic strength. Candidates were then tested with respect to breakthrough capacity of BSA in a buffer adjusted to a high conductivity (20 mM Piperazine and 0.25 M NaCl, pH 6.0). The recovery of BSA was also tested with a salt step (from 0.25 to 2.0 M NaCl using 20 mM Piperazine as buffer, pH 6.0) or with a pH-step to pH 4.0. We have found that non-aromatic multi-modal anion-exchange ligands based on primary or secondary amines (or both) are optimal for the capture of proteins at high salt conditions. Furthermore, these new multi-modal anion-exchange ligands have been designed to take advantage not only of electrostatic but also hydrogen bond interactions. This has been accomplished through modification of the ligands by the introduction of hydroxyl groups in the proximity of the ionic group. Experimental evidence on the importance of the relative position of the hydroxyl groups on the ligand in order to improve the breakthrough capacity of BSA has been found. Compared to strong anion-exchangers such as Q Sepharose Fast Flow the new multi-modal weak anion-exchangers have breakthrough capacities of BSA at mobile phases of 28 mS/cm and pH 6.0 that are 20-30 times higher. The new multi-modal anion-exchangers can also be used at normal anion-exchange conditions and with either a salt step or a pH-step to acidic pH can accomplish the elution of proteins. In addition, the functional performance of the new anion-exchangers was found to be intact after treatment in 1.0 M sodium hydroxide solution for 1 week. A number of multi-modal anion-exchange ligands based on aromatic amines exhibiting high breakthrough capacity of BSA have been found. With these ligands recovery was often found to be low due to strong non-electrostatic interactions. However, for phenol derived anion-exchange media the recovery can be improved by desorption at high pH.     

Hydrophobic interaction chromatography of Chromobacterium viscosum lipase on polypropylene glycol immobilised on Sepharose.

The fractionation of Chromobacterium viscosum lipase was performed using a polypropylene glycol-Sepharose gel. The influence of mobile phase composition on the adsorption of lipase on the gel was studied and it was found that the retention of lipase depends on the salt used and increased with increasing the ionic strength. The retention was not strongly affected by changing the pH value of the mobile phase. By using 20% (w/v) ammonium sulphate in phosphate buffer a total retention of lipase on the column was obtained and by simply decreasing the ionic strength of the buffer, desorption of lipase could be achieved. The chromatographic purification of Chromobacterium viscosum lipase by hydrophobic interaction chromatography on Sepharose CL-6B modified by covalent immobilisation of 1,4-butanediol diglycidyl ether, polyethylene glycol and polypropylene glycol was also compared.