Influence of the pore size of reversed phase materials on peptide purification processes

The influence of the pore size of a chromatographic reversed phase material on the adsorption equilibria and diffusion of two industrially relevant peptides (i.e. a small synthetic peptide and insulin) has been studied using seven different reversed phase HPLC materials having pore sizes ranging from 90 Å to 300 Å. The stationary phase pore size distribution was obtained by inverse size exclusion measurement (iSEC). The effect of the pore size on the mass transfer properties of the materials was evaluated from Van Deemter experiments. It has been shown that the lumped mass transfer coefficient increases linearly with the average pore size. The Henry coefficient and the impurity selectivity were determined in diluted conditions. The saturation capacity of the main peptides was determined in overloaded conditions using the inverse method (i.e. peak fitting). It was shown that the adsorption equilibria of the peptides on the seven materials is well described by a surface-specific adsorption isotherm. Based on this a lumped kinetic model has been developed to model the elution profile of the two peptides in overloaded conditions and to simulate the purification of the peptide from its crude mixture. It has been found that the separation of insulin from its main impurity (i.e. desamido-insulin) was not affected by the pore size. On the other hand, in the case of the synthetic peptide, it was found that the adsorption of the most significant impurity decreases with the pore size. This decrease is probably due to an increase in silanol activity with decreasing pore size.     

A Novel Approach to Reversed-Phase Preparative High-Performance Liquid Chromatography of Peptides

Abstract

In this study, we describe a novel approach to preparative liquid chromatography which takes advantage of the different relative hydrophobicities of components of a sample mixture, so that when a column is optimally loaded with an aqueous solution of the sample mixture, there is competition among the sample components for the adsorption sites on the hydrophobic stationary phase. The more hydrophobic components compete more successfully for these sites than more hydrophilic components, which are displaced and immediately eluted from the column. Thus, the major separation takes place in water. Subsequent treatment with an aqueous organic modifier is only required to wash retained components off the column and takes no part in the major separation process. This approach was applied to the preparative purification of mixtures of closely-related peptides, representing the crude peptide mixtures typically obtained from solid-phase peptide synthesis. The excellent separation profiles and high yields of pure peptide products on analytical columns reported in this study demonstrate that this methodology has great potential for preparative separation of a major component from hydrophilic and/or hydrophobic impurities.     

Comparison of the chromatography of octadecyl silane bonded silica and polybutadiene-coated zirconia phases based on a diverse set of cationic drugs

In this study, we compare the separation of basic drugs on several octadecyl silane bonded silica (ODS) phases and a polybutadiene-coated zirconia (PBD-ZrO2) phase. The retention characteristics were investigated in detail using a variety of cationic drugs as probe solutes. The ODS phases were selected to cover a relatively wide range in silanol activity and were studied with ammonium phosphate eluents at pH 3.0 and 6.0. Compared to any of the ODS phases, the PBD-ZrO2 phase showed very significant differences in selectivities towards these drugs. Due to the presence of both reversed-phase and ion-exchange interactions between the stationary phase and the basic analyte on ODS and PBD-ZrO2, mixed-mode retention takes place to some extent on both types of phases. However, very large differences in the relative contributions from ion-exchange and reversed-phase interactions on the two types of phases led to quite different selectivities. When phosphate is present in the eluent and adsorbs on the surface, the PBD-ZrO2 phase takes on a high negative charge over a wide pH range due to phosphate adsorption on its surface. On ODS phases, ion-exchange interactions result from the interactions between protonated basic compounds and ionized residual silanol groups. Since the pH of the eluent influences the charge state of the silanol groups, the ion-exchange interactions vary in strength depending on pH. At pH 6.0, the ion-exchange interactions are strong. However, at pH 3.0 the ion-exchange interactions on ODS are significantly smaller because the silanol groups are less dissociated at the lower pH. Thus, not only are the selectivities of the ODS and PBD-ZrO2 phases different but quite different trends in retention are observed on these two types of phases as the pH of the eluent is varied. More importantly, by using the large set of "real" basic analytes we show the extreme complexity of the chromatographic processes on the reversed stationary phases. Both the test condition and solute property influence the column performance. Therefore, use of only one or two probe solutes is not sufficient for column ranking.     

Capillary electrochromatography of basic compounds using octadecyl-silica stationary phases with an amine-containing mobile phase

The capillary electrochromatographic (CEC) analysis of basic compounds on octadecyl-silica stationary phases (Hypersil ODS and Spherisorb ODS I) was studied. A basic drug (fluvoxamine) and one of its possible impurities were used as test compounds. With an eluent of acetonitrile-phosphate buffer (pH 7.0), the compounds could be baseline-separated; however, broad and tailing peaks were obtained. To minimise detrimental interactions with residual silanol groups, the pH of the mobile phase was lowered to 2.5, but the plate numbers were still quite low (<2.6x10(4) plates/m). Addition of a masking agent (hexylamine or triethylamine) to the mobile phase resulted in much better peak efficiencies (ca. 1x10(5) plates/m). Therefore, the influence of the amine concentration and pH of the mobile phase on the CEC performance (peak width, peak tailing, electroosmotic flow, selectivity) was investigated in detail. Highest efficiencies (2.8x10(5) plates/m) could be obtained with the Spherisorb column, while the Hypersil column offered a better selectivity. Furthermore, the results show that the residual silanol groups are (at least partly) responsible for the separation of the basic compounds and that the amount of injected sample has an unusually large effect on the peak efficiency. The usefulness of the system for impurity profiling was demonstrated with a mixture containing fluvoxamine and its stereoisomer (a possible impurity) at the 0.1% level. The general effectiveness of amine additives in CEC was illustrated by the separation of a mixture of five structurally different basic drugs yielding plate numbers in the 1x10(5)-3x10(5) plates/m range. Comparison with capillary electrophoretic analysis revealed a unique selectivity of the CEC system which is based on both electrophoretic mobility and chromatographic partitioning.     

Steady-migration retention characteristics of peptides under gradient elution: application towards a dynamic separation method for minor-adjustments of the retention of peptides in RPLC

Minor-adjustment of the retention of peptides, induced by varying the mobile phase flow-rate(MPF-R), is a new dynamic separation method for simultaneously and rapidly identifying and improving the selectivity of hidden and overlapping peptide peaks. It can also-stabilize the reverse elution order of some pair-peaks under gradient elution in reverse phase liquid chromatography. The retention characteristics of peptides under gradient elution in RPLC was firstly found to be dominated by two variables of the steady region(SR) and migration region(MR). The changes in peptide retention induced by varying the MPF-R can be attributed to changes in the rate of bond breaking of multiple molecular interactions of peptides from the SR and of the mass transfer of peptides from the stationary phase to the mobile phase in the MR. The two dynamic variables were also found to independently depend on the type of peptide. Desirable results were obtained using six standard oligopeptides and a real sample of trypsin-digested lysozyme.It is expected that the quality control of peptide drugs, high dispersion of peptide peaks in peptide mapping and "bottom-up MS"in proteomics will be improved by this method, even enabling peptide purification on a preparative scale in industry.     

Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240000 to 460000/m, and the relative standard deviation for t0 and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated.     

Polysiloxane-encapsulated stationary phase for reversed-phase high-performance liquid chromatography

Summary Silica beads of 6-μm average diameter were silanized with methylvinyldiethoxysilane and then subjected to encapsulation with poly(methylvinylsiloxane). The resulting product is a new stationary phase for reversed-phase high performance liquid chromatography (RP-HPLC) which has superior ability for the separation of polar, non-polar and basic compounds. The chromatographic peaks are symmetric. Its stability has been studied; after continuous use for three months the carbon content and chromatographic behaviour of the phase were unchanged. on to the silica surface to given an uniform organic film. Material prepared in this way has both good chromatographic behaviour and superior selectivity. Because contact of the silica matrix with the mobile phase is avoided, the alkali-resisting ability of the stationary phase is increased. The non-specific adsorption of alkaline solutes on to the silica surface is also avoided because of the complete coverage of surface silanol groups. Reports of stationary phases encapsulated with polystyrene [6], polybutadiene [I] and octadecylsiloxane polymers have recently appeared in the literature [3]. In this paper we report the encapsulation of poly-(methylvinylsiloxane) (analogous to the phase SE-31 often used in GC) on to a silica matrix previously modified with methylvinyldiethoxysilane. The resulting phase has superior performance in reversed-phase HPLC.     

Alternative high-performance liquid chromatographic peptide separation and purification concept using a new mixed-mode reversed-phase/weak anion-exchange type stationary phase

This article describes a new complementary peptide separation and purification concept that makes use of a novel mixed-mode reversed-phase/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 microm, 100 A 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 x 4 mm i.d. RP/WAX column (5 microm) 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.     

Liposome Capillary Electrophoresis of Peptides and Proteins

Liposome capillary electrophoresis (LCE) using unilamellar liposomes composed of the zwitterionic phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a suspended pseudo stationary phase has been investigated for its capability at separating peptides and proteins in bare fused-silica capillaries. The study has explored different strategies for allowing the liposome suspension to act as a disperse pseudo stationary phase with the ability of modulating selectivity, resolution and separation performance of peptides and proteins in bare-fused silica capillaries. Such strategies comprise the use of capillaries either partially or totally filled with the liposome suspension, whereas the electrolyte solution is liposome-free, or the incorporation of the liposomes into the buffer solution employed for rinsing the capillary and as the background electrolyte. Three synthetic peptides of similar amino acid sequence and four basic standard proteins have been employed as test analytes. Varying the volume of the liposome suspension introduced in the capillary promoted differentiated variations in the migration velocity of the three peptides reflecting their selective interactions with the liposomes. Efficient separation of basic proteins was obtained at pH 7.4 in a bare fused-silica capillary with the electrolyte solution containing 60 μM POPC.     

Quantitative yields of underivatized peptides by HPLC

New methods for peptide separation are being developed. Resolution of peptide mixtures on HPLC with good reproducibility indicates the progress that has already been made in development of supports with useful separation capabilities. At present adsorptive characteristics of the stationary phases may place limitations on the applicability of the technique to underivatized peptides. Hopefully, the application of ionpairing techniques and the development of new support materials will expand the range of application for protein and peptide purification.     

Separation and purification of chlorogenic acid by molecularly imprinted polymer monolithic stationary phase

Separation and purification of chlorogenic acid by removal of the impurities compounds co-existed in the product using molecular imprinting technique was firstly reported. In this work, an in situ synthesis method was utilized for the preparation of molecularly imprinted polymer monolithic stationary phase using the impurity molecule (caffeic acid) as template, the mixture of tetrahydrofuran and isooctane as solvent, and methacrylic acid and ethyl glycol dimethacrylate as functional monomer and cross-linker, respectively. The retention behavior of the monolithic polymer to chlorogenic acid molecule, the template and several main impurities compounds in the product was studied and the adsorption capacity of compounds on the stationary phase determined by frontal chromatographic technique. A relatively weak retention of the target product molecule (chlorogenic acid) on the polymer and a strong adsorption capability of the monolith to the template and several main impurities were observed. This might mainly result from the 'shape' difference of chlorogenic acid molecule with the impurities compounds molecules. This approach was shown to be successful for the separation and purification of chlorogenic acid from the extract of Eucommia ulmodies leaves.     

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.     

Siloxane/silarylene copolymers as stationary phases for capillary gas chromatography part I: Evaluation of silanol terminated dimethyl substituted polymers

The thermal stability of silicones can be improved on replacement of certain of the oxygen atoms in the polymer backbone by phenyl groups. Such a polymer has been synthesized and evaluated for use as stationary phase in fused silica capillary gas chromatography; the polymer was dimethyl substituted and silanol terminated. A selectivity was provided by the phenyl groups in the backbone. For comparative purposes, a silanol-terminated dimethylpolysiloxane has also been evaluated. Both stationary phases gave columns of highest separation efficiency and the supporting fused silica surface was deactivated by the stationary phases on thermal treatment. Further, low column bleeding was observed at the maximum temperature tested, 370°C. The phenyl-containing phase could be immobilized to 60% by heat treatment, but the pure dimethylpolysiloxane was 10% immobilized. The influence on immobilization of factors such as nature of the supporting surface, stationary phase silanol content, reaction temperature and atmosphere in the column during reaction has been studied.     

Siloxane/silarylene copolymers as stationary phases for capillary gas chromatography. Part II: Phenylsubstituted polymers

A commercially available silanol terminated silicone stationary phase, OV-61-OH (33% phenyl), and two phenyl-substituted siloxane/silarylene copolymers, Sila 3 (27% phenyl) and 4 (35% phenyl), have been evaluated for use as stationary phases in fused silica capillary columns for gas chromatography. Ulterations in column adsorptive activity, separation efficiency, stationary phase film thickness and selectivity after column conditioning for 50 h at 370°C have been studied. A high thermal stability was experienced with the stationary phases tested here. For OV-61-OH, the best thermal stability was obtained when coated on untreated fused silica, which illustrates the importance of grafting reactions here. The heat treatment resulted in some deactivation of adsorptive sites in the column. A higher degree of column deactivation was achieved when surface silylation was performed prior to coating. High thermal stability was achieved with Sila 3 when coated on such surfaces. Sila 3 would thus be preferred in cases when high thermal stability in combination with high dsorptive inertness is desired. Sila 4 showed low column bleeding at 370 °C, but prolonged heating at this temperature resulted in the broadening of n-alkane peaks when eluted at 90 °C. This indicates that excessive crosslinking has taken place during the heat treatment and the minimum allowable column operation temperature is thereby increased to ca. 120 °C. The separation of aza-arenes and of triglycerides are shown as applications.     

Isocratic mixed mode liquid chromatographic separation of phospholipids with octadecylsilane-silica stationary phases

Molecular species of phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid were resolved by isocratic reversed phase high performance liquid chromatography (HPLC) using mobile phases of methanol-isopropanol containing para-toluenesulfonic acid (p-tsa). Separation by both non-polar fatty acid chain length and by polar head group functionality was achieved concurrently upon a commercially available octadecylsilane (C18) column endcapped with trimethylsilane (C1) groups. Using a mobile phase of 97.5:2.5 methanol:isopropanol with 7OmMpara-toluenesulfonic acid (p-tsa) at a pH of approximately 1, twelve phospholipid species comprised of four tail group classes (dilauroyl-,dimyristoyl-, dipamitoyl- and distearoyl-) and three head group speciations (phosphatidylcholine, phosphatidylethanolamine and phosphatadic acid) were separated. The column was then exposed to the acidic mobile phase for 48 hours continuously during which the bound phase underwent severe acid-induced hydrolysis, after which the separation of the twelve analytes resulted in the separation of the phospholipid species by non-polar tail group alone. The experimental results are discussed in terms of potential separation mechanisms including dependency of the separation on adsorption of the counter ion into the stationary phase, residual acidic silanol group interactions, and potential interactions of the surface active phospholipids with C1 groups.     

Peptide separation in hydrophilic interaction capillary electrochromatography

Separation of small peptides by hydrophilic interaction capillary electrochromatography (HI-CEC) has been investigated. The negative surface charge of a hydrophilic, strong-cation-exchange stationary phase (PolySULFOETHYL A) provided a substantial cathodic electroosmotic flow (EOF). The influence of acetonitrile content, ionic strength, mobile phase pH as well as applied voltage on the migration of the peptides was studied. Possible retention mechanisms of the peptides in HI-CEC were discussed. It was found that hydrophilic interaction between the solutes and the stationary phase played a major role in this system, especially when mobile phases with high acetonitrile content were used. However, an ion-exchange mechanism and electrophoretic mobility also affect the migration of the peptides in HI-CEC. Elution order and selectivity was proved to be different in HI-CEC and capillary zone electrophoresis (CZE), thus revealing the potential of HI-CEC as a complementary technique to CZE for the separation of peptides. Efficiency and selectivity of HI-CEC for the separation of peptides were demonstrated by baseline separating nine peptides in 6 min.     

LC and LC-MS Separation of Peptides on Macrocyclic Glycopeptide Stationary Phases: Diastereomeric Series and Large Peptides

Previous work on the LC separation of peptides had shown that macrocyclic glycopeptide stationary phases to be selective for peptides of five to thirteen amino acids in length. In this work, the selectivity of the teicoplanin stationary phase is compared to that of a C18 stationary phase for seven diastereomeric enkephalin peptides. The teicoplanin stationary phase separated all seven diastereomeric enkephalin peptides in a single chromatographic run. The insertion of d-amino acids into the primary enkephalin sequence produced areas of hydrophobicity that influenced retention order on the C18 stationary phase. However, analogous trends are not observed on the teicoplanin stationary phase, which is more polar and structurally diverse. Optimization of the mobile phase and the use of a step-gradient for the enkephalin separation on the teicoplanin stationary phase is discussed. Also, the selectivity of macrocyclic glycopeptide stationary phases for peptides of 14, 28, 30, and 36 amino acids also is investigated and compared to separation on a C18 stationary phase. A method for eluting peptides with multiple basic amino acids, which tend to be strongly retained on the macrocyclic glycopeptide stationary phases, is presented.     

Retention mechanism divergence of a mixed mode stationary phase for high performance liquid chromatography

Mixed mode stationary phases utilize secondary retention mechanisms to add a dimensionality to the surface of high performance liquid chromatography (HPLC) adsorbents. This approach was used by several authors to improve the separation performance of single dimension separations. We explored the magnitude of these secondary interactions by performing an off-line two-dimensional (2D)-HPLC separation with a Scherzo SM-C18 column of a β-lactoglobulin tryptic digest with a mobile phase pH of 7 in the first dimension and 2 in the second. Mechanism divergence was determined using the peak capacity and a geometric approach to factor analysis, to measure the correlation. This separation was repeated with a C18 stationary phase as a control. It was found that the C18 column had a correlation coefficient of 0.784, smaller than the mixed mode column, 0.884. This indicated that the retention mechanisms of the C18 column were more divergent under these two pH environments than the mixed mode column. However, the SM-C18 still provided alternative selectivity of the peptides to that of the C18 and could be considered as a good alternative for further 2D-HPLC separations.