Retention Behaviour Of Peptides Research Articles

Two-dimensional high-performance thin-layer chromatography for the characterization of milk peptide properties and a prediction of the retention behavior – a proof-of-principle study

High-performance thin-layer chromatography (HPTLC) is a suitable method for the analysis of peptides and proteins due to a wide selection of stationary and mobile phases and various detection options. Especially, two-dimensional HPTLC (2D-HPTLC) enables a higher resolution compared to one-dimensional HPTLC in the separation of complex peptide mixtures. Similar to 2D electrophoresis, characteristic peptide patterns can be obtained, allowing a differentiation of ingredients based on varying protein origins.The aim of this study was to evaluate 2D-HPTLC with regard to its suitability for the characterization of proteins/peptides and to verify whether it is possible to predict the retention behavior of peptides based on their properties. As models, the five most abundant milk proteins α-lactalbumin, β-lactoglobulin, α-, β-, and κ-Casein were used. In order to determine the repeatability of the peptide separation by 2D-HPTLC, each tryptic protein hydrolyzate was separated eight times. The standard deviations of the retardation factors for the separated peptides varied between 1.0 and 11.1 mm for the x-coordinate and 0.5–7.3 mm for the y-coordinate. It was also shown that after the chromatographic separation, peptides of the individual protein hydrolyzates were located in specific areas on the HPTLC plate, so that a clustering could be obtained for the whey proteins‘ as well as the caseins‘ hydrolyzates. For establishing correlations between the properties of the peptides and their retardation factors, 51 of 85 selected peptides were identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). On this basis, statistically significant correlations (α = 0.05) between the retardation factors of the peptides and their isoelectric points, as well as the percentage of anionic and non-polar amino acids in the peptides were established. Finally, it was investigated, whether the retardation factors for peptides can be predicted on the basis of a linear regression of the percentage of non-polar amino acids in a peptide. For this purpose, a mixture of artifical (synthetic) peptides (n = 14) was separated by 2D-HPTLC and the measured retardation factors were compared with the corresponding retardation factors calculated. Absolute deviations of 0.3–17.9 mm were obtained. In addition, the universal applicability of the method to other protein sources other than milk proteins (animal protein) was tested using a mixture of pea peptides (plant protein, n = 3) resulting in absolute deviations of 0.7–8.6 mm.

Accurate modelling of the retention behaviour of peptides in gradient-elution hydrophilic interaction liquid chromatography

The applicability of models to describe peptide retention in hydrophilic interaction liquid chromatography (HILIC) was investigated. A tryptic digest of bovine-serum-albumin (BSA) was used as a test sample. Several different models were considered, including adsorption, mixed-mode, exponential, quadratic and Neue–Kuss models. Gradient separations were performed on three different HILIC stationary-phases under three different mobile-phase conditions to obtain model parameters. Methods to track peaks for specific peptides across different chromatograms are shown to be essential. The optimal mobile-phase additive for the separation of BSA digest on each of the three columns was selected by considering the retention window, peak width and peak intensity with mass-spectrometric detection. The performance of the models was investigated using the Akaike information criterion (AIC) to measure the goodness-of-fit and evaluated using prediction errors. The F-test for regression was applied to support model selection. RPLC separations of the same sample were used to test the models. The adsorption model showed the best performance for all the HILIC columns investigated and the lowest prediction errors for two of the three columns. In most cases prediction errors were within 1%.

Retention of glycopeptides analyzed using hydrophilic interaction chromatography is influenced by charge and carbon chain length of ion-pairing reagent for mobile phase.

Characterization of the glycans of glycoproteins is essential for the development and production of biologics. Numerous methods are available for analyzing the glycans of glycoproteins directly and labeled glycans. Nevertheless, glycopeptides are difficult to resolve because of their exceptional complexity and the microheterogeneity of glycans. These properties represent technical challenges to efforts to insure the accurate characterization of biopharmaceuticals to comply with regulatory requirements. Therefore, we investigated the retention behavior of peptides and glycopeptides in hydrophilic interaction chromatography-mode HPLC in the presence of ion-pairing reagents. Anionic ion-pairing reagents decreased the retention times of glycopeptides and improved resolution in the presence of higher concentrations or hydrophobicities of ion-pairing reagent. Anionic ion-pairing reagents increased retention times of larger glycans because of their increased hydrophilicity. In contrast, in the presence of cationic ion-pairing reagents, the retention times of glycopeptides with greater numbers of sialic acid residues decreased. It is appropriate to add an anionic ion-pairing reagent to the mobile phase for good separation of glycopeptides. The collision cross-sectional area values of glycopeptides determined using electrospray ionization-ion mobility spectrometry-mass spectrometry correlated with retention times. These findings support the implementation of hydrophilic interaction chromatography-mode HPLC to improve the characterization of glycosylated biopharmaceuticals.

Structure‐guided RP‐HPLC chromatography of diastereomeric α‐helical peptide analogs substituted with single amino acid stereoisomers

An α-helical model peptide (Ac-EAEKAAKE-X-EKAAKEAEK-amide) was used as a template to examine the efficacy of conventional reversed-phase high-performance liquid chromatography (RP-HPLC) in separating peptide analogs with single substitutions (at position X) of diasteromeric amino acids Ile, allo-Ile, d-Ile and d-allo-Ile. We compared differences in peptide retention behavior on a C8 column and a C18 column at different temperatures. We demonstrated how subtle differences in peptide secondary structure affected by the different substitutions of amino acids with identical overall hydrophobicity enabled effective resolution of these peptide analogs. We also demonstrated the ability of RP-HPLC to separate Ile- and allo-Ile-substituted analogs of a 26-residue α-helical antimicrobial peptide (AMP), with the substitution site towards the C-terminus of the α-helix. These peptides show different values of antibacterial activity and hemolytic activity, and different selectivity against bacteria and human cells. Our results underline the ability of RP-HPLC to resolve even difficult diasteromeric peptide mixtures as well as its value in monitoring very subtle hydrophobicity changes in de novo-designed AMP.

Influence of sample and mobile phase composition on peptide retention behaviour and sensitivity in reversed-phase liquid chromatography/mass spectrometry

Because the chromatographic behaviour of peptides is totally different from that of small molecules, a good understanding of the mechanisms that occur from injection to detection in reversed-phase LC–MS is strongly recommended to successfully develop not only qualitative but also quantitative methods. In this study, design of experiments was used in order to investigate the influence of the experimental parameters, i.e. sample and mobile phase composition, on a peptide mixture covering a wide range of molecular weights, isoelectric points and hydropathies. First, a screening design was developed to identify the significant factors concerning mobile phase (ion-pairing reagent nature and concentration) and sample composition (organic modifier proportion and ion-pairing reagent nature) on retention and response intensity (sensitivity). Then, after having selected the experimental domain and the significant factors, a full factorial design was used to further investigate the role of the considered factors and their interactions. Interestingly, ion-pairing reagent nature present in the sample had a tremendous effect on retention and response intensity. Optimal conditions leading to good sensitivity and adequate peptide retention without band splitting were selected and could be used as starting point for rapid method development using classical solvents and ion-pairing reagents.

Design of peptide standards with the same composition and minimal sequence variation to monitor performance/selectivity of reversed-phase matrices

The present manuscript extends our de novo peptide design approach to the synthesis and evaluation of a new generation of reversed-phase HPLC peptide standards with the same composition and minimal sequence variation (SCMSV). Thus, we have designed and synthesized four series of peptide standards with the sequences Gly-X-Leu-Gly-Leu-Ala-Leu-Gly-Gly-Leu-Lys-Lys-amide, where the N-terminal is either Nα-acetylated (Series 1) or contains a free α-amino group (Series 3); and Gly-Gly-Leu-Gly-Gly-Ala-Leu-Gly-X-Leu-Lys-Lys-amide, where the N-terminal is either Nα-acetylated (Series 2) or contains a free α-amino group (Series 4). In this initial study, the single substitution position, X, was substituted with alkyl side-chains (Ala<Val<Ile, in order of increasing hydrophobicity) or aromatic side-chains (Phe, Tyr). Peptide series pairs 1/2 and 3/4 thus represent SCMSV peptides, with the substitution site, X, being towards the N- or C-terminal, albeit with identical adjacent residues (Gly-X-Leu) to maintain the same environment around position X. In addition, peptide series pairs 1/3 and 2/4 enable an examination of the effect of a free, positively charged α-amino group on peptide retention behaviour relative to a blocked N-terminus. Peptide mixtures were run at pH 2 on columns with a variety of stationary phase selectivity (C8, C18, polar endcapped, polar embedded, ether-linked phenyl and Phenyl-Hexyl) under linear gradient conditions with acetonitrile or methanol as organic modifier. It was interesting to note that the addition of the hydroxyl group to the aromatic ring in a 12-residue Tyr SCMSV peptide pair had a dramatic effect on resolution compared to the Phe peptide pair. In addition, SCMSV peptide pairs with the β-branched Val and Ile side-chains at position X were the most difficult to separate compared to SCMSV peptides containing the aromatic side-chains Tyr and Phe. In this initial study, SCMSV peptide pairs proved to be a potent test of the selectivity of reversed-phase packing materials. In addition, mixtures of SCMSV peptide standards to assess overall capabilities of stationary phases to resolve complex peptide mixtures underlined the useful complementarity of combinations of different columns and elution conditions to maximize flexibility in peptide applications. Finally, our controlled, de novo designed peptide approach should spur the development of more quantitative selectivity parameters for peptide separations, such as those already available for small molecules, enhancing further the universal value of utilizing peptide standards to compare column performances in the separation of peptide mixtures.

Retention behavior of peptides in hydrophilic-interaction chromatography

Selected hydrophilic interaction chromatography (HILIC) columns packed with bare silica, bridge-ethyl hybrid silica, or an amide sorbent chemistry were utilized for an investigation of chromatographic behavior and separation selectivity of tryptic peptides. Retention model was proposed allowing for retention prediction of peptides with correlation coefficient R(2)~0.92-0.97 for various columns. The values of optimized amino acid retention coefficients were compared to those obtained for reversed-phase liquid chromatography (Gilar et al., Anal. Chem. 2010, 82, 265-275) and used to elucidate the impact of different amino acid on peptide HILIC retention. In contrast to reversed-phase chromatography, where presence of Phe, Trp, Ile, and Leu amino acid residues in sequence strongly promoted, and presence of hydrophilic His, Lys and Arg residues strongly reduced peptide retention, the effects of these amino acid residues in HILIC were opposite (His, Lys and Arg promote, Phe, Trp, Ile and Leu demote peptide retention in HILIC). Retention coefficient optimized for pH experiments illustrated the impact of silanols on HILIC retention.

Modeling and prediction of retention behavior of histidine‐containing peptides in immobilized metal‐affinity chromatography

Two kinds of structural characterization method as local descriptors and global descriptors were used to parameterize peptide structures, and several quantitative structure-retention relationship models were then constructed using partial least square (PLS), least-squares support vector machine (LS-SVM) and Gaussian process (GP) coupled with genetic algorithm-variable selection. These models were validated rigorously and investigated systematically by Tropsha et al. criteria, Monte Carlo cross-validation and one-way analysis of variance. Results show that regression models constructed using nonlinear approaches such as LS-SVM and GP are more robust and predictable than those by linear PLS method. By including linear and nonlinear terms in the covariance function, the GP is capable of handling both linear and nonlinear-mixed relationship, and thus presents a better performance than LS-SVM. Investigation of the optimal GP model revealed that diversified properties contribute to the retention behavior of peptides in immobilized metal-affinity chromatography. Particularly, coordination interaction, electrostatic factor, sovlation effect and hydrogen bonding are correlated significantly with the peptide retention ability.

Comprehensive comparison of eight statistical modelling methods used in quantitative structure–retention relationship studies for liquid chromatographic retention times of peptides generated by protease digestion of the Escherichia coli proteome

In this study, we propose a new peptide characterization method that gives attention to both the amino acid composition and the residue local environment. Using this approach, structural characteristics of peptides derived from Escherichia coli proteome were parameterized and, based upon that, the performance profile of eight statistical modelling methods were validated rigorously and compared comprehensively by applying them to modelling relationship between the sequence structure and retention ability for 816 experimentally measured peptides and to predicting normalized retention times for 121,273 unmeasured peptides in liquid chromatography. Results show that the regression models constructed by nonlinear approaches are more robust and predictable but time-consuming than those by linear ones. In these modelling methods, Gaussian process and back-propagation neural network possess the best stability, unbiased ability and predictive power, thus they can be used to accurately model the peptide structure–retention relationships; multiple linear regression and partial least squares regression perform worse compared to nonlinear modelling techniques but they are computationally efficient, so they are promising candidates for solving the qualitative problems involved in massive data. In addition, by investigating the descriptor importance in different models we found that the amino acid composition presents a significantly linear correlation with the retention time of peptides, whereas the residue environment is mainly correlated nonlinearly with peptide retention. The polar Arg and strongly hydrophobic amino acids such as Leu, Ile, Phe, Trp and Val are the critical factors influencing peptide retention behavior.

Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: Implications for prediction of peptide retention behaviour

The present study set out to investigate whether observed relative hydrophilicity/hydrophobicity values of positively charged side-chains (with Lys and Arg as representative side-chains) or hydrophobic side-chains (with Ile as the representative side-chain) were context-dependent, i.e., did such measured values vary depending on characteristics of the peptides within which such side-chains are substituted (overall peptide hydrophobicity, number of positive charges) and/or properties of the mobile phase (anionic counterions of varying hydrophobicity and concentration)? Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to two series of four synthetic peptide analogues (+1, +2, +3 and +4 net charge), the only difference between the two peptide series being the substitution of one hydrophobic Ile residue for a Gly residue, in the presence of anionic ion-pairing reagents of varying hydrophobicity (HCOOH ≈ H 3PO 4 < TFA < PFPA < HFBA) and concentration (2–50 mM). RP-HPLC of these peptide series revealed that the relative hydrophilicity of Lys and Arg side-chains in the peptides increased with peptide hydrophobicity. In addition the relative hydrophobicity of Ile decreased dramatically with an increase in the number of positive charges in the peptide, this hydrophobicity decrease being of greater magnitude as the hydrophobicity of the anionic ion-pairing reagent increased. These results have significant implications in the prediction of peptide retention times for proteomic applications.

Retention behaviour of peptides in capillary electrochromatography using an embedded ammonium in dodecacyl stationary phase

The potential of a silica stationary phase bearing an embedded cationic quaternary amine in dodecacyl chain, to separate peptides by capillary electrochromatography (CEC) has been evaluated. The ability of this stationary phase, to generate a consistent anodic electroosmotic flow was first evaluated. This flow was found to be independent of pH over a wide range (2–12), of the acetonitrile percentage in the electrolyte. The stability of the stationary phase evaluated through the electroosmotic flow variations was demonstrated at extreme pH values (2.5 and 9.1). A careful examination of the influence of mobile phase conditions (acetonitrile percentage, salt concentration and nature of buffer) on the electrochromatographic retention and electrophoretic migration behaviour of different standard peptides was carried out. In acidic conditions, the electrokinetic contribution appears to be predominant compared to the chromatographic one. Several types of chromatographic interactions, reversed-phase partitioning and anion exchange, were involved in the CEC of peptides, whereas repulsive electrostatic interaction could be considered as negligible. This stationary phase affords different selectivity compared to that observed on a C 18 stationary phase. Finally, the method was applied to the peptide mapping of β-lactoglobulin and human growth hormone under unpressurized and isocratic elution.

Selectivity differences in the separation of amphipathic α-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 α-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 α-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.

Selectivity differences in the separation of amphipathic $alpha;-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0Effects 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 α-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 α-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.

Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV‐1 gp120 epitope

In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic(CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular,10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1-9, were truncated analogs of the parent peptide, 10, which is a peptidomimetic related to a HIV-1 gp120 epitope. Several of these peptides have the propensity to form alpha-helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries(25 cm packed length, 100 microm i.d.) containing 3 microm n-octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase inelectroosmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature-dependent viscosity parameter, eta, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0-5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mM ammonium acetate but containing a fixed volume fraction, psi, of acetonitrile above psi = 0.40, the CEC retention behavior of peptides 1-10 correlated with a linear relationship linking the retention coefficient, kappta(cec), and the differential frictional size-to-mass ratio parameter, Xi(fric), of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Delta(Kappa)cec, and the product term [-0.66(Delta(Sigma[Xn]) log(Mi/Mj)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, Pi and Pj. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate,using CEC procedures, the influence of the key molecular attributes of peptides on their structure-retention dependencies.Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid-state peptide synthesis under conditions of different eluent composition or temperature.

Quantitative relationship between the retention of peptides on a reversed-phase alumina support and their physicochemical parameters

The retention time of 16 peptides was determined on polyethylene-coated alumina stationary phase using mixtures of acetonitrile (MeCN) and acidic and basic phosphate buffers as mobile phases. The majority of peptides showed nonlinear retention behaviour, their retention decreased with increasing concentration of MeCN in the lower concentration range, reached a minimum and increased again with increasing concentration of MeCN in the higher concentration range. Quadratic equations described well the atypical retention behaviour of peptides, the significance level being always over 95%. Principal component analysis indicated that the steric parameters of peptides exerted the highest influence on their retention on polyethylene-coated alumina stationary phase.

Capillary electrochromatography analysis of hormonal cyclic and linear peptides.

The retention behavior of linear and cyclic peptides has been studied by capillary electrochromatography (CEC) with a variety of different n-alkyl silica reversed-phase sorbents and also with mixed-mode phases containing both strong cation-exchange (sulfonic acid) and n-alkyl groups bonded onto the silica surface, using eluents ranging from pH 2.0 to pH 5.0. Depending upon the amino acid sequence, electrochromatographic retention of the peptides was strongly affected by the composition of the eluent, its pH value, and the choice of sorbent packed into the capillaries. The dominant separation processes operating with these charged analytes could be modulated inter alia by the content of organic modifier, acetonitrile, in the eluent, with peptide resolution predominantly arising from electrophoretic migration processes at high acetonitrile content. As the concentration of acetonitrile was decreased, chromatographic retention processes became more pronounced. With the n-alkyl silica CEC columns used in this study, silanophilic interactions between the sorbents and the charged peptides could be suppressed by increasing the molarity of the buffer and by adjusting the pH of the eluent to lower values. On the other hand, electrostatic interactions between basic peptides and the surface of strong cation-exchanger, mixed-mode materials can be suppressed at low pH values by using higher ionic strength conditions in the eluent. Different selectivity behavior was achieved with desmopressin and the other peptides with Spherisorb C18/SCX and Hypersil mixed-mode materials when an identical eluent composition of 60% (v/v) acetonitrile with 7.6 mM triethylammonium phosphate, pH 3.0, was used. These findings confirm that the surface charge density of the sorbent fulfills an important role in the modulation of peptide selectivity in CEC. These studies also confirm that the dependency of the logarithm of the CEC retention coefficients, i.e., log Kcec, of a peptide separated with n-octadecyl silica sorbents under CEC conditions, on the volume fraction, psi, of the organic solvent modifier, acetonitrile, within the range of 0.20 < or = psi < or = 0.60, can be approximated by a linear relationship. Moreover, these studies show that the selectivity differences of peptides separated by CEC with nonpolar sorbents in packed capillary systems can be discussed in terms of semiempirical dependencies that link peptide retention behavior with their molecular descriptor properties, e.g., their hydrophobicity, surface charge anisotropy, surface area, molecular mass and intrinsic charge, and thus to their corresponding linear free energy relationships.

Electrochromatographic characterization of etched chemically-modified capillaries with small synthetic peptides

In this investigation, various capillary electrochromatographic (CEC) experiments have been employed to characterize the properties of etched, chemically-modified surfaces of open tubular capillary columns with peptides as solute probes and under conditions of variable voltage, temperature and solvent composition. The separation performance of etched capillaries with either n-octadecyl or liquid crystal moieties derived from a cholesterol phase bonded to the surface were compared. With the liquid crystal bonded species, interesting and significantly different variations in retention behavior of peptides are obtained compared to those observed with the corresponding n-octadecyl modified surfaces by changes in temperature, solvent composition and field strength. These peptide separations illustrate the usefulness of this CEC approach for practical applications, where both the retention characteristics of the charged analytes as well as the selectivity differences due to the surface properties of the etched chemically-modified surfaces of open tubular capillary columns can be rationally modulated. As in HPLC, appropriate choice of CEC experimental variables, including the chemical properties of the immobilized ligand(s), represents a powerful tool for optimizing resolution.

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k cec, peak symmetry coefficients, λ sym, and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 μm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 μm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 – 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors ( k cec values) of these n-alkylbenzenes, acquired with an eluent of (25 m M Tris–HCl, pH 8.0,)–acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k cec values of the n-alkylbenzenes for one set of replicates ( n=6) with one CEC capillary column was <0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60 000, whilst the observed N values for all new CEC capillary columns were ≥40 000 for n-octyl bonded silica capillary columns and ≥50 000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, λ sym, of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1000 injections were always in the range 0.95–1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.

Noncovalent immobilized artificial membrane chromatography, an improved method for describing peptide–lipid bilayer interactions

A promising approach in assessing hydrophobic peptide-membrane interactions is the use of reversed-phase high-performance liquid chromatography. The present study describes the preparation and properties of a noncovalent immobilized artificial membrane (noncovalent IAM) stationary phase. The noncovalent IAM phase was prepared by coating the C 18 chains of a reversed-phase HPLC column with the phospholipid ditetradecanoyl- sn-glycero-3-phosphocholine. Lipid coating was achieved by pumping a lipid solution in water–2-propanol through the column. The formation of a bilayer-like structure on the chromatographic surface was confirmed by calculating the phospholipid surface density of the stationary phase. The surface density was determined to be approximately 1.95 μmol m −2, which is close to that of lipid vesicles. The coating was found to be stable in chromatographic elution systems containing less than 35% of acetonitrile. Employing this new technique, we determined interaction parameters of a set of helical antibacterial magainin-2-amide peptides with pairwise substitutions of adjacent amino acids by their d-enatiomers. The results demonstrate that the chromatographic retention behavior of peptides on noncovalent IAM stationary phase shows an excellent correlation with lipid affinities to phospholipid vesicles.

Anomalous retention behaviour of peptides on porous graphitized carbon column

The retention behaviour of fourteen peptides was determined on a porous graphitized carbon (PGC) column, using acetonitrile–water mixtures as eluents. The majority of peptides showed irregular retention behaviour, their retention decreased with increasing concentration of acetonitrile in the lower concentration range, reached a minimum and increased again with increasing concentration of acetonitrile in the higher concentration range. Both quadratic and bilinear equations described well the irregular retention of peptides on the PGC column, however, the coefficients of correlation were slightly higher for the quadratic function. Calculations indicated that the impact of hydrophobicity, steric and electronic parameters on the retention of peptides on a PGC column is similar, that is a PGC column shows mixed retention mechanisms in which hydrophobic, steric, and electronic forces are equally involved.