Applicability of the critical-chromatography concept to analysis of proteins: Dependence of retention times on the sequence of amino acid residues in a chain

The BioLCCC model of the chromatographic separation of biomacromolecules, which involves the concepts of the critical chromatography of polymers, is used to describe the experimental data on the separation of proteins on different chromatographic systems. Using phenomenological parameters, i.e., effective adsorption energies of amino acid residues, we predict the effect of the sequence of these residues in the chain on retention times of proteins for reversed phases of different types (C₄, C₈, C₁₈) in the gradient of a water-acetonitrile binary solvent. It is shown that, in general, the BioLCCC model correctly represents experimental data on the separation of proteins and makes it possible to quantitatively determine the effect of the sequence of amino acid residues on the separation. We show the limits of applicability of the model and explain the universal linear dependence that relates the retention volume and the logarithm of chain length that is observed in the chromatography of peptides and proteins.     

Isocratic separation of ginsenosides by high-performance liquid chromatography on a diol column at subambient temperatures

An improved high-performance liquid chromatographic method for separation of a number of ginsenosides has been developed. The influence of temperature (from 0 to 25°C) on the retention and separation of the ginsenosides was studied by applying a binary mobile phase (acetonitrile/water, 82:18 v/v) and a diol column (LiChrospher 100 Diol). The column temperature is one of the more important parameters for the retention and separation of the components investigated. Selected thermodynamic parameters, including changes of enthalpy (ΔH°) and entropy (ΔS°), were estimated from linear van’t Hoff plots, and possible retention mechanisms were discussed. Moreover, the best separation conditions were selected based on optimization criteria including maximum retention time (t _{R max}), minimum resolution (R _{s min}), and relative resolution product (r). Temperature regions close to 14°C offered the highest selectivity and almost equal distribution of the ginsenosides peaks across the chromatogram. Under such isocratic conditions, excellent separation of chromatographic standards and selected ginseng samples was achieved in less than 16 min.     

Chromatographic separation simulation of metal‐chelating peptides from surface plasmon resonance binding parameters

Some metal‐chelating peptides have antioxidant properties, with potential nutrition, health, and cosmetics applications. This study aimed to simulate their separation on immobilized metal ion affinity chromatography from their affinity constant for immobilized metal ion determined in surface plasmon resonance, both technics are based on peptide‐metal ion interactions. In our approach, first, the affinity constant of synthetic peptides was determined by surface plasmon resonance and used as input data to numerically simulate the chromatographic separation with a transport‐dispersive model based on Langmuir adsorption isotherm. Then, chromatographic separation was applied on the same peptides to determine their retention time and compare this experimental t_R with the simulated t_R obtained from simulation from surface plasmon resonance data. For the investigated peptides, the relative values of t_R were comparable. Hence, our study demonstrated the pertinence of such numerical simulation correlating immobilized metal ion affinity chromatography and surface plasmon resonance.     

“Sock-Ball” Chromatographic Separation of Some Polycyclic Aromatic Hydrocarbons with C60-Fullerene Stationary Phase

Polycyclic aromatic hydrocarbons (PAHs) of different molecular shapes were tested and compared with a prepared C₆₀-fullerene-phase, and with the commercial Amino-phase or RP-18-phase in the mobile phase of methanol/dichloromethane (90/10 — 70/30) or n-hexane/dichloromethane (80/20). The chromatographic separation results indicate that C₆₀-fullerene-phase achieves the special selectivities and performances for the separation of PAHs. Based on the retention mechanism of “π-π” complex interaction or “Sock-Ball” shaped combination, PAHs undergo more pronounced interaction with C₆₀ ligand. For example, decacyclene, a sock-shaped PAH, the structure of which was calculated by means of semi-empirical molecular orbital methods, possesses stronger retention to give a “Sock-Ball” chromatographic separation with C₆₀-fullerene-phase. However, PAHs with bend, planar, or co-planar structures eluted on C₆₀-fullerene-phase undergo adsorption chromatography, but possess weaker retentions. The retention power of “Sock-Ball” chromatography can successfully recognize the different molecular shapes of PAHs.     

Influence of isomerism on the chromatographic behaviour of porphyrins

Summary The chromatographic behaviour of some porphyrins and their complexes with zinc has been studied by HPLC on 150×3.9 mm and 300×3.9 mm columns packed with Nova-Pak C₁₈ and μ-Bondapak C₁₈, respectively, and on a microcolumn (64×2 mm) packed with Nucleosil C₁₈. The effect of the nature and the arrangement of side substituents in the porphyrin molecules on retention is considered. It is demonstrated that HPLC can be used for the separation ofcis-andtrans-isomers (atropisomers) of the zinc complex of 5,15-di(phenyl-2-CH₃O)-3,7,13,17-tetramethyl-2,8,12,18-tetrabutylporphyrin and other porphyrins with a similar structure. The efficiency of separation has been compared on different columns.     

Structural stability–chromatographic retention relationship on exenatide diastereomer separation

In this study, the relationship of the structural stability of peptide diastereomers in elution solvents and their retention behaviors in reversed-phase chromatography (RPC) was examined to provide guidance on the solvent selection for a better separation of peptide diastereomers. We investigated the chromatographic retention behaviors of exenatide, a peptide drug for the treatment of type II diabetes mellitus and its three diastereomers using RPC and implicit molecular dynamics (MD) simulation analysis. Three diastereomers involved in the single serine residue mutation of d-form at the 11th, 32nd, and 39th residues were investigated in this study. Results show that the order of the solution structural stability of exenatide and its diastereomers is consistent with their retention order by 36?% acetonitrile/water elution. The sample loading solvent also affects the retention behaviors of exenatide peptide diastereomers in RPC column. Furthermore, a larger solution conformation energy difference of the critical pair of exenatide and its diastereomer (d-Ser39) at the elution solvent of 32?% tetrahydrofuran/water were obtained by MD simulation, and baseline separation was proved experimentally. In summary, we demonstrated that the solution structural stability–chromatographic retention relationship could be a powerful tool for elution solvent selection in peptide chromatographic purification, especially valuable for the separation of critical pair of diastereomers.

Preparation,evaluation, and application of a novel reversed-phase/zwitterionic/hydrophilic interaction liquid chromatographic mixed-mode stationary phase

The present study described the preparation and application of a reversed-phase/zwitterionic/hydrophilic interaction liquid chromatography stationary phase, named as SIL-PS. The SIL-PS was prepared through a four-step reaction, chemical bonding, nucleophilic addition, SN1 substitution, and sulfonation on the silica matrix. It was featured with C₁₂ alkyl chain, quaternary ammonium, tertiary amine, and sulfonate groups. After SIL-PS was packed into the stainless steel column (150?× 2.1 mm i.d.), chromatographic parameters, including acetonitrile content, pH, and ionic strength of the mobile phase, and the column temperature, were systematically investigated to study the retention mechanism. Electrostatic adsorptive/repulsive, partition, and hydrogen-bonding interactions were demonstrated to contribute to the retention. The stability of the SIL-PS was satisfactory, with relative standard deviations of retention factors of 1.93, 2.08, and 1.90% for loxoprofen, adenosine, and liquiritin, respectively. Additionally, to investigate the separation selectivity, the non-steroidal anti-inflammatory drugs, nucleobases/nucleotides, and alkaloids/glycosides were separated; the HPLC fingerprinting of the Cortex phellodendri extract was also conducted, and the separation performance was superior to that of the C18 column in terms of peak shape, resolution, and analytical time. The results revealed that the prepared SIL-PS possessed multifunctionalities for multiretention and could be promising for complicated samples.     

Determination of chromatographic dissociation constants of some carbapenem group antibiotics and quantification of these compounds in human urine

The dissociation constant values (_s^spK_a) of some carbapenem group drugs (ertapenem, meropenem, doripenem) in different percentages of methanol–water binary mixtures (18, 20 and 22%, v/v) were determined from the mobile phase pH dependence of their retention factor. Evaluation of these data was performed using the NLREG program. From calculated pK_a values, the aqueous pK_a values of these subtances were calculated by different approaches. Moreover, the correlation established between retention factor and the pH of the water–methanol mobile phase was used to determine the optimum separation conditions. In order to validate the optimized conditions, these drugs were studied in human urine. The chromatographic separation was realized using a Gemini NX C₁₈ column (250 × 4.6 mm i.d., 5 µm particles) and UV detector set at 220 and 295 nm. Copyright © 2013 John Wiley & Sons, Ltd.     

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.     

Comparison of various types of stationary phases in non-aqueous reversed-phase high-performance liquid chromatography–mass spectrometry of glycerolipids in blackcurrant oil and its enzymatic hydrolysis mixture

The selection of column packing during the development of high-performance liquid chromatography method is a crucial step to achieve sufficient chromatographic resolution of analyzed species in complex mixtures. Various stationary phases are tested in this paper for the analysis of complex mixture of triacylglycerols (TGs) in blackcurrant oil using non-aqueous reversed-phase (NARP) system with acetonitrile–2-propanol mobile phase. Conventional C₁₈ column in the total length of 45 cm is used for the separation of TGs according to their equivalent carbon number, the number and positions of double bonds and acyl chain lengths. The separation of TGs and their more polar hydrolysis products after the partial enzymatic hydrolysis of blackcurrant oil in one chromatographic run is achieved using conventional C₁₈ column. Retention times of TGs are reduced almost 10 times without the loss of the chromatographic resolution using ultra high-performance liquid chromatography with 1.7 μm C₁₈ particles. The separation in NARP system on C₃₀ column shows an unusual phenomenon, because the retention order of TGs changes depending on the column temperature, which is reported for the first time. The commercial monolithic column modified with C₁₈ is used for the fast analysis of TGs to increase the sample throughput but at cost of low resolution.     

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.     

Ion‐exchange vs reversed‐phase chromatography for separation and determination of basic psychotropic drugs

Ion exchange chromatography, an alternative to reversed‐phase (RP) chromatography, is described in this paper. We aimed to obtain optimal conditions for the separation of basic drugs because silica‐based RP stationary phases show silanol effect and make the analysis of basic analytes hardly possible. The retention, separation selectivity, symmetry of peaks and system efficiency were examined in different eluent systems containing different types of buffers at acidic pH and with the addition of organic modifiers: methanol and acetonitrile. The obtained results reveal a large influence of the salt cation used for buffer preparation and the type of organic modifier on the retention behavior of the analytes. These results were also compared with those obtained on an XBridge C₁₈ column. The obtained results demonstrated that SCX stationary phases can be successfully used as alternatives to C₁₈ stationary phases in the separation of basic compounds. The most selective and efficient chromatographic systems were applied for the quantification of some psychotropic drugs in fortified human serum samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of perfluorinated acids as ion-pairing reagents for reversed-phase chromatography and retention-hydrophobicity relationships studies of selected β-blockers

The addition of the homologous series of perfluorinated acids-trifluoroacetic acid (TFAA), pentafluoropropionic acid (PFPA), heptafluorobutyric acid (HFBA) to mobile phases for reversed-phase high-performance liquid chromatography (RP-HPLC) of β-blockers was tested. Acidic modifiers were responsible for acidification of mobile phase (pH 3) ensuring the protonation of the β-blockers and further ion pairs creation. The effect of the type and concentration of mobile phase additives on retention parameters, the efficiency of the peaks, their symmetry and separation selectivity of the β-blockers mixture were all studied. It appeared that at increasing acid concentration, the retention factor, for all compounds investigated, increased to varying degrees. It should be stressed that the presence of acids more significantly affected the retention of the most hydrophobic β-blockers. Differences in hydrophobicity of drugs can be maximized through variation of the hydrophobicity of additives. Thus, the relative increase in the retention depends on either concentration and hydrophobicity of the anionic mobile phase additive or hydrophobicity of analytes. According to QSRR (quantitative structure retention relationship) methodology, chromatographic lipophilicity parameters: isocratic log k and log k_w values (extrapolated retention to pure water) were correlated with the molecular (log P_o/w) and apparent (log P_app) octanol–water partition coefficients obtained experimentally by countercurrent chromatography (CCC) or predicted by Pallas software. The obtained, satisfactory retention-hydrophobicity correlations indicate that, in the case of the basic drugs examined in RP-HPLC systems modified with perfluorinated acids, the retention is mainly governed by their hydrophobicity.     

Characterization of stationary phases based on monosubstituted benzene retention indices using correspondence factor analysis and linear solvation energy relationships in RPLC

In reversed-phase liquid chromatography (RPLC), the comparison of experimental results obtained from different columns is a complex problem. A correspondence factor analysis (CFA) and a linear solvation energy relationship (LSER) were applied on retention data to characterize second-order intermolecular interactions responsible for retention on a set of RPLC columns. Seven octadecyl-C₁₈ columns with different packing materials are obtained from different manufacturers and one octyl-C₈ column. The retention data were determined under isocratic conditions using a methanol–water (65:35, v/v) mobile phase. The chromatographic retention indices based on alkan-2-ones and alkyl aryl ketones retention index scales are calculated using a multiparametric least-squares regressions iterative method. The CFA and LSER results permitted to highlight that the retention indices were appropriate for studying the second-order retention mechanisms on the eight chromatographic systems investigated and exhibited the best reproducibility. Although many earlier studies have reported the use of chemometric methods to characterize chemical factors affecting retention in RPLC using retention factors as retention parameters, this is the first study based on retention indices.     

Fast separation of flavonoids by supercritical fluid chromatography using a column packed with a sub‐2 μm particle stationary phase

The purpose of this study was to compare the effects of different chromatographic columns for the separation of seven flavonoids. Four different stationary phases are available, including bridged ethyl hybrid, BEH and the same hybrid phase modified with 2‐ethylpyridine, CSH fluorophenyl, and HSS C₁₈ SB. The analytes included calycosin, genistein, medicarpin, calycosin‐7‐O‐β‐d ‐glucoside, formononetin, formononetin‐7‐O‐β‐d ‐glucoside, and liquiritigenin. The CSH fluorophenyl column was determined to be the most suitable and provided the fastest separation within 17 min using gradient elution with carbon dioxide as the mobile phase and methanol as the co‐solvent. Good peak shapes were obtained, and the values of the peak asymmetry were close to 1.0 for all of the flavonoids. The resolution was more than 1.41 for all of the separated peaks. Baseline separation on the optimal columns was achieved by changing the co‐solvent type and adjusting the temperature and pressure. Quantitative performance was evaluated under optimized conditions, and method validation was accomplished. The validation parameters, such as linearity, sensitivity, precision, and accuracy, were satisfactory. Good repeatability of both peak area (relative standard deviation <1.02%) and retention time (relative standard deviation <0.88%) was observed. The optimized chromatographic methods were successfully used for the determination of seven flavonoids in Radix astragali . The sensitivity was sufficient for the analysis of real samples.     

Isocratic Separation of Fatty Acid Derivatives by Reversed Phase Liquid Chromatography. Influence of the Solvent on Selectivity and Rules for Elution Order

Abstract

The p-bromophenacyl esters of 16 fatty acids (C₁₂-C₂₂) have been separated by isocratic chromatography on a Radial Pak A cartridge (Reverse phase C₁₈ material). The separation factors α were measured using two solvent mixtures of comparable strength and the superiority of methanol-water to acetonitrile-water becomes evident.

Five precise rules are established, which indicates the retention of every fatty acid. They explain the chromatographic process i.e. elution order, resolution and selectivity.