Reversed-phase Stationary Phases Research Articles

Chromatographic properties of reversed-phase stationary phases under pressure- and electro-driven conditions

Seven different reversed-phase (RP) stationary phases were examined under high-performance liquid chromatographic (pressure-driven, HPLC), and capillary electrochromatographic (electro-driven, CEC) conditions. Characterization of the stationary phases was performed following well-established test procedures providing a number of distinct column descriptors: hydrophobicity, hydrophobic selectivity and silanol activity. These parameters were used to describe the behavior of the RP-columns under both HPLC and CEC conditions. It is shown that chromatographic characteristics of porous RP-phases greatly depend on the mode of operation. By contrast, column descriptors of a non-porous viz. solid RP-phase material hardly differed for HPLC and CEC conditions.

Capillary electrochromatography with macroporous particles

The performance of macro-porous particles in capillary electrochromatography is studied. Three reversed-phase stationary phases with pore diameters between 500 Å and 4000 Å have been tested for separation efficiency and mobile phase velocity. With these stationary phases, a large portion of the total flow appears to be through the pores of particles, thereby increasing the separation efficiency through a further decrease of the flow inhomogeneity and through enhancement of the mass transfer kinetics.The effects of pore size and mobile phase composition on the plate height and mobile phase velocity have been studied. With increasing buffer concentrations and larger pore diameters, higher mobile phase velocities and higher separation efficiencies have been obtained.Columns packed with 7 μm particles containing pores with a diameter of 4000 Å generated up to 430 000 theoretical plates/m for retained compounds. Reduced plate heights as low as 0.34 have been observed, clearly demonstrating that a significant portion of the flow is through the pores. For the particles containing 4000 Å pores no minimum was observed in the H-u plot up to linear velocities of 3.3 mm/s, suggesting that the separation efficiency is dominated by axial diffusion. On relatively long (72 cm) columns, efficiencies of up to 230 000 theoretical plates/column have been obtained under non-optimal running conditions. On short (8.3 cm) columns fast separations could be performed with approximately 15 000 theoretical plates generated in less than 30 s.

High-Performance Liquid Chromatography of some Bases and Nucleosides on Alkylphosphonate-Modified Magnesia-Zirconia Column

ABSTRACT The high-performance liquid chromatographic behavior of some bases and nucleosides was studied on a new reversed-phase stationary phase, alkylphosphonate-modified magnesia-zirconia. The effect of mobile phase variable such as methanol content, ionic strength and pH on their behavior was investigated. It was found that the retention behavior of the bases and nucleosides on the new stationary phase is similar to that on ODS stationary phase. The retention mechanism on the new stationary phase was also discussed. The separation of some bases and nucleosides was accomplished on the new stationary phase.

Packing and stationary phase design for capillary electroendosmotic chromatography (CEC)

Reversed-phase stationary phases based on porous silica beads in the range 0.2–3 μm have been prepared to study the dependency of electroosmotic flow (EOF) on particle diameter. Additionally, the influence of the mobile phase composition, i.e. organic modifier content, pH of buffer solution and concentration of buffer salt, on the magnitude of the electroosmotic flow velocity has been investigated. To perform separations in an aqueous acidic mobile-phase, mixed mode stationary phases containing both alkyl chains and strong cationexchange groups have been applied.

Hydrophilic Interaction Chromatography−Electrospray Mass Spectrometry Analysis of Polar Compounds for Natural Product Drug Discovery

For the drug discovery efforts currently taking place within the pharmaceutical industry, natural product extracts have been found to provide a valuable source of molecular diversity which is complementary to that provided by traditional synthetic organic methods or combinatorial chemistry. However, there exists a need for analytical tools that can facilitate the separation and characterization of components from these sources in a rapid manner. Specifically, the evaluation of highly polar compounds (i.e., compounds that cannot be retained on traditional reversed-phase stationary phases) has been challenging, and a hydrophilic interaction chromatography-electrospray ionization mass spectrometry (HILIC-ESI-MS) method was developed to meet this need. In this investigation, amide-, Polyhydroxyethyl Aspartamide-, and cyclodextrin-based packings provided superior performance for the analysis of a set of polar natural product compounds. The properties of the mobile-phase buffers also greatly impacted the separations, and relative to other volatile buffering agents, ammonium acetate at a concentration of approximately 6.5 mM was determined to facilitate optimal HILIC retention, reproducibility, and durability. An optimized HILIC-ESI-MS system was successfully applied for the analysis of complex natural product mixtures. The techniques described in this report should also prove useful for the analysis of polar compounds from synthetic sources of molecular diversity such as combinatorial chemistry.

Preparation of a cyclophane-bonded stationary phase and its application to separation of naphthalene derivatives

A cyclophane (CP44)-bonded silica gel stationary phase was prepared and elution behaviour of hydrophobic solutes was investigated in the reversed-phase mode. Aromatic compounds were retained on the stationary phases more strongly than the corresponding alicyclic compounds, as was expected by the complex-forming ability of the cyclophane. The stationary phases also showed isomer-selective separation for monomethyl- and dimethylnaphthalenes. The isomers having methyl groups at the α-position were eluted prior to those having methyl groups at the β-position, i.e., the retention order for methylnaphthalene was, α<β and that for dimethylnaphthalene, α,α<α,β<β,β. Moreover, some dimethylnaphthalene isomers which cannot be separated on ordinary reversed-phase stationary phases were separated finely on this stationary phase. The separation mechanism is discussed on the basis of the structure of the cyclophane-involved complex.

Deuterium Nuclear Magnetic Resonance Spectroscopy as a Probe For Reversed-Phase Liquid Chromatographic Bonded-Phase Solvation. 3. Tetrahydrofuran and Water Binary Systems

Deuterium nuclear magnetic resonance ((2)H NMR) spectroscopy is a useful tool in probing molecular association between components in a binary hydro-organic mixture, such as tetrahydrofuran (THF) and water. Measurements of (2)H longitudinal relaxation times (T(1)) for deuterated analogs of THF and water provide this type of information, since the magnitude of T(1) is indicative of molecular association and mobility. This technique is also useful in determining the interactions between the mobile phase and reversed-phase stationary phase. T(1) measurements of slurry samples, consisting of both mobile- and stationary-phase components, allow for changes in the longitudinal relaxation time of mobile-phase components upon contact with the stationary phase (ΔT(1)) to be monitored. These ΔT(1) values are indicative of the degree of association of the THF-water mobile-phase components with the stationary phase.

Simultaneous Separation of Diazepam and Its Chiral and Achiral Metabolites by HPLC on a Chiralcel OD-R Column

A reversed-phase HPLC method was developed for the simultaneous separation of diazepam and its achiral (nordiazepam) and chiral (oxazepam and temazepam) metabolites. Chromatographic resolution of the analytes was performed under isocratic conditions using a mobile phase of 0.3 M sodium perchlorate-acetonitrile (57:43, v/v) at a flow rate of 0.5 ml/min on a reversed-phase cellulose-based chiral stationary phase (Chiralcel OD-R column) with UV detection set at 210 nm. Disc solid-phase extraction was carried out for serum sample clean-up prior to HPLC separation. Recoveries were determined for diazepam, nordiazepam, and oxazepam and temazepam enantiomers from human serum at 300 ng/ml levels. The limits of detection were 25 ng/ml each for diazepam and nordiazepam, and about 25 ng/ml for each enantiomer of oxazepam and temazepam (S/N = 2). The method can be applicable to the in vivo analysis of human serum samples containing diazepam and its chiral and achiral metabolites.

Monitoring of new silica-based reversed-phase stationary phases for the liquid chromatographic analysis of basic pharmaceuticals using principal components analysis

In this study, fourteen commercially available reversed-phase stationary phases were compared for the high performance liquid chromatographic analysis of basic pharmaceutical compounds. The influence of silanol blocking compounds on retention and peak shape was investigated using phosphate buffers at pH values of 3 and 7. Principal components analysis was used to analyse the data set, enabling an evaluation of the various stationary phases. The commercially available stationary phases showed distinct differences in their suitability for the analysis of basic compounds, whereas addition of N,N-dimethyloctylamine to mobile phase buffer at pH 3 caused an improvement in peak shapes.

Determination of the novel topoisomerase I inhibitor NU/ICRF 505 and its major metabolite in plasma, tissue and tumour by high-performance liquid chromatography.

A high-performance liquid chromatographic technique is presented for the determination of the novel topoisomerase I inhibitor NU/ICRF 505 (a tyrosine conjugate of anthraquinone), its major metabolite (NU/ICRF 505/M) and an internal standard (NU/ICRF 513, dihydroxyphenylalanine conjugate). The method uses a reversed-phase (Apex ODS-2) stationary phase and a mobile phase consisting of 0.25 M ammonium acetate adjusted to pH 3 with 25% (v/v) trifluoroacetic acid and methanol with gradient elution. Between-day variation in retention times were less than 1% for NU/ICRF 505 and 513 and 2.4% for the metabolite. Selective detection was achieved at a wavelength of 545 nm giving a limit of detection of 2 ng on column and 50 ng/ml after sample preparation for all three components. Chromatograms were free from interfering peaks even at very high detector sensitivity. Sample preparation was based on incubation of biological specimens (0.5 ml plasma or homogenate) with dimethylsulphoxide and acetonitrile at 4 degrees C for 30 min followed by centrifugation. Liver and tumour were homogenised in phosphate buffered saline. Recoveries were consistently high (81.7-106.7% for NU/ICRF 505; 88.7-103.3% for NU/ICRF 513 and 83.7-98.7% for NU/ICRF 505/M) with between day coefficients of variation of normally less than 10%. The method will contribute significantly to the preclinical evaluation of NU/ICRF 505.

Perfusion Chromatography on Reversed-Phase Column Allows Fast Analysis of Human Globin Chains

Human globin chain analysis provides important information on the genetics and molecular pathophysiology of hemoglobinopathies. We propose using perfusion chromatography on the reversed-phase stationary phase to perform these studies. The technique, herein described, involves a high-velocity flow of the mobile phase through a porous chromatographic stationary phase made of microspheres of poly(styrene–divinylbenzene) having throughpores of 6000–8000 Å diameter with short diffusive pores of 500–1000 Å diameter connected to them. The composition of fetal hemoglobin (Gγ:Aγ ratio) is determined, using this method, as an order of magnitude faster than with conventional HPLC. Elution is performed by developing a linear gradient of acetonitrile at a flow rate of 3 ml/min (or more), easily obtained on any HPLC machine. Analyses may be done on samples containing as low as 3.0% Hb F. Results are similar to those obtained with the reference HPLC technique, which uses a C4 column. In addition, reversed-phase perfusion chromatography, using a shallow curvilinear gradient, may help in the characterization of Hb variants. This technique allowed us to discriminate several α and β chain mutants from variants that have closely similar patterns of electrophoretic mobilities.

Use of 3-(1,8-naphthalimido)propyl-modified silyl silica gel as a stationary phase for the high-performance liquid chromatographic separation of purine derivatives

The use of a packing material, 3-(1,8-naphthalimido)propyl-modified silyl silica gel (NAIP), as a stationary phase for high-performance liquid chromatography, has been studied. NAIP behaved like a reversed-phase stationary phase with some π-π interaction. Purine derivatives, i.e., xanthine, hypoxanthine, uric acid, theobromine, theophylline and caffeine, were separated by a column packed with NAIP using an eluent of borate solution (pH 6.4)-MeOH (50:50, v/v). Of these, caffeine was selected as the target of the subsequent investigation and its determination was examined in commercially available medicinal drinks and pharmaceutical preparations. The average recoveries of caffeine were 98.0–107.4% for five drinks and 99.6–107.8% for five tablets and one powder. Subsequently, determination of caffeine and its metabolites in human plasma was examined. In twelve normal human plasma, caffeine levels ranged from 0.24 to 4.26 μ/ml. Time curves of plasma caffeine concentrations and those of its demethylated metabolite, 1,7-dimethylxanthine (1,7-DMX), after an oral ingestion of caffeine (200 mg) were measured by the proposed method and it was found that the maximum concentrations of caffeine and 1,7-DMX were obtained at 1-1.5 h and 3–6 h after ingestion, respectively.

Bioavailability Estimation by Reversed-Phase Liquid Chromatography: High Bonding Density C-18 Phases for Modeling Biopartitioning Processes

There have been many attempts to estimate biological activity with either 1-octanol/water partition coefficients or chromatographic retention parameters. Bulk phases may not be appropriate, however, for modeling a partitioning process in an interphase such as biological membranes. Chromatographic stationary phases can be argued as having structure similar to a membrane because of chain organization; however, the density of the grafted stationary-phase chains in commercially available stationary phases is much too low to provide a suitable model. We have previously developed a new scheme for derivatizing silica surfaces that produces stationary phases of significantly higher chain density than traditional methods. Investigation of the molecular mechanism and thermodynamics of solute partitioning into the different phases has shown that densely bonded reversed-phase stationary phases mimic partitioning to a biomembrane better than does bulk-phase octanol. Here we report chromatographic retention for pesticides, PAHs, and barbiturates using a C-18 column with high alkyl chain density, and in all cases, correlations of log k'w with bioavailability are equivalent to or better than correlations of bioavailability with the octanol/water partition coefficient.

Retention characteristics of fluorinated bonded silica phase in reversed-phase liquid chromatography

Fluorinated bonded silica has been evaluated as a packing material in reversed-phase liquid chromatography (RPLC) with polycyclic aromatic hydrocarbons, chlorinated benzenes and substituted benzenes as sample probes. The bonded material is useful as a reversed-phase stationary phase and for polar compounds it offers a specific selectivity not found with octadecylsilica (ODS) phases. The phase also retains non-planar compounds more than planar molecules. The results can be explained by conformational differences between ODS and the fluorinated bonded phase by MM2 calculation.

Sensitive determination of 8-chloroadenosine 3',5'-monophosphate and 8-chloroadenosine in plasma by high-performance liquid chromatography.

8-Chloroadenosine 3',5'-monophosphate (8-Cl-cAMP) is progressing through clinical evaluation as an anticancer drug. There is debate as to whether 8-Cl-cAMP is the active principal or its cytotoxic metabolite 8-Cl-adenosine. Separate high-performance liquid chromatographic methods are described for (i) 8-Cl-cAMP and its nucleotide metabolites (with 8-Br-cAMP as internal standard), and (ii) 8-Cl-adenosine. Both methods use a reversed-phase (Spherisorb ODS-2) stationary phase and a mobile phase consisting of sodium phosphate buffer (10 mM, pH 3.5) and methanol but with gradient elution for the nucleotides and isocratic elution for 8-Cl-adenosine. 8-Cl-cAMP and related nucleotides are extracted from plasma using strong anion-exchange solid-phase extraction (SPE) and 8-Cl-adenosine is extracted using reversed-phase (C8) SPE. Both techniques enabled analyses to be performed at high detector sensitivity with minimal interference. Limit of detection in plasma was 10 ng/ml for both 8-Cl-cAMP and 8-Cl-adenosine. When applied to the analysis of plasma samples from a patient treated with a low dose continuous infusion of 25 micrograms/kg/h, steady-state concentrations centred around 60 ng/ml 8-Cl-cAMP were determined. In the same patient 8-Cl-adenosine was not detected. Application of this methodology will aid in the further development of 8-Cl-cAMP as a potential new form of anticancer treatment.

Reversed-phase chromatography of synthetic amphipathic α-helical peptides as a model for ligand/receptor interactions Effect of changing hydrophobic environment on the relative hydrophilicity/hydrophobicity of amino acid side-chains

To mimic a hydrophobic protein binding domain, which is a region on the surface of a protein that has a preference or a specificity to interact with a complementary surface, we have designed amphipathic α-helical peptides where the non-polar face interacts with the non-polar surface of a reversed-phase stationary phase. Two series of potentially amphipathic α-helical peptides, a native Ala peptide (AA9) and a native Leu peptide (LL9), were designed where the native peptide contains 7 residues of either Ala or Leu, respectively, in its non-polar face. This design results in an overall hydrophobicity of the non-polar face of the Leu peptide that is greater than that of the non-polar face of the native Ala peptide. Mutants of the native Ala-face peptide, AX9, and the native Leu-face peptide, LX9, were designed by replacing one residue in the centre of the non-polar face in both series of peptides. Therefore, by changing the hydrophobicity of the environment surrounding the mutated amino acid side-chain, the effect on the hydrophilicity/hydrophobicity of each amino acid side-chain could be determined. Using the substitutions Ala, Leu, Lys and Glu, it was shown that the maximum hydrophilicity of these amino acid side-chains could be determined when the environment surrounding the mutation is maximally hydrophobic; whereas its maximum hydrophobicity can be determined when the environment surrounding the mutation is minimally hydrophobic. This procedure was further extended to the remaining amino acids commonly found in proteins and it was determined that this general principle applies to all 20 amino acids. These results have major implications to understanding the hydrophilicity/hydrophobicity of amino acid side-chains and the role side-chains play in the folding and stability of proteins.

Structure-retention relationships of diastereomeric mixtures of lipidic amino acid conjugates on reversed-phase stationary phases

The retention parameters of diastereomeric mixtures of 19 lipidic amino acid conjugates have been determined on Spherisorb ODS and Supelcosil LC-ABZ stationary phases by changing the acetonitrile concentration in acetonitrile-0.1% trifluoroacetic acid-water mobile phases. In general, better resolution of the diastereomeric mixtures was obtained on the Supelcosil LC-ABZ stationary phase but in some cases the Spherisorb ODS column showed better separation. The presence of silanol groups improved the separation of the diastereomers in those cases. The log k′ values were plotted against the acetonitrile concentrations. In the case of basic derivatives parabolic relationships could be observed on the Spherisorb ODS stationary phase which contains free silanol groups, while always straight lines were obtained on the Supelcosil LC-ABZ column. The slope and the intercept values were calculated for the straight lines obtained. The octanol-water partition coefficients (log P) and the dissociation constants (p K a) of the compounds were also calculated and were related to the chromatographic parameters. It was found that the chromatographic parameters obtained on the Spherisorb ODS stationary phase showed slightly better correlations with the calculated hydrophobicity and p K a parameters than those obtained on the Supelcosil LC-ABZ column, which shows that different retention mechanism can be expected on the two stationary phases.

Effect of the chain mobility of polymeric reversed-phase stationary phases on polypeptide retention

The resolution of polypeptides in reversed-phase chromatography can be improved by inducing conformational changes in the solute. Protein unfolding can be achieved by an increased accessability of the hydrophobic stationary phase. We elucidated how far results from CP/MSS solid-state NMR relaxation measurements could be correlated with chromatographic data in terms of protein and peptide retention. The retentivity and denaturation potential of reversed stationary phases is controlled not only by their hydrophobicity but also by their ligand mobility and steric properties

Determination of oxidizable inorganic anions by reversed-phase ion-pair chromatography with amperometric detection

Operating conditions for the determination of oxidizable inorganic anions by reversed-phase ion-pair chromatography with amperometric detection were optimized. Tetrabutylammonium hydroxide was selected as the ion-pairing agent, which was modified in situ on the reversed-phase stationary phase. The present system can be used to determine nitrite, thiosulphate, iodide, sulphide and thiocyanate ions, and was applied to the determination of inorganic anions in lake water.

Deuterium nuclear magnetic resonance spectroscopy as a probe for reversed-phase liquid chromatographic bonded phase solvation: methanol and acetonitrile mobile phase components

Using the inversion-recovery method, deuterium longitudinal relaxation times, ( T 1), of [ 2H 4]methanol and [ 2H 3]acetonitrile in enriched methanol—water and acetonitrile—water mobile phases were measured for the neat binary mixtures, and for the mixtures in contact with two different monomeric C 18 reversed-phase stationary phases. Changes in the deuterium relaxation times for the organic modifiers in contact with the stationary phase compared to those observed in neat solution give a qualitative understanding of the degree of association of the organic components with the stationary phase. Both bulk solution microstructure and stationary phase C 18 bonding density play important roles in determining bonded phase solvation.