Reversed-phase Liquid Chromatographic Separation Research Articles

Reversed-phase capillary liquid chromatography coupled on-line to capillary electrophoresis immunoassays.

Capillary reversed-phase liquid chromatography (RPLC) was coupled on-line to competitive capillary electrophoresis immunoassay (CEIA) to improve concentration sensitivity of the competitive CEIA and to provide a means for detecting multiple species that cross-react with antibody. A competitive CEIA for glucagon was used for demonstration of this technique. Five-microliter samples were injected onto a 4-cm-long by 50-micron-i.d. RPLC column. Sample was desorbed by gradient elution, mixed on-line with fluorescently labeled glucagon and anti-glucagon, incubated in a continuous-flow reaction capillary, and analyzed by capillary electrophoresis with flow-gated injection and laser-induced fluorescence detection. Electrophoretic analysis of the reactor stream was performed every 1.5 s, allowing nearly continuous monitoring of the RPLC separation. Preconcentration achieved by RPLC allowed improvement in the detection limit from 760 to 20 pM. Addition of the RPLC column also allowed multiple cross-reactive species to be differentiated by first separating them chromatographically and then detecting them with the immunoassay. The technique was used to measure glucagon secretion from single islets of Langerhans and to differentiate cross-reactive forms of glucagon with one assay.

Amphoteric surfactant-modified stationary phase for the reversed-phase high-performance liquid chromatographic separation of nucleosides and their bases by elution with water

A study on surface modification of an octadecylsilica (ODS) column with amphoteric surfactants has been carried out for efficient reversed-phase liquid chromatographic separations of hydrophobic analytes only with water eluent. Two amphoteric surfactants each with different terminal groups (betaine-type and sulfobetaine-type surfactants) were employed as the stationary phase modifier, and the separation characteristics of the modified ODS columns were evaluated by comparing the elution times of nucleosides, their bases, and inorganic ions. In both surfactant-modified ODS column systems, the adsorption of amphoteric surfactant led to a decrease in the retention of nucleosides and their bases, while an increase in the retention of inorganic anions. These results indicate that the amphoteric surfactant adsorbed on the ODS surface suppresses the hydrophobicity of the reversed-phase surface, but it also generates a specific electrostatic field. In other words, the modified ODS columns exhibit hydrophobic/electrostatic mixed-mode separation properties. The degree of the contribution of each property to the separation of analytes can be varied with the amount of the adsorbed surfactant. Consequently, nucleosides and their bases were effectively separated on the ODS column coated with 0.55 mmol of betaine-type surfactant in water elution.

Development and validation of a liquid chromatographic–tandem mass spectrometric method for the determination of pibutidine in human urine

A liquid chromatographic–tandem mass spectrometric method for the rapid quantitative determination of pibutidine, an H 2-receptor antagonist, in human urine has been developed and validated over the concentration range 0.1–25.6 μg ml −1. Urine samples were prepared based on a simple dilution with 0.05% acetic acid, followed by reversed-phase liquid chromatographic separation. Pibutidine and its internal standard ( 2H 10-pibutidine) were ionized using an electrospray ionization interface and detected by tandem mass spectrometry in the selected reaction-monitoring mode. Completed validation demonstrated the method to be robust, accurate, precise and specific for the direct quantification of pibutidine in human urine. This method has enabled investigation of the urinary excretion of pibutidine following oral administration of pibutidine hydrochloride to healthy subjects.

Analysis of aliphatic alcohol ethoxylates in terms of alkyl and ethylene oxide chain lengths by reversed-phase liquid chromatography with evaporative light scattering detection

Aliphatic alcohol ethoxylates are nonionic surfactants which are incorporated in many industrial formulations as complex mixtures of alkyl homologs and ethylene oxide oligomers. Determination of both the homolog and oligomer distributions is required for product control. The proposed method consisted of three reversed-phase liquid chromatographic separation steps carried out on the same C 18-bonded silica column. The first step was a preparative one: the sample mixture was fractionated according to the alkyl chain length without discrimination between ethylene oxide oligomers by using methanol–water eluent. The even homologs (EH) were collected together in a single fraction, the odd homologs (OH) in another. In the second and third steps, respectively, EH and OH fractions were separated according to the alkyl chain length and the number of ethylene oxide units simultaneously by changing the mobile phase composition to acetonitrile–water and by using evaporative light scattering detection.

Reversed-phase liquid chromatographic determination of uranium based on its ternary complex with fluoride and 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol. New chromatographic probe for the detection of fluoride on C18 stationary phase surface

Optimum conditions for the reversed-phase liquid chromatographic separation of uranium as U(VI)–F −–(5-Br-PADAP) [5-Br-PADAP is 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol] ternary complex on the end-capped C18 column were evaluated. The developed sensitive, selective and versatile method allows to determine uranium in the wide concentration range 0.2–120.0 μg ml −1 (detection limit, DL, is 0.15 μg ml −1 for 20 μl loop). Solvent mixture, acetonitrile+water (65+35, v/v) containing fluoride in concentration 3×10 −3 mol l −1 (pH 5.5) was used as eluent. Double action of fluoride present in eluent — as stabilising agent of the ternary system and modifier of the stationary phase was evaluated. In order to prove appearance of the modifying effect of fluoride on the stationary phase the new chromatographic probe — system Zr(IV)–(5-Br-PADAP)/Zr(IV)–F −–(5-Br-PADAP) was introduced as a tool for the detection of F − presence on the surface.

Control of column temperature in reversed-phase liquid chromatography

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.

Reversed-phase high performance liquid chromatographic separation of the enantiomers of terbutaline by derivatization with 2,3,4,6-tetra-o-acetyl-beta-D-glucopyranosyl isothiocyanate.

The enantiomers of the bronchodilator terbutaline were separated by reversed-phase high performance liquid chromatography after derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate(GITC) reagent. The derivatization proceeded quantitatively within 1 h at room temperature. The corresponding diastereomeric thiourea derivatives were well resolved on an ODS column with acetonitrile-acetate buffer as a mobile phase. Elution orders of the diastereomers were confirmed by derivatization of R-(-)-terbutaline and S-(+)-terbutaline which were collected by semi-preparative chiral HPLC using Sumichiral OA-4700 column. The native fluorescence of terbutaline was quenched by derivatization with GITC. The detection limit was 25 ng when monitored at UV 278 nm.

Degree of solute inclusion in native beta-cyclodextrin: chromatographic approach

The reversed-phase liquid chromatography retention and separation of a series of D,L dansyl amino acids were investigated over a wide range of salting-out agent (sucrose) concentrations using native beta-cyclodextrin as a chiral stationary phase. An original treatment was developed to determine the number of sucrose molecules (n) excluded from the solute-beta-cyclodextrin cavity interface when the analyte transfer occurred. Using the n values, the relative degrees of compound inclusion were calculated and correlated to the steric bulkiness of the solute. Thermodynamic parameter variations are discussed in relation to the inclusion degree of the dansyl amino acids. This numerical approach is a valuable tool to explore the steric effects implied in the host-guest complex formation.

Method developments for iodine speciation by reversed-phase liquid chromatography-ICP-mass spectrometry.

Method developments are described for a iodine speciation method. This method is based on a reversed-phase liquid chromatography separation combined with inductively coupled plasma-mass spectrometry detection. Investigations are performed concerning the methanol tolerance/stability of inductively coupled plasma, postcolumn oxidation/volatilization of iodine from species, influences of buffer pH, and column temperature on separation efficiency. The experiments focused on six iodine species, namely iodine, monoiodothyrosine, di-iodothyrosine, tri-iodothyronine, reversed tri-iodothyronine, and thyroxin. Detection limits were determined between 0.08 and 1.5 microg/L (iodine related), dependent on species. Appplications on a urine sample were performed, showing iodide as the predominant iodine species.

Reversed-phase liquid chromatographic separation and simultaneous profiling of steroidal glycoalkaloids and their aglycones

Improved and simplified reversed-phase liquid chromatographic conditions for the separation and simultaneous profiling of both steroidal glycoalkaloids and their aglycones, having solanidane- or spirosolane-type structures, are described. The most reproducible retention behavior for these ionizable compounds on C 18 columns was achieved under isocratic and gradient elution conditions using acetonitrile in combination with triethylammonium phosphate buffer at pH 3.0, when basic functional groups of solutes and silanol groups on the silica are fully protonated minimizing ionic interactions. Gradient elution was the only feasible approach for the simultaneous separation of steroidal glycoalkaloids and their aglycones. A Zorbax SB C 18 column, specially designed for low-pH separations, showed good performance in critical separations. The impurities of the commercial tomatine and tomatidine standards were studied and confirmed using mass spectrometric, liquid chromatographic and thin-layer chromatographic methods.

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: II. Two-run assay procedures

By optimizing column temperature T and gradient time t G, complex samples can often be separated by means of reversed-phase high-performance liquid chromatography (RP-LC). Conclusions reached in Part I suggest that the complete separation of such samples will be difficult, however, when more than 15–20 components are present in the sample. An alternative approach is to carry out two separations with different conditions ( T, t G) in each run. The combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolved in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying complexity. Also considered is the ability of a single separation where T and t G are optimized to enable the separation and analysis of one or more individual sample components from complex mixtures (e.g., drugs in animal plasma), including the resolution of isomeric compounds from each other.

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: III. Improving the accuracy of computer simulation

Previous studies have shown that four experimental runs, where both temperature T and gradient time t G are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict “optimized” conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15–20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or t G, in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, t G or isocratic % B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time: I. Peak capacity limitations

The separation of samples that contain more than 15 to 20 analytes ( n>15–20) is typically difficult and usually requires gradient elution. We have examined the reversed-phase liquid chromatographic separation of 24 samples with 8≤ n≤48 as a function of temperature T and gradient time t G. The required peak capacity was determined for each sample, after selecting T and t G for optimum selectivity and maximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (when T and t G have been optimized). These results were also compared with literature studies of similar isocratic separations as a function of ternary-solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn provides information on the relative effectiveness of these two different method development procedures (optimization of T and t G vs. % MeOH and % THF) for changing selectivity and achieving maximum resolution.

New polymer-based prepacked column for the reversed-phase liquid chromatographic separation of peptides over the pH range 2–12

The aim of this paper was to investigate the properties of a new column, Source™ 5RPC, for the separation of peptides at pH 2, 4.5, 7, 9 and 12 and to compare this product with similar polymer-based products available on the market. All columns were prepacked with 5 μm polystyrene–divinylbenzene polymer bead matrices and had dimensions of 150×4.6 mm I.D. Separations of angiotensin peptides were achieved on these columns using different equilibration solvents in the pH range 2–12 and elution with acetonitrile gradients. The separation of the peptide mixture obtained on Source 5RPC column was compared with that of two other commercially available polymer-based matrices. Method scouting and optimisation were carried out using the novel chromatography system, ÄKTA™ purifier.

Raman Spectroscopic Characterization of RP-18-Type Chemically Bonded Stationary Phases for Liquid Chromatography

Abstract This study is the first Raman spectroscopic characterization of conventional chemically bonded liquid chromatographic (LC) stationary phases of the RP-18 type using the high-power Nd:WV04 neodymium laser. Raman spectra were obtained for octadecyl (C18) chemically bonded silica-based stationary phases, as well as for silica, aminopropyl (NH2), cyanopropyl (CN), diol (OH), octyl (C8), and methyl (C1) phases for comparison. This study demonstrates that Raman spectroscopy using the high-power neodymium laser can be used to study RP-18-type LC stationary phases and to quantitate the density of coverage of the silica matrix with octadecyl ligands. The paper presents the experimental setup and conditions usedto obtain Raman spectra of commercial RP-18-type chemically bonded stationary phases and examines the spectroscopic similarities and differences observed for different bonded ligands typically used in reversed-phase and normal-phase LC separations.

Isotope dilution high-performance liquid chromatography–electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in biological samples

A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection. The HPLC–MS approach in the single ion monitoring (SIM) mode and the HPLC–MS/MS assay in the multiple reaction monitoring (MRM) mode have been compared, using isotopically labeled [M+4] 8-oxodGuo as the internal standard. The limit of detection of 8-oxodGuo was found to be around 5 pmol and 20 fmol for the HPLC–MS and HPLC–MS/MS methods, respectively. The HPLC–MS/MS assay is sensitive enough to allow the determination of the level of 8-oxodGuo in cellular liver DNA and in urine samples.

Chiral separation of 2,4-dinitrophenyl amino acids using 3- O-methyl-β-cyclodextrin-bonded stationary phase in reversed-phase liquid chromatography

Reversed-phase liquid chromatography (RPLC) separation factors for racemic 2,4-dinitrophenyl (DNP) amino acids (Phe and Trp) having various substituents at different positions were measured on native β-CD and heptakis(3- O-methyl)-β-CD as the chiral stationary phase in order to compare enantioselectivity of these phases. Both native β-CD and heptakis(3- O-methyl)-β-CD showed good enantioselectivity for the DNP–amino acids investigated. Enantioselectivities of the two CD phases for the DNP–amino acids vary with the type and position of the substituent on the DNP moiety of the amino acids. Heptakis(3- O-methyl)-β-CD, the cavity of which is more electron-rich than that of native β-CD, showed in general much better enantioselectivity for the amino acid derivatives studied.

Investigations on Radiolabeling Substance P Derivatives and Reversed-Phase Liquid Chromatographic Separation of the Products

In investigations of radiolabeling substance P1–11 (SP) with 125I isotope, its [Tyr8]-derivative was subjected to the lactoperoxidase (LPO) technique, whereas the native substance P undecapeptide was coupled with 125I-tyrosin-N-hydroxy-succinimidyl ester to the corresponding 125I-Bolton-Hunter SP derivative. For separation of the desired components from their by-products, labeled [Tyr8]-SP was pre-purified by ion-exchange chromatography (IEC) on carboxymethyl cellulose and subsequently investigated by gradient high performance reversed-phase liquid chromatography (gRP-HPLC) with 0.1 M triethyl-ammonium phosphate in acetonitrile (pH 2.5) on an octadecylsilyl silica gel (RP-18) stationary phase. When the total amount of labeled peptide affording a specific activity of approx. 2 × 106 Ci/M was checked by gRP-HPLC, the LPO technique yields about an equimolar mixture of 125I-[Tyr8]-(Met11)-SP and 125I-[Tyr8]-(Met11O)-SP. However, in contrast, substantial enrichment of 125I-[Tyr8]-(Met11)-SP was observed when more retained IEC fractions were analyzed. The 125I-Bolton-Hunter reagent was coupled in about 54 % yield to the SP undecapeptide at slightly alkaline conditions and separated from by-products only by gRP-HPLC yielding a specific activity of approx. 2 × 106 Ci/M. Both radiolabeled derivatives of SP are further characterized by their binding properties to a specific antibody raised in rabbits against synthetic SP1–11. When individual fractions of either radioiodinated [Tyr8]-SP or the corresponding 125I-Bolton-Hunter derivative were investigated with respect to their binding properties to the antibody, only those fractions coinciding with the maximum amount of radioactivity showed a satisfactory ratio of specific vs. non-specific antibody-binding, especially in the case of the 125I-Bolton-Hunter derivative. Therefore, the observed results are in good accordance with the existence of the desired target components.

Determination of pilocarpine, isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine by high-performance liquid chromatography with tandem mass spectrometric detection

A method is described for the determination of pilocarpine and its degradation products isopilocarpine, pilocarpic acid and isopilocarpic acid in human plasma and urine. The method is based on a simple sample preparation step – ultrafiltration for plasma and dilution for urine samples – followed by a reversed-phase liquid chromatographic separation of the analytes and detection by means of tandem mass spectrometry. Parameters affecting the performance of these steps are discussed. The high sensitivity and selectivity of the method allow low ng/ml concentrations to be determined for all compounds in plasma and undiluted urine, which enables the investigation of the metabolic fate and elimination of pilocarpine after oral administration to humans.

Bonded stationary phases for reversed phase liquid chromatography with a water mobile phase: application to subcritical water extraction

Reversed phase high-performance liquid chromatography (RP-HPLC) is demonstrated for hydrophobic analytes such as aromatic hydrocarbons on a chemically bonded stationary phase and a mobile phase consisting of only water. Reversed phase liquid chromatography separations using a water-only mobile phase has been termed WRP-LC for water-only reversed phase LC. Reasonable capacity factors are achieved through the use of a non-porous silica substrate resulting in a chromatographic phase volume ratio much lower than usually found in RP-HPLC. Two types of bonded WRP-LC columns have been developed and applied. A brush phase was synthesized from an organochlorosilane. The other phase, synthesized from an organodichlorosilane, is termed a branch phase and results in a polymeric structure of greater thickness than the brush phase. A baseline separation of a mixture containing benzaldehyde, benzene, toluene, and ethyl benzene in less than 5 min is demonstrated using a water mobile phase with 12 000 plates generated for the unretained benzaldehyde peak. The theoretically predicted minimum reduced plate height is also shown to be approached for the unretained analyte using the brush phase. As an application, subcritical water extraction (SWE) at 200°C is combined with WRP-LC. This combination allows for the extraction of organic compounds from solid matrices immediately followed by liquid chromatographic separation of those extracted compounds all using a solvent of 100% water. We demonstrate SWE/WRP-LC by spiking benzene, ethyl benzene, and naphthalene onto sand then extracting the analytes with SWE followed by chromatographic separation on a WRP column. A sand sample contaminated with gasoline was also analyzed using SWE/WRP-LC. This extraction process also provides kinetic information about the rate of analyte extraction from the sand matrix. Under the conditions employed, analytes were extracted at different rates, providing additional selectivity in addition to the WRP-LC separation.