Reversed-phase Adsorption Research Articles

Parameter estimation of perillyl alcohol in RP-HPLC by moment analysis

Parameter estimations were made for the reversed-phase adsorption of perillyl alcohol (POH), a potent anti-cancer agent, on octadecylsilyl-silica gel (ODS). The average particle diame- ter of ODS was about 15 µm, and the particles were packed in the column (3.9 × 300 mm). The mobile phase used was a mixture of acetonitrile and water, in which the acetonitrile ranged be- tween 50 and 70 (v/v %). The first absolute moment and the second central moment were deter- mined from the chromatographic elution curves by moment analysis. Experiments were carried out using POH solutions within the linear adsorption range. The fluid-to-particle mass transfer coefficient was estimated using the Wilson-Geankoplis equation. The axial dispersion coefficient and the intraparticle diffusivity were determined from the slope and intercept of a plot of H vs 1/u0, respectively. The contributions of each mass-transfer step were axial dispersion, fluid-to- particle mass transfer, and intraparticle diffusion.

Capillary-electrochromatographic separations with copolymeric reversed-stationary phase and ion-exchanger-packed columns

A macroporous, spherical, 7 μm, polystyrene–divinylbenzene (PS–DVB), reversed-phase adsorbent (PRP-1) was evaluated as a stationary phase for the capillary electrochromatographic (CEC) separation of neutral, acidic, and basic analytes of pharmaceutical interest. Electroosmotic flow (EOF) for a PRP-1 packed capillary is nearly constant over the pH 2 to 10 range and is higher than for a silica-based C 18 packed capillary on the acidic side. EOF increases with an increase in buffer acetonitrile concentration or as applied potential increases. As analyte hydrophobicity increases, analyte retention and migration time increases. Increasing buffer acetonitrile concentration reduces analyte partitioning with the PS–DVB stationary phase and analyte retention and migration time decreases. When exchange sites are present on the PS–DVB copolymer, EOF (EOF is reversed for the anion-exchanger) increases as the exchange capacity increases. An increased exchange capacity also reduces partitioning of the analyte with the PS–DVB matrix and analyte retention and migration time decrease. Because of excellent stability in an acid environment, the PRP-1 packed capillary can be used in strong acid buffer solution and weak acid and base analytes depending on pK a values can be separated as neutral species and cations, respectively. CEC separations on a PRP-1 capillary of neutral steroids, weak base pharmaceuticals (separation as cations), purines and pyrimidines (as cations), fatty acids (as undissociated species), and sulfa derivatives (as cations) are described. Efficiency for the PRP-1 packed capillary for acetone or thiourea as the analyte is about 6·10 4 plates m −1.

Interpretation of the excess adsorption isotherms of organic eluent components on the surface of reversed-phase adsorbents: Effect on the analyte retention

The excess adsorption isotherms of acetonitrile, methanol and tetrahydrofuran from water on reversed-phase packings were studied, using 10 different columns packed with C 1–C 6, C 8, C 10, C 12 and C 18 monomeric phases, bonded on the same type of silica. The interpretation of isotherms on the basis of the theory of excess adsorption shows significant accumulation of the organic eluent component on the adsorbent surface on the top of “collapsed” bonded layer. The accumulated amount was shown to be practically independent of the length of alkyl chains bonded to the silica surface. A model that describes analyte retention on a reversed-phase column from a binary mobile phase is developed. The retention mechanism involves a combination of analyte distribution between the eluent and organic adsorbed layer, followed by analyte adsorption on the surface of the bonded phase. A general retention equation for the model is derived and methods for independent measurements of the involved parameters are suggested. The theory was tested by direct measurement of analyte retention from the eluents of varied composition and comparison of the values obtained with those theoretically calculated values. Experimental and theoretically calculated values are in good agreement.

Capillary electrochromatography with fused-silica capillaries packed with copolymeric reversed-phase adsorbent and ion exchangers.

Macroporous poly(styrene-divinylbenzene) (PSDVB), PRP-1, a reversed-phase adsorbent, and PSDVB-based strong acid cation exchangers and strong base and weak base anion exchangers were evaluated as stationary phases for capillary electrochromatography (CEC). Electroosmotic flow (EOF) for adsorbent and exchanger packed fused-silica capillaries for acetone as the marker increases with increasing ion exchange capacity, buffer organic solvent concentration, and applied voltage, is nearly independent of pH, and decreases with increased buffer ionic strength. For anion exchangers, EOF is reversed. Thiourea, acetone, acrylamide, nitromethane, propanal, and acetic acid were evaluated as EOF markers and undergo weak interaction with the PSDVB-based stationary phases. EOF in a basic buffer is greater than or equal to silica-based C-18 and cation exchanger packed capillaries. For an acidic buffer, EOF for a PRP-1 capillary is almost twice the C-18 packed capillary. As analyte hydrophobicity increases, retention and migration time increases for the PSDVB-based stationary phases. As exchange capacity increases, availability of the polymeric matrix for analyte partitioning decreases, causing analyte migration time to decrease. Increasing buffer organic solvent concentration decreases analyte retention. The PSDVB-based stationary phases provide good resolving power and reproducibility and are applicable to the CEC separation of neutral, weakly acidic, and basic analytes. Efficiency, however, is less than obtained with silica-based stationary phases. Because of stability in a strong acid buffer, the CEC separation of weak acids, where dissociation is suppressed, and weak bases as cations is possible. Separations of short-chain alkyl aldehydes, methyl ketones, aromatic hydrocarbons, substituted benzene derivatives, and short-chain carboxylic acids are described.

Liquid adsorption chromatography of polyethers: experiments and simulation

The adsorption behavior of poly(ethylene glycol) (PEG) in reversed-phase chromatography is studied both experimentally and theoretically and a computer simulation of chromatograms is performed on the basis of these studies. The experimental conditions were: different reversed-phase adsorbents and a solvent methanol–water system as the mobile phase. At varying mobile phase compositions highly resolved chromatograms of PEG samples were obtained, in which all peaks could be identified, and the dependencies of the distribution coefficient on the degree of polymerization for PEG molecules were evaluated by processing these chromatograms. The data were interpreted by using a theory of homopolymers based on a continuum Gaussian chain model of flexible macromolecules and a slit-like model of pores of stationary phase. The theory proved to describe well the experimental data in the whole range of studied molecular masses, and the thermodynamic parameters characterizing interactions of ethylene oxide repeating units in PEG molecules with the adsorbent pore walls have been determined from the comparison of the theory with the experimental data. The dispersion of chromatographic peaks corresponding to individual oligomer molecules is also estimated. In the system studied the peak width occurred to be proportional to the distribution coefficient of corresponding macromolecule. The theory is used to develop a computer-assisted procedure for simulation of chromatograms for samples of linear homopolymers. Using the obtained data on the thermodynamic parameters and the estimates of peak dispersion, chromatograms are simulated for PEG samples at two different chromatographic conditions. These simulated chromatograms were in good quantitative agreement with the real chromatograms.

Comparison of Chemical Composition Distributions of Poly(methyl methacrylate)-graft-polydimethylsiloxane by High-Performance Liquid Chromatography and Demixing-Solvent Fractionation

Poly(methyl methacrylate)-graft-polydimethylsiloxane (PMMA-graft-PDMS) samples with different compositions were prepared by radical copolymerization of PDMS macromonomer with methyl methacrylate. The chemical composition distributions (CCDs) of the graft copolymer samples were determined by the high-performance liquid chromatography (HPLC) based on the reversed-phase adsorption mode, using a prepacked column of butyl-modified silica gel and the linear gradient elution of acetonitrile and tetrahydrofuran. The CCD of one of the graft copolymer samples was also determined by the demixing-solvent fractionation using the demixing-solvent pair of dimethylsulfoxid-tetrachloroethylene reported by Stejskal et al. The CCD by HPLC was in good agreement with theoretical CCD, while the CCD by the demixing-solvent fractionation was narrower than those by the HPLC and the theoretical calculation.

Low-energy interactions in high-performance liquid chromatography

Liquid chromatographic systems with very weak excessive analyte–adsorbent interactions have been studied. These systems consisted of a homologous series of n-alkanes as both analytes and mobile phases with a C 18 reversed-phase adsorbent. A linear decrease of the analyte retention volume with an increase of the number of analyte carbon atoms was found. Corresponding increases of analyte retention with an increase in the number of eluent carbon atoms was also discovered. An explanation of these two effects on the basis of adsorption theory is proposed. A good correlation of column hold-up volume calculated by interpolation of the retention dependencies for above mentioned systems with that measured by the minor disturbance method has been shown. A study of the temperature dependencies of these alkane systems has shown entropy-governed retention dependencies.

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns: IV. Results obtained with Luna C 18 (2) columns

The reproducibility of retention data and band profile characteristics was investigated for a series of columns packed with Luna C 18 (2), a silica-based, reversed-phase adsorbent. High precision data were obtained and statistically compared among five columns from the same batch (column-to-column reproducibility) and nine columns from as many different batches (batch-to-batch reproducibility). These data were acquired under five different sets of chromatographic conditions, for a group of 30 neutral, acidic, and basic compounds selected as probes following an experimental protocol previously described. This work is part of a study on the precision of chromatographic analyses. Its purpose is to illustrate the contribution of the stationary phase reproducibility to this precision.

Seasonal Changes in Ethylene Oxide Chain Length of Poly(oxyethylene)alkylphenyl Ether Nonionic Surfactants in Three Main Rivers in Tokyo

Although poly(oxyethylene)alkylphenyl ether (APE) nonionic surfactants with longer ethylene oxide (EO) chain than 2 can be the important pollutants in natural aquatic environments as the potential precursor of alkylphenol, an endocrine disrupting chemical, little attention has been paid to those. Water samples were seasonally collected at five sites of each of the three main rivers in Tokyo. APE with different alkyl chains were separated and fractionated using a reversed-phase adsorption enrichment technique with gradient elution in high performance liquid chromatography (HPLC). After the concentration of each fraction, EO chain length (polymerization degree of EO) in APE was determined by electrospray ionization mass spectrometry (ESI/MS). The results indicated that, in three main river waters in Tokyo, poly(oxyethylene)nonylphenyl ether (NPE) was the dominant pollutant among APE, total NPE concentrations with different EO chain lengths were 2−6 nM in summer and 10−35 nM in winter, and its peak in the abundance of NPE components with different EO chain lengths was at 5−8 EO units in winter and shifted to 2−5 units in summer. Laboratory biodegradation experiments using filtered and NPE-spiked river water confirmed that seasonal changes in the abundance of NPE components with different EO chain lengths would be mainly caused by increased bacterial activity due to high water temperature, producing mainly the persistent nonylphenol diethoxylate.

New Method for the Direct Determination of Dissolved Fe(III) Concentration in Acid Mine Waters

A new method for direct determination of dissolved Fe(III) in acid mine water has been developed. In most present methods, Fe(III) is determined by computing the difference between total dissolved Fe and dissolved Fe(II). For acid mine waters, frequently Fe(II) ≫ Fe(III); thus, accuracy and precision are considerably improved by determining Fe(III) concentration directly. The new method utilizes two selective ligands to stabilize Fe(III) and Fe(II), thereby preventing changes in Fe reduction−oxidation distribution. Complexed Fe(II) is cleanly removed using a silica-based, reversed-phase adsorbent, yielding excellent isolation of the Fe(III) complex. Iron(III) concentration is measured colorimetrically or by graphite furnace atomic absorption spectrometry (GFAAS). The method requires inexpensive commercial reagents and simple procedures that can be used in the field. Calcium(II), Ni(II), Pb(II), Al(III), Zn(II), and Cd(II) cause insignificant colorimetric interferences for most acid mine waters. Waters con...

Comparison of microscale cleaning procedures for (Glyco) proteins prior to positive ion matrix-assisted laser desorption ionization mass spectrometry

Microscale cleaning procedures for peptides, proteins and glycoproteins prior to matrix-assisted laser desorption ionization (MALDI) mass spectrometric (MS) analysis, based on different mechanisms – drop dialysis, ultrafiltration, reverse phase adsorption and gelfiltration, are being compared. The selected standard (glyco) proteins cover the mass range of 5000–70 000 Da. Individual components and mixtures containing up to five components have been used for the investigation. Five different procedures were applied and evaluated with regard to a subsequent positive ion MALDI time-of-flight MS analysis, which requires samples with relative low salt/buffer content. All methods were adapted for protein samples in the low picomole range. Drop dialysis, ultrafiltration, reverse phase adsorption in two different forms and gelfiltration are shown to be useful for proteins and partially for peptides and glycoproteins. It turned out that glycoproteins are more difficult to handle, due to the relatively high sample losses during the desalting procedures. Gelfiltration and reverse phase adsorption are the least time consuming methods, whereas drop dialysis and ultrafiltration are well suited for working on many samples in parallel. However, reverse phase adsorption and ultrafiltration offer the highest enrichment factors. For proteins available only in large sample volumes gelfiltration and reverse phase adsorption are the methods of choice.

Reversed-phase high-performance liquid chromatography of polyethers: Comparison with a theory for flexible-chain macromolecules

Reversed-phase adsorption chromatography retention modes of polyethylene glycol (PEG), its mono and dimethyl ethers (MME and DME) and polypropylene glycol (PPG) are studied both experimentally and theoretically. The experimental conditions were: narrow- and wide-pore Spherisorb C 18 adsorbents and two mixed-solvent systems (methanol–water and acetone–water) as mobile phases. At varying compositions of the mobile phase, fully-resolved chromatograms of polyether samples were obtained, in which all peaks could be identified, and the dependencies of the distribution coefficient on the degree of polymerization for PEG and PPG molecules were extracted by processing these chromatograms. The data were interpreted by using a theory of homopolymers based on a continuum Gaussian chain model of flexible macromolecules and a slit-like model of pores of the stationary phase. Two regimes of an adsorption chromatography of macromolecules are examined in relation to the well-known Martin rule, the role of pore size and the end-group effects being discussed. The theory proved to describe the experimental data on both polymers well, in the whole range of molecular masses studied, and thermodynamic parameters characterizing interactions of ethylene oxide and propylene oxide (PO) repeating units in polyether molecules with the adsorbent pore walls have been determined from a comparison of the theory with the experimental data. The conditions corresponding to the critical chromatography mode are estimated from the thermodynamic data for PEG and methanol–water system as being at about 76% of methanol, while for the PPG and acetone–water system, it is shown that such critical conditions at the given temperature are not attainable. Additionally, the mean thickness H of adsorbed PEG and PPG macromolecules was estimated as being equal to about 3–10 Å. The parameter H proved to be a decreasing function of the water content in the mixed solvent. The same estimate of thickness H≈2.6 Å is obtained for both polymers in pure water.

Study of the Excess Adsorption of the Eluent Components on Different Reversed-Phase Adsorbents

The excess adsorption isotherms of methanol-water and acetonitrile-water mixtures are compared for C 4 , C 8 , and C 18 types of reversed-phase adsorbents with the same type of silica support (Hypersil). Adsorption behavior of the organic modifier is also measured for three different types of C 8 adsorbents (Bakerbond, Supelcosil, and Hypersil) and on the Ultracarb-ODS at different temperatures. It is shown that the adsorption behavior of acetonitrile for all types of bonded phases is similar, but that of methanol shows an obvious dependence on the alkyl chain length of the bonded phase

Compositional fractionation of poly(methyl methacrylate)- graft-polydimethylsiloxane by reversed-phase high-performance liquid chromatography

The results are reported of the compositional fractionation of poly(methyl methacrylate)- graft-polydimethylsiloxane (PMMA- graft-PDMS) by high-performance liquid chromatography (h.p.l.c.) of reversed-phase adsorption mode. Graft copolymers with different compositions were prepared by the radical copolymerization of PDMS macromonomer with MMA, using azobisisobutyronitrile as the initiator in benzene at 60°C. The gradient h.p.l.c. measurements were made using a prepacked column of octyl-modified silica gel, with tetrahydrofuran and acetonitrile as eluents. Detection was performed by an evaporative light-scattering detector. The PMMA- graft-PDMS samples were eluted from the components of higher PMMS contents to those of lower PMMA contents, according to reversed-phase adsorption. The present graft copolymers were separated according to their chemical compositions.

Column-switching high-pressure liquid chromatographic method for the determination of a new carbapenem antibiotic, L-739,428, in rat and monkey plasma

A column-switching HPLC method for determination of a new carbapenem antibiotic, L-739,428 (I), was developed that allowed direct injection of rat and monkey plasma. Following dilution with a pH 6.5 buffer, samples were injected without further cleanup into an extraction column dry-packed with 10-μm particle size Maxsil C 18 reversed-phase adsorbent. Endogenous plasma components were washed to waste for 6 min with a weak mobile phase of 0.025 M sodium phosphate, 0.005 M hexanesulfonate (pH 6.5). Compound I, retained on the extraction column, was then backflushed with a mobile phase which consisted of a mixture of the preceding buffer-ion pair solution and acetonitrile (96:4, v/v) into a Partisphere C 18 analytical column and detected by ultraviolet absorption at 299 nm. Chromatographic retention time was 11.5 min. Stability of I in plasma was maximized by use of a refrigerated autosampler which maintained plasma at 5°C until analyzed. The limit of quantification was 0.1 μg/ml using the equivalent of 75 μl plasma.

Analysis of polychlorinated biphenyls, organochlorine pesticides and chlorophenols in rain and snow

Polychlorinated biphenyls (PCBs), organochlorine pesticides and chlorophenols were measured in samples of rain and snow collected at urban and suburban/semi-rural locations in the Zagreb City area, Croatia. PCBs and organochlorine pesticides were extracted with hexane from filtered aqueous samples and from particulate matter isolated by filtration. Chlorophenols were accumulated from rain and snow water by C18 reversed-phase adsorption. All samples were analysed by capillary gas chromatography using an electron-capture detector. Polychlorinated dibenzodioxins (PCDDs) and dibenzofurans (PCDFs) in rain and snow water were checked by gas chromatographic–mass spectrometric analysis of combined hexane extracts. PCBs were detected in all water (<1–203 ng l–1) and particle (40–4155 ng g–1) samples. The only organochlorine pesticide present in all rain and snow water (1–36 ng l–1) and in particle (7–512 ng g–1) samples was γ-hexachlorocyclohexane as a consequence of the regular local usage of lindane. A positive correlation was found between its concentration in the water phase and the average air temperature during the sampling period. Compounds of the DDT-type, trace amounts of which were detected in only two rain water samples, were determined in most particle samples. The DDE : DDT median concentration ratio in particles was lower than unity and indicated a recent input of DDT into the atmosphere. The incidence and concentrations of di-, tri-, tetra- and pentachlorophenols were higher in snow (single compound concentration 11–527 ng l–1) than in rain (single compound concentration 2–171 ng l–1). A quadratic decrease in chlorophenol concentrations in snow and rain with increasing air temperature was observed. Trace amounts of PCDDs and PCDFs were detected in both rain and snow water samples and the highest concentrations were measured for octa-CDD (2 pg l–1 in snow and 6 pg l–1 in rain).

Recovery of substance P and related C-terminal fragments on solid-phase extraction cartridges for subsequent high-performance liquid chromatographic separation and radioimmunoassay.

The recoveries of substance P (SP) and five related peptides were evaluated on different types of solid-phase extraction sorbent. Best results were obtained by use of a C18 silica gel cartridge. Marked differences of extraction yields occurred for the different peptide fragments and, in general, recovery increased with increasing hydrophobicity of the peptide when reversed-phase materials like C18 and C8 cartridges were used. This observation is indicative of a sorption-desorption mechanism by prevailing solvophobic interactions. A similar trend was found when phenylpropyl silica gel (CPhenyl), generally known as a reversed-phase adsorbent of lower hydrophobicity, was used. It was concluded that a substantial participation of analyte-matrix pi-pi interactions has to be taken into account when extraction yields are compared with corresponding values obtained by use of a C8 cartridge. With CN silica gel cartridges, marked differences in extraction yields were obtained by use of acetonitrile or methanol as the organic modifier. As an attempt to explain this observation, conformational effects were assumed for the sorption-desorption behaviour of the peptides on the polar matrix.

Trace enrichment of chlorophenols in human urine by C18 reversed-phase adsorption and by n-hexane extraction

The efficiency and sensitivity of C18 reversed-phase adsorption of free chlorophenols and of n-hexane extraction of either free or acetylated chlorophenols from human urine were compared. All procedures were found to be efficient for the trace enrichment of 2,4-dichlorophenol, 2,4,6- and 2,4,5-trichlorophenols, 2,3,4,6- and 2,3,4,5-tetrachlorophenols and pentachlorophenol. The recoveries of chlorophenols from non-hydrolysed and acid hydrolysed urine samples were comparable. By treatment of 1 ml urine sample detection limits of 1–2 ng/ml were achieved, while the treatment of 5 ml samples enhanced the detection sensitivity to less than 1 ng/ml. The n-hexane extraction of acetylated chlorophenols from 1 ml urine samples is the simplest and fastest procedure because the acetylation and extraction of chlorophenols are performed simultaneously in one step. The C18 adsorption seems to be more suitable than n-hexane extraction for accumulation of chlorophenols from a urine volume of 5 ml and higher because the elution is performed always with the same small volume of acetone. Both C18 adsorption and n-hexane extraction procedures were applied for analysis of chlorophenols in general population and in persons with possible occupational exposure to organochlorine compounds.

Sample remaining in an ODS column after compositional fractionation of copolymers by high-performance liquid chromatography

High-performance liquid chromatography is very effective for the compositional fractionation of copolymers. Octadecyl-modified silica gel (ODS) has been used as a packing material for fractionation in the reversed-phase (RP) adsorption mode. In the compositional fractionation of the samples of copoly-(methyl methacrylate-styrene) by RP gradient elution through ODS column, it was found that small peaks were observed with blank elution just after the sample measurement and a perfect baseline was recovered after several blank elutions.

Adsorption of a Hydrophobic Chelating Agent and its Chelate-Metal Complex on RP-Columns

Abstract The reversed-phase adsorption of the hydrophobic chelating agent 4-dodecyldiethylenetriamine, in the presence of zinc ions, was measured at different compositions of the mobile phase. The effect of the following parameters was analyzed: proportion of metallic ion to chelating agent, concentration of chelating agent, volumic fraction of organic modifier, pH and salt concentration. Results show that, depending on the composition of the mobile phase, the chelating agent can exist in two different forms in both phases: “free” and/or forming a chelate-metal complex. The adsorption of the latter is stronger than that of the free triamine. This phenomenon, which has not been reported before, plays undoubtedly an important role on retention in Reversed-Phase Ligand Exchange Chromatography and should be considered in the study of the retention mechanism.