Silica-based Reversed-phase Columns Research Articles

Determination of the hydrogen-bond basicity descriptor by reversed-phase liquid chromatography

Except for alkanes, most organic compounds are hydrogen-bond bases. The B° descriptor of the solvation parameter model provides a convenient measure of the effective (or summation) hydrogen-bond basicity of organic compounds. A fast and convenient method to assign the B° descriptor is required to support studies of hydrogen-bonding in separation systems. A two-column system with acetonitrile-water mobile phase compositions and the measurement of up to eleven isocratic retention factors is proposed for this purpose. Several reversed-phase column chemistries and mobile phases were evaluated with the two-column system consisting of a pentafluorophenylpropylsiloxane-bonded and octadecylsiloxane-bonded silica columns recommended for this purpose. To assess the accuracy of the method values for B° were taken from the Wayne State University (WSU) compound descriptor database, which were assigned using conventional multi-technique methods and large datasets. The two-column systems provided an unbiased assignment of B° with an average deviation of 0.008 and an average absolute deviation of 0.021 compared with the target value for 55 varied compounds. The two-column system is unsuitable for assigning the other descriptors used in the solvation parameter model and results in erroneous assignments of B° for nitrogen-containing compounds capable of electrostatic interactions on silica-based reversed-phase columns.

Separation of New Coumarin Glycosides from Toddalia asiatica Using Offline Two-Dimensional High-Performance Liquid Chromatography.

Coumarins and flavonoids are the major constituents of Toddalia asiatica. The separation and purification of ingredients from T. asiatica is an important procedure to acquire high-purity compounds for subsequent pharmacological investigation to discover leading compounds. In the present work, an offline two-dimensional high-performance liquid chromatography (HPLC) method was successfully established for the separation of high-purity glycosides from T. asiatica. Based on the separation results obtained with two different chromatographic stationary phases, a phenyl-bonded silica-based reversed-phase column was employed as the first HPLC preparation, and three fractions were obtained from the sample. Then, the fractions were isolated and purified on an octadecyl-bonded silica-based reversed-phase column to obtain high-purity compounds in the second HPLC separation. As a result, three coumarin glycosides, including two undescribed and one known, along with one known flavonoid glycoside with more than 98% purity were isolated from the sample. The structures of the isolated compounds were elucidated on the basis of extensive spectroscopic evidence derived from optical rotation, mass spectrometry, and nuclear magnetic resonance experiments. Two-dimensional HPLC with different stationary phases has the potential to be an efficient method for the separation of high-purity compounds from T. asiatica.

A semi-automated LC–MS/MS method for the determination of LCI699, a steroid 11β-hydroxylase inhibitor, in human plasma

A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of LCI699 was developed and validated with dynamic ranges of 0.0500-50.0 ng/mL and 1.00-1,000 ng/mL using 0.0500 mL and 0.100mL, respectively, of human plasma. LCI699 and the internal standard, [M+6]LCI699, were extracted from fortified human plasma via protein precipitation. After transfer or dilution of the supernatant followed by solvent evaporation and/or reconstitution, the extract was injected onto the LC-MS/MS system. Optimal chromatographic separation was achieved on an ACE C18 (50 mm × 4.6mm, 3 μm) column with 30% aqueous methanol (containing 0.5% acetic acid and 0.05% TFA) as the mobile phase run in isocratic at a flow rate of 1.0 mL/min. The total analysis cycle time is approximately 3.5 min per injection. The addition of an ion-pair reagent, TFA (0.05%, v/v), to the mobile phases significantly improved the chromatographic retention and resolution of the analyte on silica based reversed-phase column. Although addition of TFA to the mobile phase suppresses the ESI signals of the analyte due to its ion-pairing characteristics in the gas phase of MS source, this negative impact was effectively alleviated by adding 0.5% acetic acid to the mobile phase. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. For the low curve range (0.0500-50.0 ng/mL), the accuracy and precision for the LLOQs (0.0500 ng/mL) were -13.0 to 2.0% bias and 3.4-19.2% CV, respectively. For other QC samples (0.100, 6.00, 20.0 and 40.0 ng/mL), the precision ranged from 1.2 to 9.0% and from 3.8 to 8.8% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from -11.3 to 8.0% and -7.2 to 1.6% bias, respectively, in the intra-day and inter-day batches. For the high curve range (1.00-1,000 ng/mL), the accuracy and precision for the LLOQs (1.00 ng/mL) were 1.0-15.0% bias and 7.4-9.2% CV, respectively. For the other QC samples (3.00, 20.0, 200 and 750 ng/mL), the precision ranged from 0.8 to 7.0% and from 1.9 to 5.2% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from -2.5 to 4.0% and 0.7-1.0% bias, respectively, in the intra-day and inter-day batches. Additional assessments of incurred sample stability (ISS) and incurred sample reanalysis (ISR) were conducted to demonstrate the ruggedness and robustness of the assay method. The absence of adverse matrix effect and carryover was also demonstrated. The validated method was successfully used to support rapid turnaround human pharmacokinetic studies.

Evaluation of a new polymeric stationary phase with reversed-phase properties for high temperature liquid chromatography

The performance of a polymeric stationary phase with reversed-phase properties (ET-RP1) was evaluated for LC separations at elevated temperature. The most significant observation was that the reduced plate height ( h) decreased from 3.4 at 25 °C (optimal flow 0.5 mL/min) to 2.4 at 150 °C (optimal flow 2.5 mL/min) which is comparable to the efficiency obtained with silica-based reversed-phase columns of 4.6 mm ID operated at 0.8 mL/min. The phase showed no deterioration after long use at 150 °C within the pH range 1–9. Catalytic activity originating from the stationary phase material, e.g. as experienced on zirconium columns operated at elevated temperature, was absent. The performance of ET-RP1 is illustrated with the analysis of some pharmaceutical samples by LC and LC–MS. Operation at elevated temperature also allows to reduce the amount of organic modifier or to replace acetonitrile and methanol by the biodegradable ethanol.

Optimized HPLC Method for Analysis of Polar and Nonpolar Heterocyclic Amines in Cooked Meat Products

A simple, precise, and specific column high-performance liquid chromatographic (HPLC) method with UV absorption diode array and fluorescence detection has been developed by optimizing a previously described method for the simultaneous quantification of 15 polar and nonpolar heterocyclic amines (HAs) in fried meat products. The HPLC determination could be improved due to the application of a silica-based reversed-phase column with octadecyl groups (TSK-gel Super ODS) and a particle size of 2 microm. The separation of HAs in the complex meat matrix was performed with a 21 min mobile phase gradient. The method was validated for instrumental precision, repeatability, and selectivity and compared with a previously published method. After liquid adsorption of the basic sample mixture on diatomaceous earth, HAs were extracted with ethyl acetate. For cleanup, solid-phase extraction (silica propylsulfonic acid and octadecyl cartridges) and different washing steps were applied. Both nonpolar and polar HAs were determined in one fraction. The calibration curves of all HAs were linear for the applied detection system (correlation coefficient = 0.990-0.995). The recoveries, with the exception of 3-amino-1-methyl-5H-pyrido [4,3-b]indole (Trp-P-2), were between 42 and 98% from meat samples spiked in a range of 1.5 to 3.3 ng/g for fluorescence-active and 4.3 to 8 ng/g for UV-active HAs. For quantification of HAs, the standard addition method was used for adjustment of different characteristics of HAs in the extraction. In fried meat samples (chicken breast and beef patties), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f] quinoxaline(4,8-DiMelQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), norharmane, and harmane were found in a concentration range of 0.02 to 14.3 ng/g.

Fast liquid chromatography–electrochemistry–mass spectrometry of ferrocenecarboxylic acid esters

Rapid liquid chromatographic separations of ferrocenecarboxylic esters of various alcohols and phenols have been achieved on reversed-phase columns of 20 mm length. After separation, the ferrocene derivatives are oxidized electrochemically under formation of the charged ferrocinium species, which are easily detected by mass spectrometry using an atmospheric pressure chemical ionization source operated in the heated nebulizer mode. While a series of nine phenol derivatives was separated within less than 1.5 min, six alcohol derivatives eluted within 1 min. Limits of detection using a single quadrupole mass analyzer ranged from 60 to 190 nmol/l. Additional work was directed on the use of a graphite in-line filter instead of a silica-based reversed-phase column to achieve the separation.

CHROMATOGRAPHIC EVALUATION OF ALKYLPHOSPHONIC ACID-MODIFIED CERIA-ZIRCONIA IN REVERSED-PHASE HPLC

Porous ceria-zirconia sperules were surface modified with alkylphosphonic acid to create reversed phase material. The chromatographic properties of the ceria-zirconia-based stationary phase were evaluated in detail with neutral, basic, and acidic compounds as probes, over a wide range of mobile phase composition and pH. Polycyclic aromatic hydrocarbons (PAHs) were well separated and their elution order were in accordance with those reported on the silica-based reversed-phase column. Typical reversed-phase chromatographic behavior was also observed with basic solutes at pH10.0, whereas dual mode retention mechanism was adopted to elucidate retention characteristics of some of the basic solutes at pH4.0. Acidic compounds exhibited strong interaction with the alkylphosphonic acid modified ceria-zirconia (PA-CeO2-ZrO2) with methanol-water mixture as mobile phases, but these interaction were reduced to different extents in the presence of competing agents, such as acetate, sulfate, and oxalate. o-hydroxybenzoic acid irreversibly adsorbed on the PA-CeO2-ZrO2 stationary phase, even in the presence of oxalate, which may be explained by the formation of chelate ring.

Retention study of inorganic and organic selenium compounds on a silica-based reversed phase column with mixed ion-pairing reagents

Separation of seven inorganic and organic selenium compounds, namely selenic acid [Se(VI)], selenous acid [Se(IV)], trimethylselenonium iodide (TMSe+), selenocystine (SeCys), selenomethionine (SeMet), selenoethionine (Seet), and selenocystamine (SeCM), has been performed on a LiChrosorb C 18 column by using mixed ion-pair reagents; 1-butanesulfonic acid and tetramethylammonium hydroxide. Flame atomic absorption spectrometry (FAAS) was used as an element-specific detector. The retention behaviors of selenium compounds in terms of several chromatographic parameters, such as pH of the mobile phase, the concentrations of ion-pair reagents, and the content of organic modifier (methanol) were investigated. It was found that the separation of both inorganic and organic selenium compounds can be achieved within 12 min with a mobile phase of 10 mM 1-butanesulfonic acid −4 mM tetramethylammonium hydroxide −4 mM malonic acid −0.05% methanol adjusted to pH 4.5 at a flow rate of 1.0 mL min−1. The results obtained in this study showed that the use of mixed ion-pair reagents is very useful to improve the separation of selenium compounds. The applicability of this technique for the speciation of selenium compounds in real samples was demonstrated by the determination of selenium compounds in a selenium nutritional supplement. The results were found to be in good agreement with those obtained by ion-exchange HPLC-ICP-MS.

Pseudo-electrokinetic packing of high efficiency columns for capillary electrochromatography

An improved and easy electrokinetic packing procedure is presented for the production of stable capillary columns suitable for capillary electrochromatography (CEC). In pseudo-electrokinetic packing a high electric field is used in conjunction with a hydrodynamic flow. The packing of silica-based reversed-phase columns can be achieved with basic, commercially available capillary electrophoresis (CE) equipment in approximately 15 min. The procedure is robust and a high success rate is achieved. No steps which might damage the stationary phase are involved and only a minimum amount of packing material is required. Columns packed according to the developed procedure are operated at high electric field strengths during the CEC separation, without the application of a stabilising pressure. Columns are stable for at least hundred runs and were tested using mixtures of polycyclic aromatic hydrocarbons and positively charged drugs. Separations were performed in a relatively high conducting ammonium acetate buffer, with efficiencies of up to 283 000 plates/m.

Analysis of higher polyamide-6 oligomers on a silica-based reversed-phase column with a gradient of formic acid as compared with hexafluoroisopropanol

The analysis of polyamide-6 oligomers and polymer is usually performed with expensive fluorinated alcohols like 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Formic acid is well known as a mobile phase additive to adjust pH in reversed-phase high-performance liquid chromatography. However, formic acid is seldom used as a modifier to perform gradient elution chromatography on octadecyl-modified silica-based columns. Here we demonstrate the determination of cyclic and linear polyamide-6 oligomers using formic acid as a modifier on an octadecyl-modified silica-based column. This column was shown to be stable for more than 5000 column volumes, even when a mobile phase of 65–95% formic acid in water at a flow of 1 ml/min is applied. With formic acid under the conditions used (65–95% formic acid in water) the oligomers are retained on the column, while the polymer does not precipitate. In comparison, during adsorption and separation with a HFIP gradient, precipitation of the polymer occurs. The implications of the different separation mechanisms, i.e., adsorption vs. precipitation chromatography are discussed. Loadability is shown to be much better with the formic acid system. However, with formic acid as a modifier UV detection below 250 nm is not feasible. The less sensitive evaporative light scattering detector is used to detect the polyamide oligomers in the formic acid phase. In addition it is shown that capillary zone electrophoresis (CZE) with UV-absorbance detection using HFIP is an attractive combination as HFIP is UV-transparent and CZE allows low modifier consumption.

Separation of erythromycin and related substances on base-deactivated reversed-phase silica gel columns

An official liquid chromatographic method for the analysis of erythromycin and related substances, which is based on a polymer reversed-phase, is described in the European Pharmacopoeia and in the United States Pharmacopeia. The pH of the mobile phase used in this system is 9.0. Recent advanced technology has led to the introduction of a new generation of silica-based reversed-phase column packings, which are claimed to be much more stable towards bases. They are useful for the analysis of basic compounds. Studies to verify the separation of erythromycin and related substances on Hypersil BDS C 18, Luna C 18(2), Inertsil ODS-2 and Supelcosil ABZ+ have been performed and the results are presented. It is shown that these base-deactivated phases give a better sensitivity and selectivity towards erythromycins than the polymer phase, provided that an adapted mobile phase is used. This is the first liquid chromatographic method described for the separation of erythromycin D from erythromycin A.

Synthesis, aggregation, and neurotoxicity of the alzheimer's Aß1-42 amyloid peptide and its isoaspartyl isomers

Amyloid Aß1-42 peptide (Aß1-42) and its isomers with an isoaspartyl residue at position 7 or 23 [Aß1-42(isoAsp7) and Aß1-42(isoAsp23)] were synthesized in high purity by the Fmoc-solid phase technique, followed by HPLC on a silica-based reversed-phase column under the basic conditions. Importantly, Aß1-42(isoAsp23) aggregated more strongly than native Aß1-42 and showed significant neurotoxicity, while the aggregation ablility and neurotoxicity of Aβ1-42(isoAsp7) vas weak. This suggests that the isomerization of the aspartyl residues plays an important role in fibril formation in Alzheimer's disease.

Evaluation procedures for reversed-phase high-performance liquid chromatographic columns in the analysis of strongly basic compounds using principal components analysis for data assessment

The performance of eight high-purity silica-based reversed-phase columns was analysed using chemometric pattern recognition. Tests were performed at pH 3.0 and 7.0, using 10 compounds, three mobile phase modifiers (methanol, tetrahydofuran and acetonitrile) and four parameters {retention factor (k′), column efficiency (N), the Dorsey–Foley efficiency [N(df)] and the asymmetry factor (As])}. Principal components analysis was performed on subsets of the data. Features of both scores and loadings plots, together with the corresponding correlation coefficients between chromatographic tests, are discussed in detail. It is concluded that methanol and acetonitrile exhibit similar properties, the relative performance at each pH differs, the number of compounds used in the test can be reduced to a recommended five, and several of the original tests are redundant. Procedures are recommended for determining a range of tests that provide information on the column performance, which can be applied to other situations.

Direct isomeric separation of a 3-hydroxyproline-containing prodrug, L-693 989, by high-performance liquid chromatography with a porous graphitic carbon column

The use of high-performance liquid chromatography (HPLC) with a porous graphitic carbon (Hypercarb) column for the direct separation of a positional isomer of L-693 989, a 3-hydroxyproline-containing prodrug, is described. The isomer peak was isolated by HPLC and analyzed by mass spectrometry and proton nuclear magnetic resonance spectroscopy. An authentic sample of the isomer was also synthesized for chromatographic comparison. The results confirm that the peak in question is a 4-hydroxyproline isomer which is difficult to separate from L-693 989 compound with the silica-based reversed-phase columns. The observed chromatographic elution supports the retention mechanism based on the unique electronic donor-acceptor interaction between the lone-pair electrons of the analyte (donor) and the delocalized electron conduction bands on the graphitized carbon stationary phase (acceptor).

Selection of buffers and of an ion-pairing agent for thermospray liquid chromatographic—mass spectrometric analysis of ionic compounds

The applicability of ammonium formate, ammonium acetate and ammonium bicarbonate as volatile electrolytes for thermospray liquid chromatographic—mass spectrometric analysis of three decomposition products of α-aspartame is demonstrated. With these buffers, the pH range of the mobile phases for silica-based reversed-phase columns can be covered. With respect to the buffer capacity and ultraviolet transparency, a buffer salt concentration of 50 m M was used in the mobile phase. The retention times of the compounds studied in the volatile buffers were comapared with those obtained in sodium acetate and sodium phosphate buffers and were found to match quite well. For the reversed-phase ion-pair separation of α-aspartame and β-aspartame in combination with thermospray mass spectrometry, a method is described using trifluoroacetate as pairing ion. Liquid chromatographic—mass spectrometric analysis of l-aspartyl- l-phenylalanine shows that the highest signal-to-noise ratio is obtained in ammonium formate. For the compounds studied, no correlation could be found between the signal-to-noise ratio and source temperature, or between signal-to-noise ratio and vaporizer temperature.

Silica versus polymer-based stationary phases for reversed-phase high-performance liquid chromatographic analyses of rat insulin biosynthesis : A comparison of resolution and recovery

Because of the problems caused by the irreversible binding of insulins and proinsulins to several silica-based reversed-phase columns, the use of polymeric reversed-phase columns was investigated for the analysis of rat islet polypeptides involved in insulin biosynthesis. No irreversible binding of insulins and proinsulins was observed for the polymeric reversed-phase columns, probably due to the absence of silanol groups. The six polypeptides involved in insulin biosynthesis in rat islets were equally well resolved in shallow trifluoroacetic acid-acetonitrile gradients on the silica-based Nucleosil 300-5C 4 column (45°C), the polymer-based Asahipak C4P-50 (25 and 45°C), and ODP-50 columns (45°C). In shallow triethylammonium phosphate-acetonitrile gradients (25°C) satisfactory resolution of the two rat proinsulins was only obtained on the polymer-based Asahipak C4P-50 and C8P-50 columns. Increasing the separation temperature to 45°C improved the separation of the two insulins and the two proinsulins in all cases. The shifts in retention times for the individual islet polypeptides observed in relation to the increased separation temperature were found to be different for the silica C 4 and the polymer C 4 columns. Recoveries of rat islet polypeptides were comparably high from the silica- and the polymer-based C 4 columns and linear load-response curves were obtained in the microgram to picogram mass range of both columns.

Matrix effect on the retention in reversed-phase liquid chromatography

A polymer-based, reversed-phase column (VA-C18), prepared by grafting octadecyl chain onto vinyl alcohol copolymer gel, was investigated for its chromatographic characteristics. n-Alkanes and n-alkyl alcohols were found to be retained only by hydrophobic interaction between the solutes and the octadecyl chain. In the case of aromatic hydrocarbons, in addition to the hydrophobic interaction, π-π interaction between the solutes and the based material was elucidated to contribute to the retention. For aromatic tertiary amines which are known to strongly interact with the residual silanol group of the silica-based reversed-phase columns to produce broadened and skewed peakes, the VA-C18 column also retained these substrates strongly by the combination of hydrophobic, π-π, and ionic interactions. In this case, however, symmetrical peaks were observed. From these results, it was determined that in the case of VA-C18, the base material was found not to produce undesirable effect although the solutes interact with the base. Further conclusion obtained was that in reversed phase liquid chromatography, chromatographic properties of base matrix is highly responsible for the overall retention.

Reversed-phase chromatography of proteins on resin-based wide-pore packings

PLRP-S, a macroporous poly(styrene—divinylbenzene) HPLC packing material, was evaluated for analysis of proteins. Materials with a particle size of 8 μm and pore sizes of 100 Å, 300 Å, and 1000 Å were tested under similar chromatographic conditions to determine the effect of pore size and surface area on retention, selectivity, and efficiency. This resin-based material has a highly homogeneous hydrophobic surface, based on phenyl moieties contributed by the copolymers of the packing. Thus, unlike silica, PLRP-S has no bonded alkyl groups or residual silanol sites. In addition, the PLRP-S material is stable over a wide range of solvents, pH, and ionic strengths. The elution order of a series of proteins on PLRP-S 300 Å was similar to that observed on a variety of alkyl-bonded silica-based reversed-phase columns. The percentage of acetonitrile in the mobile phase necessary to elute three of these proteins (ribonuclease A, cytochrome c, and ovalbumin) was determined. Protein desorption occurred over a very narrow range of 2–4% increase in concentration of the organic modifier. No apparent change in protein selectivity or peak area occurred with repetitive injections of a mixture of proteins, indicating little relative loss of protein on the column. Pore size appeared to have little effect on the selectivity or retentivity of the test proteins. It appeared that the loss of total surface area due to increased pore size was offset by the increased availability of pore surface area for protein-packing interaction. The resin-based PLRP-S 300 Å was used for analysis of wheat proteins, bovine pancreatic enzymes, high-molecular-weight proteins, and whey proteins. Over the broad range of molecular weights and hydrophobicities in these applications, proteins were eluted as sharp, symmetrical peaks. Thus, PLRP-S offers an effective alternative to reversed-phase silica for the analysis of proteines.

Characterization of silica-based reversed-phase columns with respect to retention selectivity : Solvophobic effects

Variations in retention and retention selectivity are common among different reversed-phase columns. These retention differences arise from both solvophobic and chemical effects. Solvophobic contributions were studied for seven columns differing in either surface area or composition of the bonded phase. Resulting differences in solute retention were correlated with three effects: (1) the effective phase ratio of the column as measured by the average retention of all solutes; (2) the polarity of the bonded phase; and (3) the dispersion solubility parameter of the bonded phase. These three factors largely determine differences in solvophobic selectivity among different reversed-phase columns.