Monolithic Reversed-phase Columns Research Articles

Kinetic performance comparison of superficially porous, fully porous and monolithic reversed-phase columns by gradient kinetic plots for the separation of protein biopharmaceuticals

To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and β-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.

Chromatographic Performance of an Amine/amino-bonded Column and a Monolithic Reversed-Phase Column for the Separation of Fluorescently Labeled Glycoprotein Glycans

Chromatographic Performance of an Amine/amino-bonded Column and a Monolithic Reversed-Phase Column for the Separation of Fluorescently Labeled Glycoprotein Glycans

Optimization of peptide and protein separation with a monolithic reversed-phase column and application to arsenic-binding studies

A separation method for a mixture of eight sulfur-containing peptides and proteins characterized by a wide molar mass (1–18.4 kDa) and pI range (4.5–10.7) was developed onto a monolithic phenyl phase. Based on the first optimization steps that revealed an increase of the acetonitrile content to 45 vol.% as sufficient for the elution of all biomolecules and the addition of the ion pairing reagent trichloroacetic acid (TCA) as preferable over the eluent additives formic acid or ammonium acetate buffer, the critical variables TCA concentration, gradient time, and eluent flow rate were optimized using a Box-Behnken experimental design. To achieve optimum values for separation factors of all peak pairs, a TCA content of 0.025% (m/v), a gradient time of 10 min, and a flow rate of 3.5 mL min −1 were selected. Arsenic binding studies were undertaken under conditions optimized with respect to the crucial separation factor of the nonapeptides vasotocin (Vtc) and vasopressin (Vpr) in a shortened gradient time of 7.5 min. A complete separation of phenylarsenic-substituted and unmodified forms of these peptides allowed the calculation of both consumptions and apparent equilibrium constants K from HPLC–UV peak areas. The nonapeptide consumptions by the reaction with phenylarsine oxide (PAO) increased from 7% up to 100% in dependence on the molar ratio of the reaction components. Due to an enhanced UV absorption of the phenylarsenic-substituted biomolecules, the calculation of apparent equilibrium constants led to increasing K values with rising PAO molarities from 9.6 × 10 5 to 1.2 × 10 8 in case of Vtc and from 2.2 × 10 6 to 1.4 × 10 9 in case of Vpr. For α-lactalbumin, a consumption of 59.2 ± 6.1% by the reaction with molar excesses of PAO varying from 1.4 to 21 can be derived from the chromatograms. The quantitative evaluation of the reaction of the small protein aprotinin with PAO was hindered by a pronounced peak broadening that occurred after reduction of the disulfide bridges.

Fast separation of microcystins and nodularins on narrow-bore reversed-phase columns coupled to a conventional HPLC system

Six modern narrow-bore (50 mm length × 2–2.1 mm internal diameter) reversed-phase HPLC columns were tested in the rapid separation of ten microcystins and three nodularins, cyanobacterial peptide hepatotoxins. The columns had either a sub-3 μm particle size or were of monolithic silica technology for high efficiency and rapid run times. A standard low-pressure gradient HPLC equipment with diode-array UV detector was used for the chromatography. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, and the gradient rise times were 1–4 min. Chromatographic performance was assessed by visual judgement and by calculating parameters such as capacity factors, resolution, peak width, selectivity and peak form. Several chromatographic conditions produced excellent results. We recommend sub-3 μm particle-based or monolithic reversed-phase columns for routine use in microcystin and nodularin HPLC analyses.

Development of a Simple Instrumental Setup for the Separation of Benzoic Acids by Comprehensive Liquid Chromatography with Microbore Columns and Monolithic Columns

A simple instrumental setup for the separation of benzoic acid derivatives using comprehensive liquid chromatography is presented. The separation system consists of a 1 mm i.d. microbore anion exchange column coupled to a monolithic reversed phase column, using a simple interface realized by the combination of two conventional sixport valves. Thereby, the eluate from the first dimension could be transferred to the second chromatographic dimension throughout the whole run without any losses. Employing the combination of two different separation mechanisms namely ion exchange and reversed‐phase chromatography allowed the resolution of all test analytes in less than 14 minutes.

Total pore blocking as an alternative method for the on-column determination of the external porosity of packed and monolithic reversed-phase columns

An alternative method to determine the interstitial void volume and the external porosity inside a packed or a monolithic column was developed. The method is based on the total blocking of the mesopores of a porous support by filling them with a hydrophobic solvent. The strong interaction of the latter with the hydrophobic coating inside the pores keeps the solvent in position during the subsequent measurements. With the pores of the stationary phase material completely inaccessible for any type of polar molecules, the method allows to perform interstitial void measurements using small molecular weight (MW) molecules instead of the large MW molecules that need to be used in inverse size exclusion chromatography (ISEC). These small MW molecules are able to penetrate every corner of the interstitial volume and therefore lead to a very accurate determination of the external porosity. Since only one type of molecules needs to be injected, the often troublesome regression analysis needed in ISEC is omitted as well. In the present contribution, the method has been applied to a packed bed and a monolithic column to investigate the optimal conditions of flow velocity, liquid compositions, and unretained marker selection. The robustness and the repeatability of the method are discussed as well.

Evaluation of monolithic and sub 2 µm particle packed columns for the rapid screening for illicit drugs—application to the determination of drug contamination on Irish euro banknotes

A study comparing recently available 100 x 3 mm id, 200 x 3 mm id monolithic reversed-phase columns with a 50 x 2.1 mm id, 1.8 microm particle packed reversed-phase columns was carried out to determine the most efficient approach (using traditional van Deemter analysis and a modern kinetic plot approach) for the rapid screening of samples for 16 illicit drugs and associated metabolites. A plot of column backpressure versus plate number (N) showed a significant advantage of using the monolithic phases, with the 20 cm monolithic column exhibiting a maximum 15,000 plates at a column backpressure of approximately 70 bar, compared to approximately 7000 plates at 150 bar for the 5 cm 1.8 microm particle packed column. Optimum linear velocities were found to be 0.40 mm s(-1), 0.52 mm s(-1) and 0.98 mm s(-1) for the three above columns, respectively. The 20 cm monolithic column was subsequently applied to the separation and determination of illicit drug contamination on Irish euro banknotes, using methanol extraction followed by LC-MS/MS. Method performance data showed that the new LC-MS/MS method was significantly more sensitive than previous GC-MS/MS based methods for this application, with detection limits in the pg note(-1) region, based upon a 20 microL standard injection. All of the notes examined tested positive for trace quantities of cocaine, with benzoylecgonine detected on 12 of the 45 notes sampled. Traces of heroin were also detected on three of the 45 notes.

Determination of phenolic compounds in fennel by HPLC and HPLC–MS using a monolithic reversed-phase column

A reversed-phase high-performance liquid chromatography (HPLC) method for analyzing phenolic compounds in fennel ( Foeniculum vulgare) has been developed. The use of a monolithic column with short dimensions in combination with optimized chromatographic conditions allows over 100 samples per day to be analyzed. Chromatographic parameters such as column temperature and injection volume, were found to be crucial in obtaining adequate selectivity and resolution, consequently allowing short run times. The method was validated for the major phenolic compounds present in fennel plant material: 3- O-caffeoylquinic acid (3-CQA), chlorogenic acid, 4- O-caffeoylquinic acid (4-CQA), eriocitrin, rutin, miquelianin, 1,3- O-dicaffeoylquinic acid (1,3-diCQA), 1,5- O-dicaffeoylquinic acid (1,5-diCQA), 1,4- O-dicaffeoylquinic acid (1,4-diCQA) and rosmarinic acid. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.05 to 1.0 μg/mL and from 0.15 to 2.5 μg/mL, respectively. With some adaptation, the extraction procedure could be even less invasive, which is useful in screening work.

Determination of phenolic compounds in river water with on-line coupling bisphenol A imprinted monolithic precolumn with high performance liquid chromatography

The bisphenol A (BPA) imprinted monolithic precolumn has been prepared by in situ polymerization using 4-vinylpyridine (4-VP) and ethylene dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The column with good flow-through property was obtained by changing the molar ratio of the porogens (toluene and dodecanol). The selectivity and retention properties of the monolith for the BPA and other phenolic compounds were evaluated. The results show that the hydrophobic and hydrogen-bonding interaction plays important roles in the recognition process. The determination of BPA and other phenolic compounds with on-line solid-phase extraction (SPE) by monolithic precolumn coupled with conventional particulates packed and monolithic reversed-phase columns, respectively, was performed. The method was successfully applied to the analysis of phenolic compounds in river water.

Development and optimization of a stability indicating method on a monolithic reversed-phase column for Colchicum dry extract

This article presents a method developed on a monolithic reversed-phase column for separation of related alkaloids and degradation products from colchicine in dry extract preparations from Colchicum seeds. Optimization was performed using an optimization software with variation of the pH of the mobile phase and the percentage of organic modifier. This method permits for the first time a fast separation of all alkaloids including colchiceine without prior derivatization or complexation with excellent linearity, precision and low limits of quantitation which makes it a suitable method for stability testing of extracts or drug formulations containing extracts of Colchicum seeds.

Comparison of the performance of conventional microparticulates and monolithic reversed-phase columns for liquid chromatography separation of eleven pollutant phenols

The performance of isocratic separations of 11 pollutant phenols (PP) using monolithic (Chromolith RP-18e) and conventional reversed-phase 5 μm (Luna and Purospher C 18) and 4 μm (Synergi C 12) particulate size columns, selected from high purity silica materials, has been compared. The separations have been optimized based on a previously optimized separation in which a reversed-phase C 18 Luna column and acetonitrile as organic modifier were used, allowing the separation of all phenols tested in 23 min. The optimization process was carried out for each column by studying the effect of the mobile phase (acetonitrile as organic modifier, pH, flow-rate) on phenols separation. Under the optimized separation conditions, all phenols were separated in less than 23 min for all columns tested. Asymmetry factors were further evaluated and used to estimate column efficiency using the Dorsey–Foley equation. The efficiency and asymmetry factors were lower for Chromolith than for Purospher and Luna columns respectively. The Chromolith column was finally selected, due to its lower flow resistance, analysis time and good efficiency and asymmetry factors. The PPs separation was achieved in 3 min. The asymmetry factors were in the range 0.9–1.5 using 50 mM acetate buffer (pH = 5.25)–ACN (64:36, v/v) as mobile phase, T = 45 °C and 4.0 ml min −1 flow-rate.

Optimized suppression of adducts in polymerase chain reaction products for semi-quantitative SNP genotyping by liquid chromatography-mass spectrometry

While electrospray ionization mass spectrometry has shown great potential for the identification of genotypes in DNA sequences amplified by polymerase chain reaction (PCR), the quantitative determination of allele frequencies remains challenging because of the presence of cationic adducts in the mass spectra which severely impairs accuracy of quantitation. We report here on the elaboration of an optimized desalting protocol for ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS) of PCR amplicons which facilitates the genotyping of single nucleotide polymorphisms (SNPs). Chromatographic purification at temperatures between 50 and 70 degrees C using monolithic reversed-phase columns and acetonitrile gradients in aqueous, 20-30 mmol/l butyldimethylammonium bicarbonate enabled the mass spectrometric analysis of nucleic acid solutions containing up to 1.7 mol/l sodium chloride. A further improvement in removal of metal cations was achieved upon the addition of 5-10 mmol/l ethylenediaminetetraacetic acid (EDTA) to the sample solution prior to liquid chromatography. ICEMS was used for the semi-quantitative genotyping of SNPs amplified from the tetraploid genome of potato cultivars. Using a quadrupole ion trap mass spectrometer, allele frequencies were determined with an accuracy of 2-9% by measuring the relative intensities of the signals corresponding to the molecular mass of each of the alleles in the deconvoluted mass spectra. ICEMS results correlated well with those obtained by pyrosequencing, single nucleotide primer extension, and conventional dideoxy sequencing.

Characterization of the acidity of residual silanol groups in microparticulate and monolithic reversed-phase columns

The residual silanol acidity and activity of several microparticulate and monolithic C18 columns has been measured from the retention of LiNO 3 in the columns with a methanol/buffer (1 mM in Na +) (60:40 v/v) mobile phase buffered to different pH values. For Luna C 18 (2) and LiChrospher RP-18 columns, at least two different types of silanols with different acidity for each packing, were observed. Purospher RP-18e and Chromolith RP-18e packings present evidence of some active silanols only at pH values close to their basic pH stability limit or higher. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MSC 18 and Underivatized XTerra. A modification of an equation previously proposed has been applied to all columns studied and the results obtained have been used to classify the columns according to their silanol acidity and activity. The method allows the prediction of the extent of the silanol activity of the columns studied at a particular mobile phase pH.

Characterization of the acidity of residual silanol groups in microparticulate and monolithic reversed-phase columns*1

The residual silanol acidity and activity of several microparticulate and monolithic C18 columns has been measured from the retention of LiNO3 in the columns with a methanol/buffer (1 mM in Na+) (60:40 v/v) mobile phase buffered to different pH values. For Luna C18 (2) and LiChrospher RP-18 columns, at least two different types of silanols with different acidity for each packing, were observed. Purospher RP-18e and Chromolith RP-18e packings present evidence of some active silanols only at pH values close to their basic pH stability limit or higher. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MSC18 and Underivatized XTerra. A modification of an equation previously proposed has been applied to all columns studied and the results obtained have been used to classify the columns according to their silanol acidity and activity. The method allows the prediction of the extent of the silanol activity of the columns studied at a particular mobile phase pH.

Applications of Monolithic Silica Capillary Columns in Proteomics

The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography-mass spectrometry (LC-MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing very good resolution and reproducibility in addition to the known advantages of a low pressure drop over a time period of 6 months. Monoliths were subsequently tested for their suitability to separate proteins and peptides from samples typically encountered in proteomic research such as in-gel digested tryptic peptide mixtures or fractions of proteolytically digested human serum. The monolithic capillaries also proved useful in the analysis of phospholipid species in bronchoalveolar lavage fluid. Compared to particle-filled conventional capillary columns, rapid and highly efficient separation of peptides and proteins was achieved using these bimodal pore size distribution columns, and good quality collision induced dissociation (CID) mass spectra were obtained on an ion trap mass spectrometer. These novel monolithic separation media are thus a promising addition to the methodological toolbox of proteomics research.

Analysis of ecstasy with a monolithic reversed-phase column

A new, rapid, and precise liquid chromatographic method has been developed for simultaneous identification and quantification of amphetamine,N-methyl-3,4-methylenedioxymetamphetamine,N-ethyl-3,4-methylenedioxymetamphetamine, andN-methyl-1-(3,4-methyl-enedioxyphenyl)-2-butanamine in the presence of other constituents. The compounds were separated on a monolithic column with a gradient prepared from acetonitrile and 20mm monobasic potassium buffer at a flow rate of 1.5 mL min−1. Quantitation was performed with metoclopramide as internal standard. Use of different flow rates was investigated and enabled reduction of the separation time from 11 to 3.5 min for seven substances. The method was then applied to ten seized tablets to identify and quantify their active ingredients.

Comparison of conventional microparticulate and a monolithic reversed-phase column for high-efficiency fast liquid chromatography of basic compounds

The effect of mobile phase flow on column efficiency for a neutral compound together with weak and strong bases was compared for conventional microparticulate (3/3.5 μm and 5 μm) silica RP columns and a monolithic silica RP. For benzene, the minimum plate height (Hmin) at optimum flow-rate (uopt) for weak bases was similar for the 5 μm and the monolith phases. However, the monolith generated much flatter Van Deemter curves, such that at high flow-rate (5 ml min−1) the plate height was nearly 3.5 times lower on the monolith. For weak bases analysed in unbuffered mobile phases, and stronger bases with acid phosphate buffer, increased tailing was obtained on the monolith compared with the conventional phases. Nevertheless, Van Deemter plots on the monolith still showed some advantages over particulate phases, even when asymmetry factor was included in the calculation of the plate height. However, at pH 7 considerable tailing of strong bases was found using the monolith; it is not clear whether this results from unique features of the monolith structure, or whether it is due merely to usual problems of silica activity. Van Deemter plots for conventional phases may be improved considerably by operating the column at elevated temperatures. At pH 3, these improvements are influenced to a considerable extent by increases in Dm, as shown by measurements of Dm using the Taylor–Aris procedure. However, at pH 7.0, improvements are much too substantial to be explainable wholly on this basis.