Fast analysis of soy isoflavones by high-performance liquid chromatography with monolithic columns

A fast method using high-performance liquid chromatography based on two monolithic columns has been developed for the simultaneous determination of isoflavones extracted from soybeans and derived foods. The 12 main isoflavones were resolved in 10 min in two coupled monolithic columns working at 35 °C using a elution gradient of acidified water (0.1% acetic acid) and methanol (0.1% acetic acid) at a flow rate of 5 mL min⁻¹. Retention time and relative area standard deviations were below 1% for all isoflavones. The method developed was successfully applied to several soy food samples and spiked samples. Total isoflavone concentration in sampled soy foods ranged from 34.28 mg L⁻¹ to 4.29 mg g⁻¹.     

Polydimethylsiloxane-functionalized monolithic silica column for reversed-phase capillary liquid chromatography

A polydimethylsiloxane (PDMS)-modified monolithic silica column was prepared for performing reversed-phase capillary liquid chromatography. The prepared PDMS column has a permeability of 6.4×10(-14) m(2) with a plate height <9.2 μm. Alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) were well separated with the PDMS stationary phase, which exhibited similar selectivity and separation mechanism to that of octadecyl stationary phase. The hydrophobic interactions between the analytes and the PDMS stationary phase mainly play the roles for the separation of alkylbenzenes and PAHs. The characteristics of the PDMS column for the separation of alkylbenzenes and PAHs demonstrated that it would be a promising alternative to the octadecyl column.     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.     

Intact-protein trapping columns for proteomic analysis in capillary high-performance liquid chromatography

A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol–gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Monolithic trapping columns were fabricated by entrapping C8 reversed-phase particles into the capillary columns through a sol–gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to 300 bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8 monolithic trapping columns was 99.3% on average. The loading capacity of 5 mm × 320 μm i.d. C8 trapping columns for the protein mixtures was 30 μg. Day-to-day relative standard deviation (RSD) values for recoveries of protein mixtures on the same C8 trapping column ranged from 2.34 to 5.87%, column-to-column RSD values were from 3.01 to 6.81%. The C8 trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.     

Simple and rapid method for cis- and trans-resveratrol and piceid isomers determination in wine by high-performance liquid chromatography using chromolith columns

A simple and rapid HPLC method has been developed for simultaneous determination of the four resveratrol forms (aglycon and glycosidic) in a Grenache wine from Chateauneuf du Pape (Vaucluse). These analyses were achieved by using two commercial monolith HPLC columns and diode array detection. The method provided reliable separations at low pressure with a short analysis time. The limit of detection (LD) and limit of quantification (LQ) were calculated for each standard. The molecules were separated and quantified in a single run without any purification of the sample.     

Electron beam triggered, free radical polymerization-derived monolithic capillary columns for high-performance liquid chromatography

Monolithic capillary columns were prepared via electron beam triggered free radical polymerization within the confines of 0.2 and 0.1mm I.D. capillary columns using ethyl methacrylate and trimethylolpropane triacrylate as monomers as well as 2-propanol, 1-dodecanol and toluene as porogenic system. The influence of column diameter on reproducibility and separation performance was investigated. For evaluation, a protein standard consisting of five proteins in the range of 5800-66,000 g mol(-1) was used. Reproducibility was checked by determining the relative standard deviations in retention times, peak widths at half height, asymmetry and resolution. Excellent run-to-run reproducibility was found for both 0.2 and 0.1mm I.D. columns; batch-to-batch reproducibility was good for both column types. In order to enhance the non-polar character of the monolithic columns, lauryl methacrylate-based capillary columns were prepared. These were successfully used for the separation of proteins and a cytochrome c digest.     

Rapid analysis of pyrethroids in whole urine by high-performance liquid chromatography using a monolithic column and off-line preconcentration in a restricted access material cartridge

A rapid high-performance liquid chromatography (HPLC) method using a monolithic column with UV detection at 238 nm was developed for the determination of fenpropathrin, betacyfluthrin, deltamethrin, and permethrin (cis and trans isomers) in whole urine. The method is based on the use of a monolithic chromatographic column and a restricted access material (RAM) cartridge for sample preparation. The mobile phase was water/acetonitrile (42:58 v/v), the flow rate was 3 mL min^–1, and chromatographic separation was carried out in 10 min. The separation of cis and trans isomers of permethrin was also possible under the above-mentioned conditions. Detection limits in reconstituted whole urine samples were between 0.9 g L^–1 for betacyfluthrin and 4.4 g L^–1 for fenpropathrin and trans-permethrin. Recoveries for urine samples spiked with different amounts of pyrethroids (between 19 g L^–1 and 75 g L^–1) were in the 70±6 to 90±7% range.     

Performance evaluation of long monolithic silica capillary columns in gradient liquid chromatography using peptide mixtures

A systematic study is reported on the performance of long monolithic capillary columns in gradient mode. Using a commercial nano-LC system, reversed-phase peptide separations obtained through UV-detection were conducted. The chromatographic performance, in terms of conditional peak capacity and peak productivity, was investigated for different gradient times (varying between 90 and 1320min) and different column lengths (0.25, 1, 2 and 4m) all originating from a single 4m long column. Peak capacities reaching values up to n=10(3) were measured in case of the 4m long column demonstrating the high potential of these long monoliths for the analysis of complex biological mixtures, amongst others. In addition, it was found that the different column fragments displayed similar flow resistance as well as consistent chromatographic performance in accordance with chromatographic theory indicating that the chromatographic bed of the original 4m long column possessed a structural homogeneity over its entire length.     

Rapid analysis of triazolopyrimidine sulfoanilide herbicides in waters and soils by high-performance liquid chromatography with UV detection using a C18 monolithic column

In this work, the simultaneous analysis of five triazolopyrimidine sulfoanilide herbicides (flumetsulam, florasulam, metosulam, cloransulam-methyl, and diclosulam) by HPLC using UV detection and a C18 monolithic column is proposed. The mobile phase which was composed of ACN, water, and formic acid was pumped at a high flow rate (5 mmL/min) providing an analysis time of all the compounds in less than 2.3 min. The LODs were in the low microg/L range (i.e. between 60 microg/L for flumetsulam and 90 microg/L for florasulam) and the calibration curves showed good linearity (R2 > 0.9949). The method was applied to the analysis of these compounds in spiked mineral and tap waters and soils after an SPE preconcentration procedure using C18 cartridges. Mean recovery values ranged between 35 and 110% for water samples providing LODs of the whole procedure in the low ng/L level, down to 280 ng/L, and between 77 and 92% for soil samples with LODs down to 9.38 microg/kg. This is the first time that this family of pesticides is simultaneously analyzed in both types of samples by HPLC and also using a monolithic column.     

The application of monolithic columns in pesticides analysis

HPLC and HPLC/MS are the most widely used analytical techniques in the field of pesticides analysis. In recent years, there has been considerable focus on fast separations in HPLC in order to reduce analysis time as well as cost. Monolithic columns, consisting of continuous beds with macropores and mesopores, can meet this requirement and have been widely used in the medical and biological fields. However, it has seldom been used when analyzing pesticides. In this work, the application of monolithic columns in pesticides analysis and their advantages are evaluated and compared with those obtained using conventional packed columns.     

Liquid-liquid-liquid phase microextraction of aromatic amines in water using crown ethers by high-performance liquid chromatography with monolithic column

Liquid-liquid-liquid phase microextraction (LLLME) coupled with high-performance liquid chromatography (HPLC) for the analysis of some aromatic amines is described. These compounds were extracted from 4.0 mL aqueous sample that adjusted to pH 13 with, NaOH-NaCl buffer solution (donor phase, P₁) into an organic phase (P₂) 150 μl benzyl alcohol and ethyl acetate (2:1) and then back extracted into a microdrop of aqueous acceptor phase (P₃), adjusted at pH 2, with Na₂HPO₄-H₃PO₄ buffer solution. The extraction time, T₁ (from P₁ to P₂) was 20 min and T₂ (from P₂ to P₃) was 1 min. Different crown ethers as complexing agents for amines were added to the acceptor phase to improve the extraction time. Factors such as organic solvents, extraction times, and addition of crown ethers to acceptor phase and stirring rate were optimised. The method was applied for determination of aromatic amines in wastewater samples. Enrichment factors ranged from 184.5 to 389.7. The linearity range was from 3 to 1000 ng/ml and the detection limits varied from 0.8 to 1.80 ng/ml. Relative standard deviations (%, n = 5) were found (at S/N 3) in the range of 1.9 to 10.1. All experiments were carried out at room temperature, 22 ± 0.5 °C.     

Hydrothermal preparation of hybrid carbon/silica monolithic capillary column for liquid chromatography

A simple, easy and economical approach for the preparation of a hybrid carbon/silica monolithic capillary column was described for the first time by using silica monolith as framework in combination with hydrothermal carbonization at 180°C. During the preparation process, formamide was introduced to the reaction solutions to reduce the dissolution rate of monolithic silica skeleton and its optimal concentration was 1.5 M. Fourier transform infrared spectrometry, scanning electron microscopy, energy dispersive X‐ray spectrometry, and inverse size exclusion chromatography were carried out to characterize the as‐prepared column. The results demonstrated that carbon spheres ranging from 150 to 1000 nm were successfully attached to the surface of silica skeleton. The prepared hybrid carbon/silica column had a permeability of 4.4 × 10^?14 m². Chromatographic performance of the column was evaluated by separation of various compounds including alkylbenzenes, nucleosides and bases, and aromatic acids. The column exhibited an efficiency of 75 000 plates/m for butylbenzene at the optimal linear velocity of 0.23 mm/s. The successful separation of these compounds and the study on mechanism indicated that the column can be applied in mixed‐mode chromatography.     

Preparation and application of hydrophilic monolithic columns

Hydrophilic interaction chromatography (HILIC) has experienced increasing attention in recent years. Much research has been carried out in the area of HILIC separation mechanisms, column techniques and applications. Because of their good permeability, low resistance to mass transfer and easy preparation within capillaries, hydrophilic monolithic columns represent a trend among novel HILIC column techniques. This review attempts to present an overview of the preparation and applications of HILIC monolithic columns carried out in the past decade. The separation mechanism of various hydrophilic monolithic stationary phases is also reviewed.     

Electrically assisted capillary liquid chromatography using a silica monolithic column

A silica monolithic capillary column was linked to an open capillary of the same internal diameter via a Teflon sleeve to form a duplex column to investigate the combination of chromatography and electrophoresis in the mode of electrically assisted capillary liquid chromatography (eCLC). Using a commercial CE instrument with an 8.5 cm long, 100 μm i.d. reversed phase silica monolithic section and a window 1.5 cm beyond the end of this in a 21.5 cm open section, a minimum plate height of 9 μm was obtained in capillary liquid chromatography (CLC) mode at a low driving pressure of 50 psi. In eCLC mode, high speed and high resolution separations of acidic and basic compounds were achieved with selectivity tuning based on the flexible combination of pressure (0–100 psi) and voltage. Taking advantage of the excellent permeability of silica monolithic columns, use of a step flow gradient enabled elution of compounds with different charge state.     

Characterization of plant oils on a monolithic silica column by high-performance liquid chromatography-atmospheric pressure chemical lonization-mass spectrometry

Summary Five plant oils (peanut, pumpkin seed, sesame seed, soybean, and wheat germ) have been analyzed by high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS). Gradient elution was performed with acetone-acetonitrile mobile phases on a short monolithic silica column (SilicaROD, RP-18e, 50 mm×4.6 mm). Identification of plant oil triacylglycerols (TAG) was based on the pseudomolecular ion [M+H]⁺ and the diacylglycerol [M−RCO₂]⁺ fragments. Positional isomers of triacylglycerols were identified from the relative intensities of the [M-RCO₂]⁺ fragments. Principal-component analysis, used to find similarities and differences between the different oils, indicated that the different plant oils could be clearly differentiated according to their triacylglycerol composition. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Determination of the purity of phenoxymethylpenicillin by micellar electrokinetic chromatography and reversed phase liquid chromatography on a monolithic silica column

A micellar electrokinetic chromatographic and a fast reversed‐phase liquid chromatographic method have been developed for determination of the purity of phenoxymethylpenicillin. The optimized running buffer composition was 40 mM phosphate–borate–125 mM SDS–3.5% (v/v) methanol. The HPLC method employed a monolithic silica C18 column and a mobile phase composed of phosphate buffer, pH 3.5, and ACN, the flow‐rate being 3.5 mL/min. Both methods were successfully validated. Linearity, intermediate precision, limits of quantitation, accuracy, and a good correlation of the HPLC and MEKC results were demonstrated. Both methods proved to be fast and reliable and sufficiently sensitive. A combination of the two methods can be very useful in impurity profiling.     

Rapid and simultaneous determination of imidazolium and pyridinium ionic liquid cations by ion-pair chromatography using a monolithic column

A method for rapid and simultaneous determination of imidazolium and pyridinium ionic liquid cations by ion-pair chromatography with ultraviolet detection was developed.Chromatographic separations were performed on a reversed-phase silica-based monolithic column using 1-heptanesulfonic acid sodium-acetonitrile as mobile phase.The effects of ion-pair reagent and acetonitrile concentration on retention of the cations were investigated.The retention times of the cations accord with carbon number rule.The method has been successfully applied to the determination of four ionic liquids synthesized by organic chemistry laboratory.     

Selectivity comparisons of monolithic silica capillary columns modified with poly(octadecyl methacrylate) and octadecyl moieties for halogenated compounds in reversed-phase liquid chromatography

Stationary phase selectivities for halogenated compounds in reversed-phase HPLC were compared using C18 monolithic silica capillary columns modified with poly(octadecyl methacrylate) (ODM) and octadecyl moieties (ODS). The preferential retention of halogenated benzenes on ODM was observed in methanol/water and acetonitrile/water mobile phases. In selectivity comparison of selected analytes on ODM and ODS, greater selectivities for halogenated compounds were obtained with respect to alkylbenzenes on an ODM column, while similar selectivities were observed with a homologous series of alkylbenzenes on ODM and ODS columns. These data can be explained by greater dispersive interactions by more densely packed octadecyl groups on the ODM polymer coated column together with the contribution of carbonyl groups in ODM side chains. For the positional isomeric separation of dihalogenated benzenes (ortho-, meta-, para-), the ODM column also provided better separation of these isomers for the adjacently eluted isomers that cannot be completely separated on an ODS column in the same mobile phase. These results imply that the ODM column can be used as a better alternative to the ODS column for the separation of other halogenated compounds.     

Purification of genomic DNA by short monolithic columns

The isolation and purification of nucleic acids is essential for many procedures in molecular biology. After showing that bacterial and eukaryotic genomic DNA can be specifically bound to the CIM DEAE monolithic column, this characteristic was exploited in development of a simple and fast chromatographic procedure for isolation and purification of genomic DNA from cell lysates that does not include the usage of toxic organic solutions. The purity and the quality of the isolate as well as the duration of the procedure was similar to other chromatographic methods used today for isolation of genomic DNA, but the initial sample volume was not restricted.     

Fast high-performance liquid chromatographic analysis of mianserin and its metabolites in human plasma using monolithic silica column and solid phase extraction

A fast high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of mianserin (MIAN) and its metabolites desmethylmianserin (DMM), 8-hydroxymianserin (HM) and mianserin-N-oxide (MNO) in human plasma. Each compound, together with internal standard (propranolol) was extracted from the plasma matrix using solid phase extraction. Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column ( mm i.d.) under isocratic conditions using a mobile phase of 74:26 (v/v) 25 mM phosphate buffer (pH 5.3 adjusted with phosphoric acid): acetonitrile. The elution of the analytes were monitored at 292 mm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 3.5 ml/min. The total run time of the assay was 5 min. The method was validated over the range of 10-200 ng/ml for MIAN, 10-150 ng/ml for DMM, 20-300 ng/ml for HM and 25-500 ng/ml for MNO. The method proved to be precise (within-run precision ranging from 1.6 to 6.9% R.S.D. and between-run precision ranging from 1.3 to 7.2% R.S.D.) and accurate (within-run accuracies ranged from 1.4 to 6.4% and between-run accuracies ranging from 1.5 to 4.5%). The mean absolute recoveries were 95.7, 94.8, 99.6, and 102.6% for MIAN, DMM, HM and MNO, respectively. The limit of quantitation (LOQ) for MIAN and DMM was 10 ng/ml and for HM and MNO were 20 and 25 ng/ml in human plasma, respectively. The limit of detection (LOD) for MIAN, DMM, HM and MNO was 5, 2.5, 10 and 15 ng/ml, respectively. The described method demonstrates the feasibility for employing monolithic columns to effect rapid bioanalytical HPLC analysis for the quantitative determination of MIAN and its major metabolites in human plasma.