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 microm and 5 microm) silica RP columns and a monolithic silica RP. For benzene, the minimum plate height (Hmin) at optimum flow-rate (mu(opt)) for weak bases was similar for the 5 microm 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.     

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.     

Study of triacontyl-functionalized monolithic silica capillary column for reversed-phase capillary liquid chromatography

A novel stationary phase triacontyl-functionalized monolithic silica capillary column was successfully prepared for reversed-phase capillary liquid chromatography. The performance of the monolithic silica capillary column coated with triacontyl chain for the separation of alkylbenzenes, xylene isomers, polycyclic aromatic hydrocarbons, and mixture of α- and β-carotenes was studied, which was compared to that using the monolithic silica capillary column coated with octadecyl chain. The comparison results showed that triacontyl-functionalized monolithic silica capillary column would be a promising media to be used for the separation of isomeric solutes with long chain in reversed-phase capillary liquid chromatography.     

Preparation and characterization of lauryl methacrylate-based monolithic microbore column for reversed-phase liquid chromatography

Poly(lauryl methacrylate-co-ethylene dimethacrylate) monoliths were in situ synthesized within the confines of a silicosteel tubing of 1.02 mm i.d. and 1/16" o.d. for microbore reversed-phase HPLC. In order to obtain practically useful monoliths with adequate column efficiency, low flow resistance, and good mechanical strength, some parameters such as total monomer concentration (%T), cross-linking degree (%C) and polymerization temperature were optimized. High-efficiency monoliths were successfully obtained by thermal polymerization of a monomer mixture (40%T, 10%C) with a binary porogenic solvent consisting of 1-propanol and 1,4-butandiol (7:4, v/v) at a high temperature of 90 °C. The morphology and porous structure of the resulting monoliths were assessed by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC), while the column performance was evaluated through the separations of a series of alkylbenzenes in acetonitrile-water (50:50, v/v) eluent. At a normal flow rate of 50 μL/min (corresponding to 1.66 mm/s), the optimized monolithic columns typically exhibited theoretical plate numbers of 6000 plates/10 cm-long column for amylbenzene (k>40), and the pressure drop was always less than 1 MPa/10 cm. The monoliths, which were chemically anchored to the tube inner wall surface using a bifunctional silylation agent, exhibited adequate mechanical strength of up to 12-13 MPa, and were properly operated at 10 times higher flow rate than normal, reducing the separation time to one tenth. The lauryl methacrylate-based monolithic column was applied to a rapid and efficient separation of ten common proteins such as aprotinin, ribonuclease A, insulin, cytochrome c, trypsin, transferrin, conalbumin, myoglobin, β-amylase, and ovalbumin in the precipitation-redissolution mode. Using a linear CH(3)CN gradient elution at a flow rate of 500 μL/min (10-times higher flow rate), 10 proteins were baseline separated within 2 min.     

Comparison of reversed-phase column materials for high-performance liquid chromatography of proteins

Nine reversed-phase materials with various bonded phases from different suppliers were studied for the separation of hydrophilic proteins with two solvent systems. Protein retention, resolution and recovery were not correlated with the nature of the hydrocarbonaceous ligand. Peak volumes increased with molecular weight, which led to broad, irregular peaks for the larger proteins on some columns. Four columns that performed equally well were selected for the purification of hydrophobic Sendai virus membrane proteins. In this case, more distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent systems.     

Comparison of high-performance liquid chromatography separation of red wine anthocyanins on a mixed-mode ion-exchange reversed-phase and on a reversed-phase column

Anthocyanins, which confer the characteristic color to red wine, can be used as markers to classify wines according to the grape variety. It is a complex separation that requires very high chromatographic efficiency, especially in the case of aged red wines, due to the formation of pyranoanthocyanins. A coelution between these kinds of compounds can affect the R_ac/coum ratio of aged wines, and might lead to false results when classifying the wine variety. In 2007, the use of a novel mixed-mode ion-exchange reversed-phase column was reported to separate anthocyanins extracted from grapes of Vitis labrusca with different selectivity than C-18 columns. In the present work, the separation of anthocyanins including pyranoanthocyanins in young and aged Cabernet Sauvignon wines and other varieties is evaluated. The most interesting contributions of this research are the different elution order and selectivity obtained for anthocyanins and pyranoanthocyanins (only formed in wine), compared with those observed in C-18 stationary phases. Also interesting is the separation of the polymeric fraction, which elutes as a clearly separated peak at the chromatogram's end. However, a comparison with a high efficiency C-18 column with the same dimensions and particle size demonstrated that the tested mixed-mode column shows broader peaks with a theoretical plate number below 8000, for malvidin-3-glucoside peak, while it can be up to 10 times higher for a high efficiency C-18 column, depending on the column manufacturer. Under the tested conditions, in mixed-mode phase, the analysis time is almost twice that of a C-18 column with the same dimensions and particle size. A mixed-mode phase with increased efficiency should provide an interesting perspective for separation of anthocyanins in wine, due to its improved selectivity, combined with a useful role in a second-dimension separation in preparative anthocyanin chromatography.     

High-performance liquid chromatography analysis of polyacetylenes and polyenes in Echinacea pallida by using a monolithic reversed-phase silica column

In this study, a RP-HPLC method for the analysis of polyacetylenes and polyenes in Echinacea pallida roots and phytopharmaceuticals was developed. The reference compounds used for quantification were isolated from the plant material and their structures were determined on the basis of the analysis of UV, IR, NMR and MS data. The complete structure elucidation of three compounds, namely 8-hydroxy-tetradec-(9E)-ene-11,13-diyn-2-one (1), tetradec-(8Z)-ene-11,13-diyn-2-one (6) and pentadec-(8Z)-en-2-one (9) is described. In the analysis of the n-hexane extracts of E. pallida roots, the comparison between conventional and monolithic columns showed that the elution order in both cases is identical and the selectivity is equivalent. However, the retention times achieved by the monolithic column are shorter, resulting in a faster separation (20 min). Therefore, the analyses were carried out on a Chromolith Performance RP-18e (100 mm x 4.6 mm i.d.), with a gradient mobile phase composed by H(2)O and ACN at the flow rate of 2 mL/min. The column was thermostatted at 20 degrees C. The photodiode array detector monitored the eluent at 210 nm. The validation procedure confirmed that this technique affords reliable analysis of these components and is appropriate for the quality control of complex matrices, such as E. pallida roots and phytopharmaceuticals.     

Control of column temperature in reversed-phase liquid chromatography

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

Retention equilibrium and mass transfer characteristics in reversed-phase liquid chromatography using methanol-water mixtures

Pulse response experiments (i.e., elution chromatography) were made in reversed-phase liquid chromatography (RPLC) using a C18 silica gel column and methanol-water mixtures of different compositions (phi). The moment analysis of the elution peak profiles measured in the RPLC system provided some items of information about four parameters characterizing the retention equilibrium and the mass transfer kinetics in the column, i.e., adsorption equilibrium constant, isosteric heat of adsorption, surface diffusion coefficient and activation energy of surface diffusion. Characteristics of the chromatographic behavior were studied by analyzing the dependence of the four parameters on phi and the correlation between them. It was found that surface diffusion was one of the important processes of molecular migration having a significant contribution to the mass transfer kinetics in the column. Both the adsorption equilibrium constant and the surface diffusion coefficient varied depending on phi. The direction of their changes was approximately opposite, suggesting that the mass transfer in the manner of surface diffusion was restricted owing to the retention of the sample molecules on the stationary phase.     

Capillary monolithic titania column for miniaturized liquid chromatography and extraction of organo-phosphorous compounds

A new sol?Cgel protocol was designed and optimized to produce titanium-dioxide-based columns within confined geometries such as monolithic capillary columns and porous-layer open-tubular columns. A surface pre-treatment of the capillary enabled an efficient anchorage of the monolith to the silica capillary wall during the synthesis. The monolith was further synthesized from a solution containing titanium n-propoxide, hydrochloric acid, N-methylformamide, water, and poly(ethylene oxide) as pore template. The chromatographic application of capillary titania-based columns was demonstrated with the separation of a set of phosphorylated nucleotides as probe molecules using aqueous normal-phase liquid chromatography conditions. Capillary titania monoliths offered a compromise between the high permeability and the important loading capacity needed to potentially achieve miniaturized sample preparations. The specificity of the miniaturized titania monolithic support is illustrated with the specific enrichment of 5??-adenosine mono-phosphate. The monolithic column offered a ten times higher loading capacity of 5??-adenosine mono-phosphate compared with that of the capillary titania porous-layer open-tubular geometry.     

Comparison of column properties in reversed-phase chromatography: monolithic, cholesterolic and mixed bonded stationary phases

A commercial Chromolith C18 column and two new stationary phases with mixed ligands bonded on the Kromasil silica gel support, SG-MIX and SG-Chol, were characterized using simple tests based on the retention of non-polar, basic and acidic compounds. Polar and methylene selectivity tests in acetonitrile-water and methanol-water mobile phases revealed lower hydrophobicities of the SG-MIX and SG-Chol columns in comparison to the Chromolith column. The columns were further characterized using new test criteria - gradient oligomer capacity and isomeric selectivity and peak symmetry of naphthalene di-sulphonic acids in aqueous mobile phases. The cholesterolic column shows greater gradient oligomer selectivity for the separation of oligoethylene glycol samples than the SG-MIX and the Chromolith columns. Increased retention and peak tailing, but decreased isomeric selectivity for naphthalene-di-sulphonic acids was observed with the SG-MIX column, because of interactions with various polar bonded groups.     

Comparison of the performance of a few packing materials designed to minimize the thermodynamic band tailing of basic compounds in reversed-phase liquid chromatography

The adsorption isotherms of phenol, caffeine, propranolol chloride, and amitriptyline chloride were measured on three new brands of C(18)-bonded silica that have been designed to be more resistant than conventional C18-bonded silica at high pHs (>8). These columns were the 4 microm Bidendate Cogent-C18 (Microsolv Technology Corporation, Long Branch, NJ, USA), the 3.5 microm Zorbax Extend-C18 (Agilent Technologies, Palo Alto, CA, USA), and the 5 microm XTerra-C18 (Waters, Milford, MA, USA). The originality of these adsorbents is due to their surface chemistry, which protects them from rapid hydrolysis or dissolution at extreme pH conditions. Their adsorption properties were compared to those of the 3 microm Luna-C18 (Phenomenex, Torrance, CA), which is a more conventional monofunctional material. The adsorption data were acquired by frontal analysis (FA) and the adsorption energy distributions (AEDs) of all systems studied were calculated by the expectation-maximization (EM) method. The experimental results show that neither a simple surface protection (Extend-C18) nor the elimination of most of the silanol groups (Cogent-C18) is sufficient to avoid a peak tailing of the basic compounds at pH 8 that is of thermodynamic origin. The incorporation of organic moieties in the silica matrix, which was achieved in XTerra-C18, the first generation of hybrid methyl/silica material, reduces the silanols activity and is more successful in reducing this peak tailing.     

High-speed liquid chromatography/tandem mass spectrometry using a monolithic column for high-throughput bioanalysis

With the ever-increasing workload from a variety of in vitro and in vivo screening procedures, new analytical methodologies to perform bioanalysis in an accurate and high-throughput manner are in great demand. In this work, monolithic columns were used instead of conventional particulate HPLC columns to perform chromatographic separations. Because the pressure drop on a monolithic column was considerably lower than that on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 x 50 mm monolithic column with a total backpressure of about 61 bar measured using acetonitrile/water (50:50). The capability of using a regular column length at high flow rates, combined with the extremely small dependency of separation efficiency on linear flow velocity, allowed for the generation of sufficient chromatographic resolving power in a significantly reduced runtime. As demonstrated in this work, a plasma extract of a mixture of tempazepam, tamoxifen, fenfluramine, and alprozolam were baseline separated within a total analysis time of one minute. An average peak width at half maximum of approximately one second was noted using a generic broad gradient. It was also found that the separation efficiency and signal/noise (S/N) ratios for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min, respectively. The ruggedness of the separation was evaluated by injecting 600 plasma extracts containing the replicates of a standard curve of the above mixture during an overnight run. The chromatographic retention time, separation quality, peak response and sensitivity were highly reproducible throughout the run. This high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been used routinely in the authors' laboratory to support drug discovery programs.     

Comparison of the adsorption mechanisms of pyridine in hydrophilic interaction chromatography and in reversed-phase aqueous liquid chromatography

The adsorption isotherms of pyridine were measured by frontal analysis (FA) on a column packed with shell particles of neat porous silica (Halo), using water–acetonitrile mixtures as the mobile phase at 295 K. The isotherm data were measured for pyridine concentrations covering a dynamic range of four millions. The degree of heterogeneity of the surface was characterized by the adsorption energy distribution (AED) function calculated from the raw adsorption data, using the expectation-maximization (EM) procedure. The results showed that two different retention mechanisms dominate in Per aqueous liquid chromatography (PALC) at low acetonitrile concentrations and in hydrophilic interaction chromatography (HILIC) at high acetonitrile concentrations. In the PALC mode, the adsorption mechanism of pyridine on the silica surface is controlled by hydrophobic interactions that take place on very few and ultra-active adsorption sites, which might be pores on the irregular and rugose surface of the porous silica particles. The surface is seriously heterogeneous, with up to five distinct adsorption sites and five different energy peaks on the AED of the packing material. In contrast, in the HILIC mode, the adsorption behavior is quasi-homogeneous and pyridine retention is governed by its adsorption onto free silanol groups. For intermediate mobile phase compositions, the siloxane and the silanol groups are both significantly saturated with acetonitrile and water, respectively, causing a minimum of the retention factor of pyridine on the Halo column.     

Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.     

反相硅胶整体柱离子色谱法快速分析BrO-3

研究了基于反相硅胶整体柱的离子色谱快速分析BrO;的方法.以氢氧化四丁铵(TBA)为流动相动态修饰反相硅胶整体柱,采用直接电导检测,在普通高效液相色谱仪上实现了BrO3-和常见阴离子的快速分析.实验考察并讨论了流动相、色谱柱温度和流速对所测阴离子保留和分离的影响.确定分离测定BrO3-的最佳色谱条件是:以0.75 mm...     

The influence of wettability on the equilibrium wall flow of liquid in a packed column

It is well known that when a packed column is irrigated by a liquid and is operated in the trickle flow regime, part of the liquid flows preferentially along the column wall. The liquid distribution reaches equilibrium if the column is tall enough. In the present study the equilibrium wall flow in a column packed with rasching rings was studied experimentally. The effects of the wettability of the wall and the packing, and the total liquid flow rate on the equilibrium wall flow were investigated. It was found that the equilibrium wall flow depends strongly on the wettability of the wall, but is almost independent of the wettability of the packing. These results were used to gain some insight into the mechanism of the development of wall flow in packed columns.     

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.     

Comparison of provitamin A determination by normal-phase gravity-flow column chromatography and reversed-phase high performance liquid chromatography

Summary The provitamin A content of some food samples was determined by methods involving MgO: Hyflosupercel gravityflow column chromatography (GFCC) and reversed phase high performance liquid chromatography (HPLC), the quantitation being done by external standardization (HPLC-ES) or internal standardization (HPLC-IS) with Sudan. The results obtained with - and -carotene in carrots, -carotene and -cryptoxanthin in papaya and -carotene in tomato and kale agreed well, showing that any of the these techniques can be used, provided the analysis is done under optimum conditions. Good separation of the different provitamins using GFCC depends on the analyst's skill and visual acuity. HPLC-ES required a constant supply of provitamin standards, thus the varying purity of commercially available standards and the high instability of these compounds could pose grave problems. Due to the stability of Sudan, HPLC-IS appeared to be the method of choice although passage of the extract through a MgO: Hyflosupercel minicolumn was required prior to injection to separate chlorophylls, dihydroxy- and polyoxycarotenoids which would otherwise elute with Sudan. Nonconformity of the Sudan structure to those of the provitamins did not effect the quantitative results. The chromatographic separation, identity and quantification of the provitamins could be more easily established by using HPLC-IS, complemented with GFCC.