Comparison of the performance of different reversed-phase columns for liquid chromatography separation of 11 pollutant phenols

A systematic optimization of the HPLC separation of a mixture containing 11 pollutant phenols (PPs) using a Hypersil ODS (250 mm x 4.6 mm, 5 microm) column and UV-DAD detection has been carried out. The binary mobile phases used were obtained by mixing 50 mM phosphate (pH = 3.0) and methanol, ACN, or THF as organic modifiers. After selecting ACN as an organic modifier, the effects of pH and temperature on PPs separation were studied. A mobile phase of 50 mM acetate (pH = 5.0)-ACN (60:40 v/v) at 50 degrees C allowed the separation of 11 phenols but not to baseline in 17 min. To improve the performance of this separation, the following RP columns were tested: Luna C18 (2), Purospher C18, Synergi C12, Synergi Fusion C18, Gemini C18, Luna Cyano, Lichrospher C8, and Envirosep-PP (polymeric). In all the cases, the performance (analysis time, retention, selectivity, resolution, asymmetry factors, and efficiency) was evaluated. A further reoptimization of the mobile phase was carried out for all the columns by studying the ACN content and pH, with the aim of improving the above-mentioned separations and selecting the most suitable one for PPs analysis.     

An assessment of the retention behaviour of polycyclic aromatic hydrocarbons on reversed phase stationary phases--thermodynamic behaviour on C18 and phenyl-type surfaces

The thermodynamic retention behaviour of a linear series of polycyclic aromatic hydrocarbons (PAHs) was investigated on C18 and selected phenyl-type reversed-phase stationary phases, namely C18, C18 Aqua, Propyl-phenyl and Synergi polar-RP stationary phases, using methanol mobile phases. The Propyl-phenyl stationary phase, despite having the lowest surface coverage, was found to exhibit significantly larger enthalpic interactions to the other Phenyl-type phase (Synergi polar-RP) even though this had a much higher surface coverage. This indicated that stronger interactions between the PAHs and the stationary phase ligands were occurring on the Propyl-phenyl phase. Evaluation of the elution band profile of the PAHs in the aqueous methanol mobile phase revealed fairly symmetrical bands for the C18, C18 Aqua and Synergi polar-RP, but severe peak tailing on the Propyl-phenyl phase. A change in mobile phase from methanol to acetonitrile improved the peak shape of the PAHs on the Propyl-phenyl phase, leading to the assumption that unfavourable pi-pi interactions were occurring between the electron-rich PAHs and the electron-rich phenyl rings of the Propyl-phenyl phase.     

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 microm (Luna and Purospher C18) and 4 microm (Synergi C12) 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 C18 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 50mM acetate buffer (pH = 5.25)-ACN (64:36, v/v) as mobile phase, T=45 degrees C and 4.0 ml min(-1) flow-rate.     

液相色谱-质谱法分析黑碳的分子标志物——苯多羧酸和硝基苯多羧酸

苯多羧酸法是定量检测黑碳和溶解态黑碳的重要分子标志物法,其中苯多羧酸和硝基苯多羧酸的分离和定量是关键环节。本文建立了苯多羧酸和硝基苯多羧酸的液相色谱-质谱分析方法,利用质谱的定性能力解决了部分苯多羧酸和硝基苯多羧酸化合物在缺乏标准品情况下的定性问题。用Phenomenex Synergi Polar RP分离柱,柱温为35 ℃,流动相为0.5%(体积分数)甲酸水溶液和甲醇,流速为0.5 mL/min,梯度洗脱,以电喷雾离子源负离子全扫描和选择扫描模式进行离子阱质谱检测,同时利用串联的二极管阵列检测器采集三维紫外光谱数据。在实际样品检测中,14个含3个及以上羧基的苯多羧酸和硝基苯多羧酸化合物获得良好分离。此方法简化并加快了苯多羧酸法定量黑碳和溶解态黑碳的分析过程,可满足环境样品中黑碳和溶解态黑碳的分析,促进了苯多羧酸法应用的普及。     

Determination of the porosities of monolithic columns by inverse size-exclusion chromatography

Retention data of polystyrene samples of narrow molecular size distribution and known average molecular mass were measured on several monolithic columns (Chromolith Performance, Merck) and one conventional packed column (Luna C18, Phenomenex) by size-exclusion chromatography. These data were used to determine the external, the internal, and the total porosities of these columns. These data provided also information on the pore-size distribution of the adsorbent medium. The external and the total porosities of these columns are much higher than those of conventional packed columns. The results illustrate the profound changes brought by monolithic columns to the balance of the hydrodynamic and the mass transfer kinetic properties of chromatographic columns. Classical methods of comparison between column performance must be re-evaluated.     

Simultaneous quantification of Panax and Epimedium species using Rapid Resolution Liquid Chromatography (RRLC)

Two Rapid Resolution Liquid Chromatography (RRLC) methods have been developed and validated for simultaneous quantification of eight major ginsenosides from Panax species, namely, R1, Rg1, Re, Rf, Rb1, Rb2, Rc, and Rd, and flavonoids from Epimedium species, namely, epimedins A, B, and C and icariin. The analyses were performed using an Agilent 1200 series RRLC system with Phenomenex Luna C18-HST and Zorbax Eclipse XDB columns. The separation was performed with a gradient mobile phase of A (pure water) and B (acetonitrile) at a flow rate of 1.0 mL/min and 2.5 mL/min, respectively. Both columns were kept at 40 degrees C with the detection wavelength set at 203 nm. Specific eluted compounds were identified by using reference samples of ginsenosides R1, Rg1, Re, Rf, Rb1, Rc, Rb2, and Rd, and epimedins A, B, C and icariin. Baseline separation was achieved in less than 15 minutes for the Phenomenex Luna column and 4 minutes for the Zorbax Eclipse column. Characteristic RRLC profiles were established for complex mixtures of ginsenosides from Panax species and flavonoids from Epimedium species. Both methods developed here are effective for the quality control of formulated products containing both Panax and Epimedium varieties.     

Development of capillary column packed with thiol-modified gold-coated polystyrene particles and its selectivity for aromatic compounds

Three types of thiol compounds (n-octadecanethiol, thiophenol, and 2-phenylethanethiol) were used to modify the gold-coated polystyrene particles (dp. 5microm) to prepare a stationary phase for capillary liquid chromatography through the formation of self-assembled monolayer. The column with n-octadecanethiol-modified gold-coated polystyrene particles (C18-Au) demonstrated the higher affinity to phenanthrene and anthracene than small aromatics compared to the ODS column. In addition, the shape selectivity between phenanthrene and anthracene in the C18-Au column was much higher than that in the ODS column (separation factors: 1.82 and 1.14, respectively). The relationship between the retention factor and acetonitrile content in the mobile phase revealed that the retention behaviors in the C18-Au column was more sensitive on the acetonitrile content than those in the ODS column. Relatively higher affinity for phenanthrene and anthracene was commonly observed in all the three thiol-modified Au columns than that for the conventional ODS column, whereas separations of benzene and nitro- and chlorobenzenes were quite different among the three thiol-modified Au and ODS columns.     

Identification of the factors that influence the reproducibility of chromatographic retention data

Principal component analysis was used to identify the parameters that influence the column-to-column and batch-to-batch reproducibility of retention times and retention factors measured on Symmetry C18, Kromasil C18, Luna C18 (2) and Vydac RP C18, all reversed-phase silica columns. We devised a procedure that allows the determination of the differences in column volume and packing density between two columns, provided that these columns are packed with identical stationary phases (i.e., phases that originate from the same batch). Principal component analysis of the retention times confirmed that the column-to-column variations of the column volume and the total porosity of the bed are the factors that influence the reproducibility of the retention times, the column volume being the major factor. For the fluctuations of the retention factors, the column phase ratios (or the bed porosities) and some specific, secondary retention mechanisms are responsible. All the C18 columns investigated proved to behave in a very similar fashion. Two principal components were always sufficient to characterize the variations of either the retention times or the retention factors.     

Behavior of packing materials in axially compressed chromatographic columns

The behavior of a packing material (Luna C18 from Phenomenex, Torrance, CA, USA) was studied during the consolidation of a column bed under axial compression stress. The kinetics of this consolidation, the permeability and efficiency of the columns obtained, and the reproducibility of these column properties were measured under different conditions. The consolidation process and the column properties are considerably affected by the friction between the packing material in the bed and the column wall. Clear evidence of this wall effect was demonstrated. The apparent permeability of columns consolidated under the same axial stress increases with increasing column length. The apparent modulus of elasticity of the beds increases with increasing column length. The shear resistance between the packed bed and the column wall was measured for columns of different lengths. It increases rapidly with increasing bed length. The column efficiency for thiourea (unretained) and phenyloctane (retention factor, k' approximately 1) was much poorer after recompression than after the first compression. It depended little on the compression stress. The effect of the column length was small.     

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.     

Characterization of the acidity of residual silanol groups in immobilized artificial membranes

Residual silanol acidity and activity of one immobilized artificial membrane (IAM) column have been measured from the retention of LiNO(3) in the column with a methanol/buffer (1mM in Na(+)) (60:40, v/v) mobile phase buffered to different pH values. Just one type of silanol with pssK(a)=7.61 (near the pH limit recommended by the manufacturer) was found, although these silanols show large activity. The results obtained have been compared with those obtained previously for Resolve C18, Resolve Silica, Symmetry C18, Symmetry Silica, XTerra MS C18, underivatized XTerra, Lichrospher 100 RP-18, Purospher RP-18e, Luna C18 (2) and Chromolith Perfomance RP-18e, showing that the IAM column is similar to Luna C18 and Symmetry C18 in terms of silica quality, as measured by Li(+) retention. A warning about the use of IAM columns for emulation of biological systems at physiological pH 7 is given because the ionized silanols may contribute to the retention of some drugs at this pH.     

Repeatability in column preparation of a reversed-phase C18 monolith and its application to separation of tocopherol homologues

This work investigated the repeatability of column preparation for a reversed-phase C18 monolith, namely stearyl methacrylate-co-ethylene glycol dimethacrylate (SMA-EDMA). The columns were thermally polymerised using three commonly available heating devices (GC oven, hot air oven and water bath) and their chromatographic performance evaluated using micro-liquid chromatography for separation of five test compounds. Precision in terms of %RSD of retention times were 9.0, 6.5, and 12.5 using GC oven, hot air oven and water bath, respectively. Between-batch precision for the hot air oven (n = 3 days) was less than 10.4% for retention time. The SMA-EDMA monolith was applied to the separation of tocopherol homologues by capillary electrochromatography. Usually tocopherol homologues cannot be completely separated by conventional reversed-phase C8- or C18-packed bed or C18-silica based monolithic columns. Polymer monolith has been shown to give remarkable selectivity towards the tocopherols compared to the conventional microparticulate phase and silica based monolith. Successful separation of the tocopherol isomers was achieved on the SMA-EDMA monolith without any column modification.     

An isocratic fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children receiving E. colil-asparaginase for the treatment of acute lymphoblastic leukaemia

A novel assay for the determination of l-asparaginase activity in human plasma is described that is based on the HPLC quantitation of l-aspartic acid produced during enzyme incubation. Methods for monitoring l-asparagine depletion are also described. Chromatography of l-aspartic acid, l-asparagine and l-homoserine (the internal standard) involved derivatization with o-pthaldialdehyde, then separation from other amino acids on a Phenomenex Luna C(18) column using a 1 mL/min flow rate and a mobile phase consisting of di-potassium hydrogen orthophosphate propionate buffer, pH 6, with 10% methanol and 10% acetonitrile. Fluoresence detection was at excitation/emission wavelengths of 357/455 nm. Under these conditions l-aspartic acid, l-asparagine and l-homoserine had retention times of 3.5, 9.8 and 17.7 min, respectively. The l-asparaginase assay was linear from 0.1 to 10 U/mL activity and interday precision and accuracy were less than 13%. The limit of quantitation was approximately 0.03 U/mL. The assay utility was established in 12 children who received E. coli l-asparaginase as treatment for acute lymphoblastic leukaemia.     

Comparison between the loading capacities of columns packed with partially and totally porous fine particles. What is the effective surface area available for adsorption?

The adsorption isotherms of phenol, caffeine, insulin, and lysozyme were measured on two C(18)-bonded silica columns. The first one was packed with classical totally porous particles (3 microm Luna(2)-C(18)from Phenomenex, Torrance, CA, USA), the second one with shell particles (2.7 microm Halo-C(18) from Advanced Materials Technology, Wilmington, DE, USA). The measurements were made at room temperature (T=295+/-1K), using mainly frontal analysis (FA) and also elution by characteristic points (FACP) when necessary. The adsorption energy distributions (AEDs) were estimated by the iterative numerical expectation-maximization (EM) procedure and served to justify the choice of the best adsorption isotherm model for each compound. The best isotherm parameters were derived from either the best fit of the experimental data to a multi-Langmuir isotherm model (MLRA) or from the AED results (equilibrium constants and saturation capacities), when the convergence of the EM program was achieved. The experiments show than the loading capacity of the Luna column is more than twice that of the Halo column for low-molecular-weight compounds. This result was expected; it is in good agreement with the values of the accessible surface area of these two materials, which were calculated from the pore size volume distributions. The pore size volume distributions are validated by the excellent agreement between the calculated and measured exclusion volumes of polystyrene standards by inverse size exclusion chromatography (ISEC). In contrast, the loading capacity ratio of the two columns is 1.5 or less with insulin and lysozyme. This is due to a significant exclusion of these two proteins from the internal pore volumes of the two packing materials. This result raises the problem of the determination of the effective surface area of the packing material, particularly in the case of proteins. This area is about 40 and 30% of the total surface area for insulin and for lysozyme, respectively, based on the pore size volume distribution validated by the ISEC method. The ISEC experiments showed that the largest and the smallest mesopores have rather a cylindrical and a spherical shape, respectively, for both packing materials.     

Accessible silanol sites - beneficial for the RP-HPLC separation of constitutional and diastereomeric azaspirovesamicol isomers

Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C(18) and special end-capped C(8) and C(18) phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20mM NH(4)OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20mM NH(4)OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH(4)OAc. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism.     

Development of a quantitative high-performance liquid chromatography-photodiode array detection measurement system for phenolic acids

A quantitative high-performance liquid chromatography-photodiode array detection method separating 16 phenolic acids was achieved. Six columns and several mobile phases were investigated. Resolution was achieved with a high-purity silica Phenomenex Luna C18 column (150 mm x 4.6 mm, 5 microm) and a binary gradient consisting of CH3OH-water (with 0.1% formic acid) and flow rate set at 0.7 ml/min. Acids were detected and quantitation performed at wavelength representing the lowest energy lambda(max) for individual acids. Extraction procedure from wine was optimized and yields ranged from 79 to 87% based on internal standard recovery. To confirm our quantitative results, identical samples were analyzed both in-house and by a collaborating laboratory. Correlation of two data sets generated linear regression equations that approached unity (0.93-0.98) and R2 values ranging from 0.990 to 0.999.     

Quantitative screening for steroids in animal feeding water using reversed phase LC with gradient elution

Several isocratic separations for the determination of 20 steroids (STER) in animal feeding water samples (AFWS) from drinking-trough by LC using a mobile phase ACN/H(2)O (35:65 v/v) and different RP columns (Hypersil C18, Gemini C18 (GM), Purospher Star C18, Synergi Max C12, and Synergi Fusion) and UV detection were obtained. The elution order was the same: a first group of corticoids (CC) was early eluted, a second group of CC and anabolics (AAS) exhibited intermediate retention, and a third group constituted by AAS was strongly retained. To improve the separation performances of the isocratic separations an ACN gradient elution optimization was carried out for each column. The most satisfactory results were obtained using a Purospher column which allowed the separation of 19 STER in an analysis time close to 26 min. After sample preparation using SPE, method validation was performed in an AFWS spiked with STER according to the EC decision criteria established for quantitative screening methods. For this purpose calibration graphs, extraction efficiencies, decision limits, detection capabilities, precision (repeatability and within-laboratory reproducibility), accuracy, selectivity, and robustness were evaluated. The proposed method was applied to other AFWS with satisfactory results.     

Evaluation of the separation characteristics of application-specific (volatile organic compounds) open-tubular columns for gas chromatography

The solvation parameter model is used to characterize the separation characteristics of two application-specific open-tubular columns (Rtx-Volatiles and Rtx-VGC) and a general purpose column for the separation of volatile organic compounds (DB-WAXetr) at five equally spaced temperatures over the range 60-140 degrees C. System constant differences and retention factor correlation plots are then used to determine selectivity differences between the above columns and their closest neighbors in a large database of system constants and retention factors for forty-four open-tubular columns. The Rtx-Volatiles column is shown to have separation characteristics predicted for a poly(dimethyldiphenylsiloxane) stationary phase containing about 16% diphenylsiloxane monomer. The Rtx-VGC column has separation properties similar to the poly(cyanopropylphenyldimethylsiloxane) stationary phase containing 14% cyanopropylphenylsiloxane monomer DB-1701 for non-polar and dipolar/polarizable compounds but significantly different characteristics for the separation of hydrogen-bond acids. For all practical purposes the DB-WAXetr column is shown to be selectivity equivalent to poly(ethylene glycol) columns prepared using different chemistries for bonding and immobilizing the stationary phase. Principal component analysis and cluster analysis are then used to classify the system constants for the above columns and a sub-database of eleven open-tubular columns (DB-1, HP-5, DB-VRX, Rtx-20, DB-35, Rtx-50, Rtx-65, DB-1301, DB-1701, DB-200, and DB-624) commonly used for the separation of volatile organic compounds. A rationale basis for column selection based on differences in intermolecular interactions is presented as an aid to method development for the separation of volatile organic compounds.     

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.