Aqueous Acetonitrile Mobile Phase Research Articles

Microchip electrospray: Cone-jet stability analysis for water–acetonitrile and water–methanol mobile phases

Changes in mobile phase composition during high performance liquid chromatography (HPLC) gradient elution coupled to mass spectrometry (MS) sensitively affect electrospray operation modes. In this work, we identify the influences of dynamic changes in bulk conductivity on the cone-jet stability island for aqueous acetonitrile and aqueous methanol mobile phases commonly used in reversed-phase HPLC. Bulk conductivities of the mobile phases were varied by adding different amounts of formic acid. A commercial microchip-HPLC/ESI-MS configuration was modified to enable in situ electrospray diagnostics by frequency analysis of the microchip emitter current and spray imaging. This approach facilitated the detection of different spray modes together with their onset potentials. The established spray modes are described and the differences in onset potentials and stability regions explained by the physicochemical properties of the electrosprayed liquid.

Liquid chromatography and characterization of ether-functionalized imidazolium ionic liquids on mixed-mode reversed-phase/cation exchange stationary phase

Abstract A new series [CnOmmim]Cl of imidazolium cation-based ionic liquids (ILs), with an ether functional group on the alkyl side-chain, has been prepared. The possibility of analyzing the ionic liquids by high performance liquid chromatography (HPLC) was investigated on mixed-mode reversed/cation exchange stationary phase with the aqueous-acetonitrile mobile phase. Elution parameters, such as retention factor, selectivity and column efficiency, were studied as functions of mobile phase composition and pH. The ILs were characterized by elemental analysis, and infrared, UV and 1H, 13C NMR spectroscopy.

A thin-layer contactless conductivity cell for detection in flowing liquids

The design is described of a thin-layer contactless conductivity detector suitable for liquid chromatography and flow-injection analysis. Its principal analytical parameters have been determined using a potassium chloride solution: the linear dynamic range extends from 7.5 × 10 −6 to 1.5 × 10 −2 S m −1, corresponding to the KCl concentration range from 0.5 to 1000 μM, the limit of detection equals 3.5 × 10 −6 S m −1 (0.2 μM KCl), the detection repeatability, expressed in terms of the relative standard deviation, amounts to 1.13% and the detection volume is 0.6 μL. The detector was applied to detection of ionic compounds, benzoic, lactic and octanesulfonic acids, and sodium capronate, after their separation by liquid chromatography in a Biospher PSI 100C 18 columns using a 60% aqueous acetonitrile mobile phase. The frequency characteristics of the detector are reasonably theoretically described on the basis of a simple model which is commonly used in the field of contactless impedance detectors.

Simultaneous Analysis of a Mixture of Iridoids in Water Extracts of Plantago asiatica L. by LC

A simple liquid chromatographic method using an ultraviolet detector was developed for the simultaneous quantification of a mixture of iridoid glycosides, catalpol, aucubin, and geniposidic acid. By adding a minute amount of trifluoroacetic acid into the conventional aqueous acetonitrile mobile phase, the unstable resolution arising from the tautomeric rearrangements in the carboxylic group at the 11 position of geniposidic acid could be solved, resulting in a clear separation profile of the catalpol, aucubin, and geniposidic acid peaks. We were able to quantify the contents of catalpol, aucubin, and geniposidic acid contained in the leaves and seeds of P. asiatica.

Chromatographic behaviour of steroidal saponins studied by high-performance liquid chromatography–mass spectrometry

The chromatographic behaviour of steroidal saponins found in Anemarrhena asphodeloides, Asparagus officinalis, Convallaria majalis, Digitalis purpurea and Ruscus aculeatus was studied by HPLC–MS using a C-18 reversed-phase column and aqueous acetonitrile or aqueous methanol mobile phase gradients, with or without the addition of 1% acetic acid. The behaviour was compared to that of triterpene saponins found in Aesculus hippocastanum, Centella asiatica, Panax notoginseng and Potentilla tormentilla. Inclusion of methanol in the mobile phase under acidic conditions was found to cause furostanol saponins hydroxylated at C-22 to chromatograph as broad peaks, whereas the peak shapes of the spirostanol saponins and triterpene saponins studied remained acceptable. In aqueous methanol mobile phases without the addition of acid, furostanol saponins chromatographed with good peak shape, but each C-22 hydroxylated furostanol saponin was accompanied by a second chromatographic peak identified as its C-22 methyl ether. Methanolic extracts analysed in non-acidified aqueous acetonitrile mobile phases also resolved pairs of C-22 hydroxy and C-22 methoxy furostanol saponins. The C-22 methyl ether of deglucoruscoside was found to convert to deglucoruscoside during chromatography in acidified aqueous acetonitrile, or by dissolving in water. Poor chromatography of furostanol saponins in acidified aqueous methanol is due to the interconversion of the C-22 hydroxy and C-22 methoxy forms. It is recommended that initial analysis of saponins by HPLC–MS using a C-18 stationary phase is performed using acidified aqueous acetonitrile mobile phase gradients. The existence of naturally-occurring furostanol saponins methoxylated at C-22 can be investigated with aqueous acetonitrile mobile phases and avoiding methanol in the extraction solvent.

Retention behavior of oligomeric proanthocyanidins in hydrophilic interaction chromatography

A novel method was developed for the separation of proanthocyanidins (PAs; oligomeric flavan-3-ols) by hydrophilic interaction chromatography (HILIC) using an amide–silica column eluting with an aqueous acetonitrile mobile phase. The best separation was achieved with a linear gradient elution of acetonitrile–water at ratios of 9:1 to 5:5 (v/v) for 60min at a flow rate of 1.0ml/min. Under these HPLC conditions, a mixture of natural oligomeric PAs (from apple) was separated according to degree of polymerization (DP) up to decamers. The DP of each separated oligomer was confirmed by LC/electrospray ionization MS. In further HILIC separation studies of 15 different flavan-3-ol and oligomeric PA (up to pentamer) standards with an isocratic elution of acetonitrile–water (84:16), a high correlation was observed between the logarithm of retention factors (logk) and the number of hydroxyl groups in their structures. The coefficient of this correlation (r2=0.9501) was larger than the coefficient (r2=0.7949) obtained from the correlation between logk and logPo/w values. These data reveal that two effects, i.e. hydrogen bonding between the carbamoyl terminal on the column and the hydroxyl group of solute oligomer and hydrophilicity based on the high-order structure of oligomeric PAs, corporately contribute to the separation, but the hydrogen bonding effect is predominant in our HILIC separation mode.

Highly Selective Determination of Total Iron in Environmental Water Samples by HPLC

A simple and selective high performance liquid chromatographic method has been developed for the determination of total iron in water samples. 2‐(8‐quinolylazo)‐4,5‐diphenylimidazole (QAI) was used as a precolumn chelating reagent for iron(II). The iron(II)‐QAI chelate was separated on a Cosmosil 5C8‐MS column with an aqueous acetonitrile mobile phase containing tetrabutylammonium bromide and spectrophotometrically detected at 710 nm. Iron(III) was determined as the iron(II) chelate with QAI after it was reduced to iron(II) using ascorbic acid. Detection limit, defined as three times of standard deviation of a blank signal, was 18 pg of iron(II) in 100 µL injection. Total iron in environmental water samples was selectively determined without any pretreatment.

Application of liquid chromatography–mass spectrometry to the investigation of poisoning by Oenanthe crocata

Liquid chromatography-mass spectrometry (LC-MS) analysis of methanol extracts of Oenanthe crocata roots revealed that oenanthotoxin co-eluted with another major polyalkyne, 2,3-dihydro-oenanthotoxin, using the existing high performance liquid chromatography (HPLC) method (isocratic elution from C18 with aqueous methanol) for investigating Oenanthe poisoning. Positive ES or APCI gave [(M+H)-H(2)O](+) and its methanol adduct as major ion species for oenanthotoxin, whereas 2,3-dihydro-oenanthotoxin formed [M+H](+) and its methanol adduct. The two polyalkynes could be chromatographically resolved on C18 by gradient elution with aqueous acetonitrile. This provides superior analysis for oenanthotoxin using HPLC with photodiode array (PDA) detection alone, but for LC-MS/MS aqueous acetonitrile was less suitable due to poor ionisation and, with APCI, an increase in the relative abundance of a [M-1](+) species, which could confuse compound assignment. HPLC-PDA and LC-MS/MS methods using an aqueous acetonitrile or aqueous methanol mobile phase, respectively, were successful when applied to the analysis of the stomach contents of a pony suspected to have eaten O. crocata. Relevant product ion spectra, by ion trap MS/MS, accurate mass data and complete sets of (1)H and (13)C NMR spectral assignments are given for the two compounds.

Retention and selectivity tests of silica‐based and metal‐oxide bonded stationary phases for RP‐HPLC

Chromatographic properties of silica-, zirconia- and alumina-based columns with octadecyl-, polyethylene glycol- and pentafluorophenylpropyl-bonded stationary phases were tested. Selectivities of nine columns for LC were characterized using chromatographic methods including Walters, Engelhardt, Tanaka and Galushko hydrophobicity and silanol activity tests, measurements of methylene selectivity in various aqueous-methanol and aqueous-acetonitrile mobile phases and of gradient lipophilic capacity as a measure of the effect of the sample hydrophobicity on gradient-elution separations. A semi-empirical interaction indices model, assuming a predominant role of the solvophobic interactions of test compounds with different polarities, was compared with the linear free energy relationships approach taking into account selective polar interactions. The interaction indices model was applied to both non-polar stationary phases bonded on silica, alumina and zirconia supports, and to the non-modified adsorbents in the normal-phase LC. The retention data of isomeric naphthalene disulfonic acids were used to compare the attractive and repulsive ionic interactions of the columns in purely aqueous mobile phases. The results of the hydrophobicity and polarity tests were consistent, and allowed column characterization and classification. Silanol activity was important with octadecyl silica columns, but was relatively insignificant with bonded polyethylene glycol and pentafluorophenylpropyl phases on silica gel support. Polar interactions with the alumina and zirconia support materials significantly affect the retention.

Reversed-phase liquid chromatography and argentation chromatography of the minor capsaicinoids

An investigation of the liquid chromatography of the minor capsaicinoids in a commercial capsaicinoid mixture is reported. Twelve stationary phases including C8, C18, C30, phenyl, and cation-exchange chemistries were examined in combination with isocratic aqueous methanol and aqueous acetonitrile mobile phases. A phenyl stationary phase and aqueous acetonitrile mobile phase baseline-resolved 7 of 11 capsaicinoids, and selected ion chromatograms (LC-ESI-MS) demonstrated this was the most effective reversed-phase separation. Argentation chromatography with an alkyl or phenyl column and aqueous silver nitrate-methanol mobile phase revealed the presence of the 6-ene-8-methyl and 6-ene-9-methyl homocapsaicin isomers and the absence of 7-ene-9-methyl homocapsaicin. A mixed phenyl-cation-exchange stationary phase (charged with silver ion) enabled unique and useful separations of the capsaicinoids.

Evaluation of a Semi-Empirical Approach to Correlation of Retention with Octanol-Water Partition Coefficients by Use of Adjustable Hydrogen-Bonding Indicator Variables

An improved model with adjustable hydrogen-bonding (HB) descriptors is evaluated for correlation of LogP with the retention of neutral and ionizable solutes in reversed-phase liquid chromatography (RPLC). This model is developed by adding adjustable secondary HB variables to the main linear Logk-LogP relationship to improve the quality of the correlation. The HB variables are not fixed, as is usual, but determined by nonlinear least-squares optimization from the retention of reference solutes. Retention times obtained for 56 solutes (neutral, acidic, and basic) by use of aqueous-acetonitrile (ACN) mobile phase gradients at different pH on a Zorbax StableBond C18 column are used to evaluate the method and the capability of the model. It is concluded that the simple model, together with the proposed data-processing scheme, describes the retention of both neutral and ionizable compounds very well. Accordingly, the model will simplify prediction of the retention of complex molecules and assist in chromatographic optimization, including selectivity optimization and internal standard selection. The other potential of the model is for simultaneous determination of both LogP and pKa by RPLC.

Preparation and Evaluation of a Series of Reversed‐Phase Monolithic Columns for Capillary Electrochromatography

A series of methlyacrylamide‐based, reversed‐phase micromonolithic columns were prepared by in situ copolymerization in fused‐silica capillaries for capillary electrochromatography (CEC). Different alkenes were used as functional monomers to tune chromatographic behaviors and porous properties of monoliths. The performance of the monolithic column was evaluated in the aqueous acetonitrile mobile phase with respect to separation selectivity, efficiency, and reproducibility. The baseline separation of 6 neutral compounds can be obtained. The separation mechanism in this system was further investigated. Furthermore, the high column efficiency (up to 250,000 plates/m) at the optimum condition can be achieved. In addition, the reproducibility for the monolithic column was also very satisfactory. The study showed that this series of monoliths possess potential as an effective separation media in CEC.

Sulfamethoxazole-imprinted polymer for selective determination of sulfamethoxazole in tablets

A sulfamethoxazole (SMO)-imprinted polymer (MIP) was prepared in acetonitrile using the mixture of acrylamide and 4-vinylpyridine as functional monomers. The molecular recognition properties of the polymer was evaluated in both acetonitrile and aqueous acetonitrile mobile phases. SMO contents in two kinds of tablets were determined satisfactorily using the MIP packed HPLC column with aqueous mobile phase.

Retention mechanism of fatty alcohol ethoxylates in reversed-phase liquid chromatography

Fatty alcohol ethoxylates (FAEs) are widely used nonionic surfactants that have distributions in both alkyl and poly(ethylene oxide) (PEO) chain length. Generally, two-dimensional liquid chromatography technique is required for the complete characterization of both distributions. By selecting a proper stationary and mobile phase condition, however, we can obtain fully resolved chromatograms of a FAE sample (Brij 30) with respect to both alkyl and PEO chain length by using a single reversed-phase C 18 column and aqueous acetonitrile mobile phase. FAEs show a peculiar reversed-phase liquid chromatography (RPLC) retention behavior with an aqueous–organic mobile phase, the retention mechanism of which has not been fully elucidated. For a fixed alkyl chain length, FAEs with higher-molecular-mass PEO block elutes first and the van’t Hoff plot of the retention factor shows a curvature. The unique retention behavior can be understood from the opposite thermodynamic characteristics associated with RPLC retention of PEO block and alkyl chain: the sorption process of PEO to the non-polar stationary phase shows Δ H o>0 and Δ S o>0 while the alkyl chain shows Δ H o<0 and Δ S o<0 in contrast. The relative magnitude of the two contributions can change the elution order of the FAE. Therefore the often found, inverted elution order of FAEs (the early elution of FAEs with longer PEO block) is due to the positive enthalpic interaction of PEO blocks, which is a characteristic of the hydrophobic interaction. And the curvature of the van’t Hoff plots was analyzed assuming the temperature dependent thermodynamic variables.

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (∼1–2 μm) and through-pores (∼2–8 μm) in a fused-silica capillary (50–200 μm I.D.). The columns were evaluated in HPLC after derivatization to C 18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 μm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (>80%) and large through-pores resulted in high permeability ( K=8·10 −14–1.3·10 −12 m 2) that was 2–30 times higher than that of a column packed with 5-μm silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8–12 μm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-μm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.

Hydrophilic interaction chromatography using amino and silica columns for the determination of polar pharmaceuticals and impurities

Hydrophilic interaction chromatography (HILIC) is described as a useful alternative to reversed-phase chromatography for applications involving polar compounds. In the HILIC mode, an aqueous–organic mobile phase is used with a polar stationary phase to provide normal-phase retention behavior. Silica and amino columns with aqueous–acetonitrile mobile phases offer potential for use in the HILIC mode. An examination of the retention and separation of several pyrimidines, purines, and amides on silica and amino columns from three manufacturers revealed that mobile phases should contain a buffer or acid for pH control to achieve similar and reproducible results among columns from different sources. Amino columns may also be used in an anion-exchange mode, which provides an advantage for some applications. In some cases, silica can provide different selectivity and better separation than an amino column. Example applications include: low-molecular-mass organic acids and amides as impurities in non-polar drug substances, 5-fluorouracil in 5-fluorocytosine, guanine in acyclovir, and different selectivity for polar basic compounds compared to an ion-pairing system.

Monolithic columns containing sol–gel bonded octadecylsilica for capillary electrochromatography

A new method for preparing monolithic capillary columns is reported. A fused-silica capillary packed with porous octadecylsilica (ODS) particles using a CO 2 slurry was partially filled with a siliceous sol formed by hydrolysis and polycondensation of tetramethoxysilane and ethyltrimethoxysilane. After gelling and aging of the siliceous sol at room temperature, the column was dried with supercritical CO 2. The scanning electron micrograph of a cross-section of the capillary column revealed that the ODS particles were bonded to each other and to the column inner wall by the sol–gel, forming a monolith. The performance of the monolithic column was evaluated for capillary electrochromatography using small aromatic compounds and polycyclic aromatic hydrocarbons with Tris buffer in aqueous acetonitrile mobile phase. The electroosmotic flow velocity increased with a decrease in Tris buffer concentration from 10 to 5 m M. The electroosmotic flow velocity increased significantly with an increase in Tris buffer pH from 5 to 7.5, and then remained nearly constant with Tris buffer pH between 7.5 to 9.0. The electroosmotic flow velocity increased with an increase in acetonitrile content from 20 to 50%, and then remained nearly constant with acetonitrile content between 50 to 90%. No bubble formation was observed during CEC operation, and all of the test compounds eluted as symmetrical peaks. The logarithms of the retention factors of the aromatic compounds and polycyclic aromatic hydrocarbons decreased linearly with an increase in acetonitrile content. For a 21/29 cm×75 μm I.D. monolithic column containing 9% sol–gel bonded 5 μm ODS, approximately 2.7×10 4 theoretical plates (plate height 7.8 μm; 1.3×10 5 theoretical plates per meter) were measured. It was found that the resistance to mass transfer of unretained thiourea was small, which was attributed to partial filling of the small pores in the particulate packing and partial filling of the interstitial space by the porous sol–gel network. The procedure for column fabrication was simple, and three columns were prepared without failure.

Determination of solute lipophilicity, as log P(octanol) and log P(alkane) using poly(styrene–divinylbenzene) and immobilised artificial membrane stationary phases in reversed-phase high-performance liquid chromatography

A number of RP–HPLC systems have been characterized by the linear free energy relationship: (i) log SP=c+r.R 2+s.π H 2+a.∑α H 2+b.∑β 2+v.V x Here, SP is either log k′ or log k w for a series of solutes in a given system, where k′ is the capacity factor and k w is the capacity factor extrapolated to l00% water, and the solute descriptors are, R 2 an excess molar refraction, π 2 H the dipolarity/polarizability, ∑ α 2 H and ∑ β 2 the overall or effective hydrogen-bond acidity and basicity, and V x the McGowan characteristic volume. Comparison of the coefficients in Eq. (1)with those for water-solvent partitions confirms that the modified electrostatically coated C 18 phase of Pagliara et al. (J. Liq. Chromatogr., 18 (1995) 1721) can be used to obtain solute lipophilicities, as log P oct. For RP–HPLC systems based on poly(styrene–divinylbenzene), the coefficients in Eq. (i)are nearer those for the correlation of water–alkane partition coefficients, as log P alk, than for the correlation of log P oct, suggesting that the RP–HPLC systems with poly(styrene–divinylbenzene) phases could be used as a rapid method for determination of solute lipophilicity, as log P alk or as log P cyc, where the latter is the water–cyclohexane partition coefficient. Eq. (i)has also been applied to RP–HPLC log k′ values obtained with an immobilized artificial membrane (IAM) phase. A good regression equation was obtained, but the coefficients in this equation are substantially different to those for regressions with log P oct, log P alk, or log P cyc as the dependent variable. On the other hand, log k′ values from the RP–HPLC system of Miyake al. [J. Chromatogr., 389 (1987) 47], consisting of silica gel coated with dipalmitoyl phosphatidyl choline as a stationary phase, with aqueous acetonitrile mobile phases, yielded coefficients in Eq. (i)very similar to those for log P oct.

Comparison of Solvent Modulation with Premixed Mobile Phases for the Separation of Corticosteroids by Liquid Chromatography

The separation of eight common corticosteroids is optimized by solvent modulation and by conventional premixed solvents. The separation is achieved on an octadecylsilica column using aqueous acetonitrile and methanol mobile phases. The optimized separations are compared with respect to accuracy, total analysis time, critical resolution, and overall quality of the separation. The solvent modulation approach compares favorably to premixed solvents in all of these respects and, hence, is demonstrated to be a very promising optimization strategy.

Solvent modulation in liquid chromatography: extension to serially coupled columns

The concept and theory of solvent modulation are extended to serially coupled columns in liquid chromatography. Because the mobile phases are maintained in separate zones and the stationary phases in separate columns, solute retention is a simple, time-weighted average of the individual environments to which the solute is exposed. Thus, optimization of complex multidimensional chromatographic separations is more accurate and predictable. This approach is demonstrated by application to the separation of isomeric polynuclear aromatic hydrocarbons using octadecylsilica and β-cyclodextrin silica stationary phases with aqueous methanol and acetonitrile mobile phases.