Reversed-phase Liquid Chromatography Mode Research Articles

A novel two-dimensional liquid-chromatography method for online prediction of the toxicity of transformation products of benzophenones after water chlorination.

Benzophenone-type UV filters (BPs) are ubiquitous in the environment. Transformation products (TPs) of BPs with suspected toxicity are likely to be produced during disinfection of water by chlorination. To quickly predict the toxicity of TPs, in this study, a novel two-dimensional liquid-chromatography (2D-LC) method was established in which the objective of the first dimension was to separate the multiple components of the BPs sample after chlorination, using a reversed-phase liquid-chromatography mode. A biochromatographic system, i.e. bio-partitioning micellar chromatography with the polyoxyethylene (23) lauryl ether aqueous solution as the mobile phase, served as the second dimension to predict the toxicity of the fraction from the first dimension on the basis of the quantitative retention-activity relationships (QRARs) model. Six BPs, namely 2,4-dihydroxybenzophenone, oxybenzone, 4-hydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid, 2,2'-dihydroxy-4,4'-dimethoxybenzophenone and 2,2'-dihydroxy-4-methoxybenzophenone, were the target analytes subjected to chlorination. The products of these BPs after chlorination were directly injected to the 2D-LC system for analysis. The results indicated that most TPs may be less toxic than their parent chemicals, but some may be more toxic, and that intestinal toxicity of TPs may be more obvious than blood toxicity. The proposed method is time-saving, high-throughput, and reliable, and has great potential for predicting toxicity or bioactivity of unknown and/or known components in a complex sample. Graphical Abstract The scheme for the 2D-LC online prediction of toxicity of the transformation products of benzophenone-type UV filters after chlorination.

Hydrophilic interaction chromatography: A promising alternative to reversed-phase liquid chromatography systems for the purification of small protonated bases.

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed-phase liquid chromatography mode (BEH-C18), an electrostatic repulsion reversed-phase liquid chromatography C18 column (BEH-C18+), a poly(styrene-divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm(3)). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed-phase liquid chromatography like conditions (with BEH-C18, BEH-C18+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed-phase liquid chromatography columns.

Preparation and evaluation of surface-bonded tricationic ionic liquid silica as stationary phases for high-performance liquid chromatography

Two tricationic ionic liquids were prepared and then bonded onto the surface of supporting silica materials through “thiol-ene” click chemistry as new stationary phases for high-performance liquid chromatography. The obtained columns of tricationic ionic liquids were evaluated respectively in the reversed-phase liquid chromatography (RPLC) mode and hydrophilic interaction liquid chromatography (HILIC) mode, and possess ideal column efficiency of 80,000 plates/m in the RPLC mode with naphthalene as the test solute. The tricationic ionic liquid stationary phases exhibit good hydrophobic and shape selectivity to hydrophobic compounds, and RPLC retention behavior with multiple interactions. In the HILIC mode, the retention and selectivity were evaluated through the efficient separation of nucleosides and bases as well as flavonoids, and the typical HILIC retention behavior was demonstrated by investigating retention changes of hydrophilic solutes with water volume fraction in mobile phase. The results show that the tricationic ionic liquid columns possess great prospect for applications in analysis of hydrophobic and hydrophilic samples.

Quantitative analysis of five antiviral drugs by hydrophilic interaction liquid chromatography-charged aerosol detection

Antiviral drugs are widely used for human and animals. However, the analysis of the mixture of antiviral drugs is a challenge for high performance liquid chromatography, since some of the antiviral drugs have weak UV absorbance and poor retention in reversed phase liq- uid chromatography. A method of hydrophilic interaction liquid chromatography-charged aerosol detection (HILIC-CAD) was optimized for the qualitative and quantitative analysis of five antiviral drugs. In this study, Click TE-Cys was used as the stationary phase and CAD was used as the detector. Various chromatographic conditions including the kind of detector, chromatographic mode, column and mobile phase composition were investigated. Compared to UV-Vis, more antiviral drugs could be detected by CAD, since it is a universal detector. HILIC mode is an alternative to reversed phase liquid chromatography mode. HILIC provides higher sensitivity and unique selectivity to target compounds. After the optimized parameters were obtained, the developed method was used for the quantitative analysis of the five antiviral drugs. As a result, the current method has good repeatability, a wide linear range (0.07-2.28 mg/mL) and good sensitivity (LOQ ≤ 0.04 mg/mL). The RSDs of intra-day and inter-day peak areas were less than 3. 06% and 5. 38% respectively. The above results demonstrated that the current method is sensitive, robust and effective for the separation and determination of these five antiviral drugs.

Preparation and characterization of surfactin-modified silica stationary phase for reversed-phase and hydrophilic interaction liquid chromatography

Surfactants are good candidates as selectors in mixed-mode reversed-phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC) because they contain both a hydrophobic and a hydrophilic moiety. Surfactin, a cyclic heptapeptide, is an efficient biosurfactant produced by Bacillus subtilis that comprises seven amino acids and a β-hydroxyl fatty acid. A surfactin-modified silica (SMS) stationary phase was prepared by amide bond formation between amino groups on aminopropyl silica and the carboxylic acid groups of L-Glu and L-Asp residues in surfactin. The resulting SMS stationary phase was characterized in both RPLC and HILIC mode using different mobile phases. The SMS column was found to separate analytes in both modes. The retention of polar solutes exhibited “U-shaped” curves, depending on the acetonitrile content. “U-shaped” curves are an indicator of RPLC/HILIC mixed-mode retention behavior. The presence of hydrophobic and hydrophilic moieties in surfactin provides unique properties that allow the SMS column to be used for both RPLC and HILIC separations, simply by changing the mobile phase composition.

Hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry: Effect of electrospray ionization source geometry on sensitivity

In this study, the influence of electrospray ionization (ESI) source design on the overall sensitivity achieved in hydrophilic interaction chromatography (HILIC) and reversed phase liquid chromatography (RPLC), was investigated. State-of-the-art triple quadrupole mass analyzers from AB Sciex, Agilent Technologies and Waters equipped with brand specific source geometries were tested with various mobile phase pH on 53 pharmaceutical compounds. The design of the ESI source showed to strongly influence the gain in sensitivity that can be achieved in HILIC compared to RPLC mode. The 6460 Triple Quadrupole LC/MS system from Agilent Technologies was particularly affected by mobile phase settings. Indeed, compared to RPLC conditions, 92% of the compounds had an increased signal-to-noise ratio at a flow rate of 300μL/min in HILIC mode at pH 6, while this percentage dropped to only 7% at 1000μL/min and pH 3. In contrast, the influence of flow rate and mobile phase pH on the gain in sensitivity between RPLC and HILIC was found very limited with the API 5000™ LC/MS/MS system from AB Sciex, as only 15 to 36% of the tested compounds showed an enhanced sensitivity in HILIC mode. With the Xevo TQ-S instrument from Waters, superior sensitivity in HILIC was noticed for 85% of the compounds with optimal conditions (i.e., pH 3 and 1000μL/min), whereas at sub-optimal conditions (i.e. pH 6 and 300μL/min), it represented less than 50%. The gain in sensitivity observed in HILIC was found less significant with the recent LC–MS platforms used in this study than for old-generation instruments. Indeed, the improved ESI sources equipping the recent mass analyzers allow for enhanced evaporation efficiency, mainly for RPLC mobile phases containing high proportion of water and this even at high flow rates

Hydrophilic Interaction Chromatography Using a Meter-Scale Monolithic Silica Capillary Column for Proteomics LC-MS

A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

High Submicellar Liquid Chromatography

Surfactant addition above the critical micellar concentration (CMC), in reversed-phase liquid chromatography (RPLC), was proposed as a way to modify the selectivity and analysis time, giving rise to a chromatographic mode called micellar liquid chromatography (MLC). However, solutions containing only surfactant are too weak and yield poor peak shape. This was remediated by the addition of a small amount of organic solvent. To preserve the existence of micelles, in MLC high contents of organic solvent are avoided. Nevertheless, there is no reason to neglect the potentiality of mobile phases containing a surfactant above its CMC in water and a high organic solvent content (without micelles). This chromatographic mode has been called high submicellar liquid chromatography (HSLC). Several reported procedures show that the combination of stronger elution strength, larger selectivity and improved peak shape, with respect to MLC and conventional RPLC, makes HSLC a promising chromatographic mode to achieve in practical times separations of compounds unresolved or highly retained with other RPLC modes. Some insights on the interactions that occur inside the chromatographic column, the modification of the stationary and mobile phases, retention modeling, and chromatographic performance in HSLC are here offered, in comparison to MLC and conventional RPLC.

Comparison between the intra-particle diffusivity in the hydrophilic interaction chromatography and reversed phase liquid chromatography modes. Impact on the column efficiency

The effective diffusion coefficients of five low molecular weigh compounds (naphthalene, uracil, uridine, adenosine, and cytosine) were measured at room temperature in a 4.6mm×100mm column packed with 3.5μm XBridge HILIC particles. The mobile phase was an acetonitrile–water mixture (92.5/7.5, v/v) containing 10mM ammonium acetate and 0.02% acetic acid. Using a physically reliable model of effective diffusion in binary composite media (Torquato's model), accurate estimates of the intra-particle diffusivities in the HILIC particles were obtained as a function of the retention of these analytes. The HILIC diffusion coefficients were compared to those previously obtained for endcapped RPLC-C18 particles (5.0μm Gemini-C18). The experimental results confirm that adsorption sites are not localized in RPLC whereas they are so in the HILIC mode. In contrast to RPLC columns, HILIC columns provide longitudinal diffusion B/u terms that increase very little with increasing retention factors. This confirms the absence of surface diffusion in HILIC. The impact of intra-particle diffusivity on the column efficiency was projected in HILIC and RPLC on the basis of the measured intra-particle diffusivities and on the well established theory of band broadening in particulate columns. Accordingly, RPLC columns generate short-range eddy dispersion and solid–liquid mass transfer resistance Cu terms that increase less than do HILIC column with increasing retention factors. The HETP contribution caused by the trans-column structure heterogeneity is smaller in the HILIC than in the RPLC modes because the transverse excursion length is smaller in HILIC. Even though the overall column efficiencies are comparable in HILIC and RPLC, this study shows that the individual mass transfer phenomena are inherently different in the HILIC and the RPLC retention modes.

Purification of amide alkaloids from Piper longum L. using preparative two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography

A comprehensive off-line two-dimensional liquid chromatography (2D-LC) method coupling normal phase liquid chromatography (NPLC) and reversed phase liquid chromatography (RPLC) was developed for separation and purification of amide alkaloids from Piper longum L. In the first dimension, the crude alkaloid fractions were separated in NPLC mode and 20 fractions were collected. Then fractions 5-20 were selected for further purification in RPLC mode in the second dimension. The purities of RPLC fractions with similar structures were all identified accurately by ultra performance liquid chromatography (UPLC). In total, 28 compounds with high purity were obtained and their structures were comprehensively characterized by electrospray ionization-mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. The results demonstrate that this 2D NPLC × RPLC method with good orthogonality (58.3%) was effective for the preparative separation and purification of amide alkaloids from Piper longum L.

CHROMATOGRAPHIC FINGERPRINTS OF SEED EMBRYO OF NELUMBO NUCIFERA GAERTN BY REVERSED-PHASE AND HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY

Reversed-phase liquid chromatography (RPLC) is the most common method to construct a fingerprint analysis for quality control of traditional Chinese medicine (TCM). However, many polar compounds in TCM nearly have no retention at RPLC mode. Therefore, it is inadequate for the quality control of TCM. In this study, an approach was proposed to develop chromatographic fingerprints by means of RPLC and hydrophilic interaction chromatography (HILIC) to gain more chemical information about polar compounds and weakly polar compounds from seed embryo of Nelumbo nucifera GAERTN (SENN). The two chromatographic methods demonstrated good precision, repeatability, and stability, with relative standard deviations of <2% for retention time and 7% for peak area. Different chemical information regarding the same sample can be obtained by two chromatographic modes. Similarity analysis of 19 samples showed a fair consistence, but the similarities of samples in HILIC mode were relatively lower than in RPLC mode. Combination use of the two chromatographic fingerprints can comprehensively and properly reveal the quality characteristics of the SENN.

Preparation and Evaluation of Poly-l-Lysine Stationary Phase for Hydrophilic Interaction/Reversed-Phase Mixed-Mode Chromatography

In this work, a poly-l-lysine-grafted stationary phase was synthesized by polymerization of N-carboxyanhydride of l-lysine initiated by 3-aminopropylated silica. The resulting material was characterized by FT-IR spectra, elemental analysis and thermogravimetric analysis, which clearly indicated that the new phase had been prepared successfully. The retention of polar solutes depending on acetonitrile content in mobile phase exhibited “U-shaped” curves, which was an indication of hydrophilic interaction liquid chromatography (HILIC)/reversed-phase liquid chromatography (RPLC) mixed-mode retention behavior. The retention mechanisms in HILIC and RPLC modes also were investigated. Phenol compounds, aniline compounds and hydrophilic compounds were separated in RPLC or HILIC mode on the new stationary phase, respectively. This result shows that the new phase could be used for both RPLC and HILIC applications, providing greater flexibility for real sample analysis.

Imidazoline type stationary phase for hydrophilic interaction chromatography and reversed-phase liquid chromatography

An imidazoline was prepared by solvent-free microwave-assisted organic synthesis and immobilized on porous silica particles by polymerization. The resulting material was composed of both hydrophobic alkyl ester chains and hydrophilic imidazoline rings, which gave it both hydrophilic interaction and reversed-phase characteristics. The titration curve suggests that the new material has buffering capacity and acquires increasing positive charge over the pH range 9–4, and is “zwitterionic” in the upper part of this pH range. Through investigating the effect of column temperature, the water content, pH and ion strength of mobile phase on the retention time of polar compounds in highly organic eluents, it was found that the new material could be used as a hydrophilic interaction liquid chromatography (HILIC) stationary phase which involved a complex retention process consisting of partitioning, surface adsorption and electrostatic interactions. In addition, the retention behavior of aromatic compounds in different mobile phase conditions was also studied, which showed the new material mainly exhibited a partitioning mechanism in the reversed-phase liquid chromatography (RPLC) mode. The separation of six water-soluble vitamins and five aromatic compounds were achieved by using the new material in the HILIC and RPLC modes, respectively.

Orthogonal separation on one beta-cyclodextrin column by switching reversed-phase liquid chromatography and hydrophilic interaction chromatography

A dual retention combined with reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) has been observed on beta-cyclodextrin (beta-CD) bonded stationary phase. A typical U-shaped retention curve was achieved owing to dual retention mechanism. Based on this observation, a beta-CD column can be operated under reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) modes. Two-dimensional liquid chromatography (2D-LC) analysis can be realized on just a beta-CD column by switching these two different separation modes. In this study, off-line 2D-LC analysis for a natural product was carried out to prove the orthogonal separation between RP-LC and HILIC modes on a Click beta-CD column. Herba Hedyotis Diffusae, the whole grass of Hedyotis Diffusae wild was extracted with water, pretreated with macroporous resin and then first separated at RP-LC mode on the Click beta-CD column to obtain successive fractions, which were then reanalyzed at HILIC mode on the same Click beta-CD column. The result proved that both separation modes on the Click beta-CD column have good retention and peak shape, and these two separation modes have good orthogonality. 2D-LC analysis revealed abundant information in the natural product. Especially numerous minor components were enriched and separated. The mobile phase used in RP-LC and HILIC modes can be same and the switch between these two separation modes is easily realized by changing the ratio of the acetonitrile and water. Hence the mobile phase in this 2D-LC system is completely compatible. This advantage makes this combination is an appropriate 2D-LC method for the solutes having retention at both separation modes.

Capillary liquid chromatography using a hydrophilic/cation-exchange monolithic column with a dynamically modified cationic surfactant

A novel form of reversed-phase liquid chromatography (RPLC) by the dynamically modified hydrophilic interaction monolithic column has been described in this paper. A porous poly(SPMA-co-PETA) monolith with strong cation-exchange (SCX) was prepared and the resulting monolith showed a typical hydrophilic interaction chromatography (HILIC) mechanism at higher organic solvent content (ACN% > 50%). The good selectivity for neutral, basic and acidic polar analytes was observed in the HILIC mode. In order to increase the hydrophobic interaction, the monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX monolithic material, and the resulting hydrophobic layer was used as the stationary phase. Using the dynamically modified SCX monolithic column, neutral, basic and acidic hydrophobic analytes were well separated with the RPLC mode.

Chromatographic retention prediction and octanol–water partition coefficient determination of monobasic weak acidic compounds in ion-suppression reversed-phase liquid chromatography using acids as ion-suppressors

Although simple acids, replacing buffers, have been widely applied to suppress the ionization of weakly ionizable acidic analytes in reversed-phase liquid chromatography (RPLC), none of the previously reported works focused on the systematic studies about the retention behavior of the acidic solutes in this ion-suppression RPLC mode. The subject of this paper was therefore to investigate the retention behavior of monobasic weak acidic compounds using acetic, perchloric and phosphoric acids as the ion-suppressors. The apparent octanol–water partition coefficient (K″ow) was proposed to calibrate the octanol–water partition coefficient (Kow) of these weak acidic compounds, which resulted in a better linear correlation with log kw, the logarithm of the hypothetical retention factor corresponding to neat aqueous fraction of hydroorganic mobile phase. This log K″ow−log kw linear correlation was successfully validated by the results of monocarboxylic acids and monohydrating phenols, and moreover by the results under diverse experimental conditions for the same solutes. This straightforward relationship not only can be used to effectively predict the retention values of weak acidic solutes combined with Snyder–Soczewinski equation, but also can offer a promising medium for directly measuring Kow data of these compounds via Collander equation. In addition, the influence of the different ion-suppressors on the retention of weak acidic compounds was also compared in this RPLC mode.

MECHANISM OF IONIZATION OF POLYCYCLIC AROMATIC HYDROCARBONS BY A TOLUENE/ANISOLE MIXTURE AS A DOPANT IN LIQUID CHROMATOGRAPHY/DOPANT-ASSISTED ATMOSPHERIC-PRESSURE PHOTOIONIZATION/MASS SPECTROMETRY

Ionization mechanisms of 16 polycyclic aromatic hydrocarbons (PAHs) by dopant-assisted atmospheric-pressure photoionization (DA-APPI) with a toluene/anisole mixture as a dopant were examined in the reversed-phase liquid chromatography mode with a methanol/water to acetonitrile linear gradient mobile phase. When toluene/anisole mixtures were used as dopants, their characteristics were governed by anisole. Since the IE of anisole (8.20 eV) is below that of toluene (8.83 eV), anisole is ionized not only by photons from the krypton lamp but also by charge transfer from toluene radical cations. In the latter half of chromatogram, when acetonitrile was dominant in the eluent, ions originating from both toluene and anisole decreased while acetonitrile-cluster ions began to increase. Because acetonitrile-cluster ions are not formed with anisole alone, but are formed with toluene alone, toluene radical cations would be consumed to form acetonitrile-cluster ions.

Preparation of pH-responsive stationary phase for reversed-phase liquid chromatography and hydrophilic interaction chromatography

Novel pH-responsive polymer-grafted silica was successfully synthesized through the radical “grafting from” polymerization on azo initiator-immobilized silica. The immobilization of azo initiator onto the silica surface was achieved by the reaction of surface amino groups with 4,4′-azobis(4-cyanovaleric acid chloride). The polymer-grafted silica was prepared by stirring suspension of the azo initiator-immobilized silica in anhydrous dioxane containing acrylic acid (AAc) and butyl acrylate (BA). The resulting polymer-grafted silica was demonstrated to be pH responsive to hydrophobic/hydrophilic property by reversed-phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC). In RPLC mode, the retention of aromatic compounds decreased with the increase in the pH of mobile phase. However, the opposite result was obtained in HILIC mode; the retention of soybean isoflavones was stronger with the mobile phase at higher pH. Finally, the separations of sulfonamides and soybean isoflavones were carried out in RPLC mode and the separation of some nucleotides was achieved in HILIC mode.

In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy

The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate- co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.