Hyphenation of atmospheric pressure chemical ionisation mass spectrometry to supercritical fluid chromatography for polar car lubricant additives analysis

Car lubricant additives are added to mineral or synthetic base stocks to improve viscosity and resistance to oxidation of the lubricant and to limit wear of engines. As they belong to various chemical classes and are added to a very complex medium, the base stock, their detailed chromatographic analysis is very difficult and time consuming. In a previous paper, it was demonstrated that supercritical fluid chromatography (SFC) allows the elution of common low-molecular-weight additives. Since their total resolution could not be achieved owing to the limited peak capacity of packed columns, the hyphenation of selective and informative detection methods such as atomic emission detection (AED) was required. Further to results obtained in SFC-AED, this work describes the hyphenation of SFC to atmospheric pressure chemical ionisation ion trap mass spectrometry (MS). SFC–MS hyphenation is detailed: temperature, flow rates of gas and mobile phase introduced in the source, position of the restrictor, ionisation additives and conditions of autotune are studied. Car lubricant monitoring requires negative and positive ionisation modes with or without the addition of ionisation auxiliary solvent according to the nature of additives. Moreover, when sensitivity is of major concern for a selected additive, the autotuning routine of the MS has to be performed in conditions as close as possible to analytical conditions, i.e. under subcritical conditions. Unambiguous identification and structure elucidation of several additives in formulated car lubricants are also presented.     

Stationary and mobile phases in hydrophilic interaction chromatography: a review

Hydrophilic interaction chromatography (HILIC) is valuable alternative to reversed-phase liquid chromatography separations of polar, weakly acidic or basic samples. In principle, this separation mode can be characterized as normal-phase chromatography on polar columns in aqueous-organic mobile phases rich in organic solvents (usually acetonitrile). Highly organic HILIC mobile phases usually enhance ionization in the electrospray ion source of a mass spectrometer, in comparison to mobile phases with higher concentrations of water generally used in reversed-phase (RP) LC separations of polar or ionic compounds, which is another reason for increasing popularity of this technique. Various columns can be used in the HILIC mode for separations of peptides, proteins, oligosaccharides, drugs, metabolites and various natural compounds: bare silica gel, silica-based amino-, amido-, cyano-, carbamate-, diol-, polyol-, zwitterionic sulfobetaine, or poly(2-sulphoethyl aspartamide) and other polar stationary phases chemically bonded on silica gel support, but also ion exchangers or zwitterionic materials showing combined HILIC-ion interaction retention mechanism. Some stationary phases are designed to enhance the mixed-mode retention character. Many polar columns show some contributions of reversed phase (hydrophobic) separation mechanism, depending on the composition of the mobile phase, which can be tuned to suit specific separation problems. Because the separation selectivity in the HILIC mode is complementary to that in reversed-phase and other modes, combinations of the HILIC, RP and other systems are attractive for two-dimensional applications. This review deals with recent advances in the development of HILIC phase separation systems with special attention to the properties of stationary phases. The effects of the mobile phase, of sample structure and of temperature on separation are addressed, too.     

A unified classification of stationary phases for packed column supercritical fluid chromatography

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.     

Characterization of gasolines using on-line multidimensional supercritical fluid chromatography/ capillary gas chromatography

Summary Using packed column supercritical fluid chromatography on-line with capillary gas chromatography, gasolines have been characterized with respect to the separation of hydrocarbon classes as a group as well as a detailed fingerprinting of individual components within the classes. Using selective heartcutting, specific gasoline peaks were transferred to the capillary gas chromatograph as they eluted from the supercritical fluid chromatograph. Quantitative information was collected simultaneously from the flame ionization detectors that were used on both chromatographs. This report will describe the operational chromatographic conditions that were used for gasoline separations as well as provide results of quantitative heartcut reproducibility experiments of gasolines and hydrocarbon standards.

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Charakterisierung von Benzinen durch on-line-Kombination von multidimensionaler Chromatographie mit überkritischen fluiden Phasen und Capillar-Gas-Chromatographie

     

Stationary phases for hydrophilic interaction chromatography, their characterization and implementation into multidimensional chromatography concepts

Hydrophilic interaction chromatography (HILIC) is becoming increasingly popular for separation of polar samples on polar columns in aqueous-organic mobile phases rich in organic solvents (usually ACN). Silica gel with decreased surface concentration of silanol groups, or with chemically bonded amino-, amido-, cyano-, carbamate-, diol-, polyol-, or zwitterionic sulfobetaine ligands are used as the stationary phases for HILIC separations, in addition to the original poly(2-sulphoethyl aspartamide) strong cation-exchange HILIC material. The type of the stationary and the composition of the mobile phase play important roles in the mixed-mode HILIC retention mechanism and can be flexibly tuned to suit specific separation problems. Because of excellent mobile phase compatibility and complementary selectivity to RP chromatography, HILIC is ideally suited for highly orthogonal 2-D LC-LC separations of complex samples containing polar compounds, such as peptides, proteins, oligosaccharides, drugs, metabolites and natural compounds. This review attempts to present an overview of the HILIC separation systems, possibilities for their characterization and emerging HILIC applications in 2-D off-line and on-line LC-LC separations of various samples, in combination with RP and other separation modes.     

Multi-modal applicability of a reversed-phase/weak-anion exchange material in reversed-phase,anion-exchange,ion-exclusion,hydrophilic interaction and hydrophobic interaction chromatography modes

We recently introduced a mixed-mode reversed-phase/weak anion-exchange type separation material based on silica particles which consisted of a hydrophobic alkyl strand with polar embedded groups (thioether and amide functionalities) and a terminal weak anion-exchange-type quinuclidine moiety. This stationary phase was designed to separate molecules by lipophilicity and charge differences and was mainly devised for peptide separations with hydroorganic reversed-phase type elution conditions. Herein, we demonstrate the extraordinary flexibility of this RP/WAX phase, in particular for peptide separations, by illustrating its applicability in various chromatographic modes. The column packed with this material can, depending on the solute character and employed elution conditions, exploit attractive or repulsive electrostatic interactions, and/or hydrophobic or hydrophilic interactions as retention and selectivity increments. As a consequence, the column can be operated in a reversed-phase mode (neutral compounds), anion-exchange mode (acidic compounds), ion-exclusion chromatography mode (cationic solutes), hydrophilic interaction chromatography mode (polar compounds), and hydrophobic interaction chromatography mode (e.g., hydrophobic peptides). Mixed-modes of these chromatographic retention principles may be materialized as well. This allows an exceptionally flexible adjustment of retention and selectivity by tuning experimental conditions. The distinct separation mechanisms will be outlined by selected examples of peptide separations in the different modes.     

Glycolipid class profiling by packed-column subcritical fluid chromatography

The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.     

Silica modified with a thiourea derivative as a new stationary phase for hydrophilic interaction liquid chromatography

Nowadays there are limited types of commercially available stationary phases for hydrophilic interaction liquid chromatography and therefore new ones with unique selectivity are urgently in demand to meet the need of separations of various polar and hydrophilic analytes. The present study describes the preparation and evaluation of a new stationary phase based on thiourea derivative modified silica for hydrophilic interaction liquid chromatography. Thiourea derivative was bonded onto the surface of silica particles via a mild addition reaction between –NH₂ and –SCN, and the result of elemental analysis together with infrared analysis and solid‐state NMR spectroscopy proved that the synthesis method was feasible. The new stationary phase succeeded in fast separations of a wide range of polar and hydrophilic analytes and exhibited excellent separation performance, especially unique selectivity. Furthermore, the effects of water content, buffer pH, and salt concentration on retention indicated that a complicated separation mechanism rather than partitioning was involved in the stationary phase and hydrogen bonding interaction between analytes and thiourea functional group could play a very important role in its selectivity. For sure, the new stationary phase is of a great potential as a new type of hydrophilic interaction liquid chromatographic stationary phase.     

High throughput screening or drug impurity profiles using gradients in normal phase chromatography

Summary Developmental drug substances and a variety of precursors and process-related impurities were separated using mobile phase gradients under normal phase conditions. These separation conditions produced efficient separations in a fraction of the time necessary for comparable reversed-phase separations. The use of elevated temperatures, polar modifiers and moderate levels of acidic and basic additives allowed highly polar analytes to be eluted with high efficiency and selectivity using fast flow rates. Reproducibility for retention time and peak areas was demonstrated to be very good despite very short equilibration times.     

咪唑键合硅胶固定相微柱液相色谱分离酚类和胺类化合物

由于微柱液相色谱(μ-LC)具有高检测灵敏度、低溶剂消耗、可以与质谱等多种检测器联用的优点,近年来受到广泛关注。将咪唑键合硅胶固定相填充到毛细管中,在自制的微柱液相色谱系统下利用此键合相具有的弱疏水作用,采用不同的流动相对酚类和胺类化合物进行了分离。结果表明,流动相中只需添加少量的有机溶剂就可以实现对一些有机化合物的分离,甚至可以只用纯水作流动相就能分离一些弱疏水性化合物,如酚类。微柱液相色谱的流动相用量少,避免或大大减少了对环境的污染。自制微柱液相色谱系统为下一步微柱液相色谱-质谱联用奠定了一定的基础。     

Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography

A stationary phase composed of silica-bonded sulfonated cyclofructan 6 (SCF6) was synthesized and evaluated for hydrophilic interaction chromatography (HILIC). The separation of a large variety of polar compounds was evaluated on different versions of the stationary phase and compared with the same separations obtained with commercially available HILIC columns. The new columns successfully separate polar and hydrophilic compounds including β blockers, xanthines, salicylic acid related compounds, nucleic acid bases, nucleosides, maltooligosaccharides, water soluble vitamins and amino acids. The separation conditions were optimized by changing the composition and the pH of the mobile phase. The dependence of analyte retention on temperature was studied using van't Hoff plots. The newly synthesized stationary phase showed broad applicability for HILIC mode separations.     

Thin-layer chromatography of chlorinated anisoles and veratroles

Summary The thin-layer chromatography of chlorinated anisoles (methoxybenzenes) and veratroles (1,2-dimethoxybenzenes) has been examined on silica gel G60 and RP-18 thin-layer plates. More than fifty solvent systems were screened and some of them recommended for particular separations. Acetone was shown to be suitable for group separation of both chlorinated anisoles and veratroles on a silica gel G60 stationary phase having a very narrow range of R_F values. On the other hand, benzene, dichloromethane and the various mixtures of light petroleum (b.p. 40–60°C) and a more polar eluent (such as diethyl ether, acetone or ethyl acetate) were recommended for separation of certain individual isomers. The best separation of chloroanisoles was achieved using RP-18 plates and methanol-water (9010) as the solvent system.     

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.     

Clinical and pharmaceutical applications of packed-column supercritical fluid chromatography

Packed-column supercritical fluid chromatography (pSFC) is a fast separation technique that combines the properties of HPLC and GC. pSFC with carbon dioxide as the mobile phase and packed silica column as the stationary phase possesses the properties of normal phase mechanism; however, the addition of modifiers to the mobile phase allows the separation of relatively polar compounds. In spite of its many positive attributes, pSFC has not been widely used in areas such as proteomics, where methods such as HPLC dominate. Packed column SFC has been extensively used in clinical and pharmaceutical laboratories, especially for separation of nonpolar and chiral drugs. This review will discuss recently published applications of pSFC, with a specific focus on its advantages and limitations for the analysis of pharmaceuticals with varying chemical properties.     

Comparison of ultra high performance supercritical fluid chromatography,ultra high performance liquid chromatography,and gas chromatography for the separation of synthetic cathinones

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite‐5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended.     

Feasibility of ultra high performance supercritical neat carbon dioxide chromatography at conventional pressures

The implementation of columns packed with sub-2 μm particles in supercritical fluid chromatography (SFC) is described using neat carbon dioxide as the mobile phase. A conventional supercritical fluid chromatograph was slightly modified to reduce extra column band broadening. Performances of a column packed with 1.8 μm C18-bonded silica particles in SFC using neat carbon dioxide as the mobile phase were compared with results obtained in ultra high performance liquid chromatography (UHPLC) using a dedicated chromatograph. As expected and usual in SFC, higher linear velocities than in UHPLC must be applied in order to reach optimal efficiency owing to higher diffusion coefficient of solutes in the mobile phase; similar numbers of theoretical plates were obtained with both techniques. Very fast separations of hydrocarbons are presented using two different alkyl-bonded silica columns.     

Imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase for hydrophilic interaction/reversed‐phase mixed‐mode chromatography

A novel imidazolium‐embedded iodoacetamide‐functionalized silica‐based stationary phase has been prepared by surface radical chain‐transfer polymerization. The stationary phase was characterized by Fourier transform infrared spectrometry, thermogravimetric analysis, and element analysis. Fast and efficient separations of polar analytes, such as nucleosides and nucleic acid bases, water‐soluble vitamins and saponins, were well achieved in hydrophilic interaction chromatography mode. Additionally, a mixed mode of hydrophilic interaction and reversed‐phase could be also obtained in the analysis of polar and nonpolar compounds, including weak acidic phenols, basic anilines and positional isomers, with high resolution and molecular‐planarity selectivity, outperforming the commercially available amino column. Moreover, simultaneous separation of polar and nonpolar compounds was also achieved. In conclusion, the multimodal retention capabilities of the imidazolium‐embedded iodoacetamide‐functionalized silica‐based column could offer a wide range of retention behavior and flexible selectivity toward hydrophilic and hydrophobic compounds.     

Overview of the retention in subcritical fluid chromatography with varied polarity stationary phases

Retention and separation of achiral compounds in supercritical fluid chromatography (SFC) depend on numerous parameters: some of these parameters are identical to those encountered in HPLC, such as the mobile phase polarity, while others are specific to SFC, as the density changes of the fluid, due to temperature and/or pressure variations. Additional effects are also related to the fluid compressibility, leading to unusual retention changes in SFC, for instance when flow rate or column length is varied. These additional effects can be minimised by working at lower temperatures in the subcritical domain, simplifying the understanding of retention behaviours. In these subcritical conditions, varied modifiers can be mixed to carbon dioxide, from hexane to methanol, allowing tuning the mobile phase polarity. With nonpolar modifiers, polar stationary phases are classically used. These chromatographic conditions are close to the ones of normal-phase LC. The addition of polar modifiers such as methanol or ACN increases the mobile phase polarity, allowing working with less polar stationary phases. In this case, despite the absence of water, retention behaviours generally follow the rules of RP LC. Moreover, because identical mobile phases can be used with all stationary phase types, from polar silica to nonpolar C18-bonded silica, the classical domains, RP and normal-phase, are easily brought together in SFC. A unified classification method based on the solvation parameter model is proposed to compare the stationary phase properties used with the same subcritical mobile phase.     

Polarizable biphenyl polysiloxane stationary phase for capillary column gas chromatography

A crosslinkable biphenylmethylpolysiloxane stationary phase was synthesized for capillary column gas chromatography and compared with methyl, phenyl, and cyanopropyl polysiloxane stationary phases for the separation of isomeric polycyclic aromatic compounds. While the new phase gave similar separations of nonpolar isomers when compared to the nonpolar phases, separations of polar isomers were greatly improved because of the induced polarity of the biphenyl group of the stationary phase by the solute molecules. This polarizable stationary phase offers a unique selectivity which is not available in other stationary phases.     

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.