Narrow-bore Column Research Articles

Fast gas chromatography-tandem mass spectrometry under milder electron ionization conditions for the assay of vitamin D metabolites in human serum

The proposed research was focused on the development of a gas chromatography-tandem mass spectrometry (GC-QqQ-MS) method under milder electron ionization (EI) conditions for the assay of vitamin D metabolites in human serum. Efficiency of three different silylation agents was evaluated for the conversion of vitamin D species into trimethylsilyl (TMS) derivatives, among which N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) proved to be the most effective. Indeed, the MSTFA reagent was able to convert in TMS ether even the 25-hydroxyl vitamin D derivative that, as known, possesses steric hindrance problems. The separation of vitamin D compounds was obtained in about 11.5 min using a narrow-bore column of dimensions 30 m × 0.25 mm ID × 0.10 μm df with a poly(5% diphenyl/95% dimethyl siloxane) stationary phase. The mass spectrometry ionization of the silylated derivatives was performed under milder EI conditions (20-eV energy) that, respect to common 70-eV energy, generated scan mass spectra with higher relative and absolute intensities of high-mass diagnostic ions, along with a reduced abundance of the low-mass. The signals of the ionized compounds were acquired in multi-reaction-monitoring (MRM) mode, thus enabling the obtainment of highly-sensitive and selective quantitative data. The developed method was validated in term of linearity, accuracy, limits of detection (LoD) and quantification (LoQ). In detail, regression coefficients of the calibration curves were between 0.9959 and 0.9999; LoDs ranged from 0.06 ng mL−1 to 0.73 ng mL−1 and LoQs from 0.16 ng mL−1 to 2.45 ng mL−1. With respect to accuracy, the serum SRM 972a certified reference material (Vitamin D metabolites in frozen human serum) (Levels 1–4) was analyzed.

Development of Narrow-Bore C18 Column for Fast Separation of Peptides and Proteins in High-Performance Liquid Chromatography.

Separation with high efficiency and good resolution is constantly in demand in the pharmaceutical industry. The fast and efficient separation of complex samples such as peptides and proteins is a challenging task. To achieve high efficiency with good resolution, chromatographers are moving towards small particles packed into narrow-bore columns. Silica monolith particles (sub-2 µm) were derivatized with chlorodimethyl octadecyl silane (C18) and packed into stainless steel columns (100 mm × 1.8 mm i.d) by a slurry-packing method. The developed columns were used for the separation of peptides and proteins. A separation efficiency (N) of 40,000 plates/column (400,000 plates/m) was achieved for the mixture of five peptides. Similarly, the fast separation of the peptides was carried out using a high flow rate, and the separation of the five peptides was achieved in one minute with high efficiency (N ≅ 240,000 plates/m). The limit of detection (DL) and the limit of quantification (QL) for each analyte were determined by developing a linear regression curve with relatively very low concentrations of the target compound. The average values of the QL for the peptide and proteins were 0.55 ng and 0.48 ng, respectively, using short C18 column (1.8 mm × 100 mm) UV (at 214 nm). The fast analysis of peptides and proteins with such high efficiency and good resolution has not been reported in the literature yet. Owing to high efficiency, these home-made columns could be used as an alternative to the expensive commercial columns for peptide and protein separation.

Determination of Haloacetic Acids, Disinfection Byproducts, in Tap Water with Reversed-Phase Ultra-Performance Liquid Chromatography-High Resolution Mass Spectrometry

To establish a method for quantitative analysis of haloacetic acids (HAAs), disinfection byproducts, in tap water with reversed-phase ultra-performance liquid chromatography-quadrupole-orbitrap high resolution mass spectrometry. Tap water samples were collected and 0.70 g/L ascorbic acid was added to eliminate residual chlorine. Then, the water samples were directly injected into the instrument for analysis after filtration. After separation on a pentafluorobenzene (PFP) column with an inner diameter of 1.0 mm at a higher linear velocity and a lower volume flow rate compared with those of a narrow-bore column, nine HAAs, namely, monochloroacetic acid (MCAA), monobromoacetic acid (MBAA), dichloroacetic acid (DCAA), bromochloroacetic acid (BCAA), dibromoacetic acid (DBAA), trichloroacetic acid (TCAA), bromodichloroacetic acid（BDCAA), chlorodibromoacetic acid (CDBAA) and tribromoacetic acid (TBAA), were examined by negative electrospray ionization and full MS/dd-MS 2 acquisition mode. In order to adjust for the matrix effect, matrix matching calibration curves were used to quantitate the nine HAAs. Good linearity was obtained for each of the nine HAAs within their respective linear ranges. The detection limits and quantification limits of the method were 0.020-1.0 μg/L and 0.060-3.0 μg/L. The recoveries were 69.8%-119%. The proposed method showed strengths in separation speed and qualitative accuracy. It did not require for complicated pretreatment procedures and can meet the need of tap water sample analysis.

A comparative study of two HPLC methods for dissolved monosaccharide analysis in seawater using 2-amino benzamide and 2-amino pyrazine as pre-column derivatization reagents

Two reversed-phase HPLC methods for molecular analysis of dissolved free monosaccharides in seawater were investigated comparatively by optimizing chromatographic separations and pre-column derivatization reactions. Monosaccharides derivatized with 2-amino benzamide or 2-amino pyrazine, and reduced with 2-picoline borane or 2-dimethylamino borane. According to results of optimization, separation performance, and detection limits, 2-amino benzamide method gave better results than 2-amino pyrazine method. Among 12 monosaccharides tested, it was possible to quantify glucose + galactose, galacturonic acid, glucuronic acid, xylose + arabinose, ribose, mannose, and N-acetyl-d-glucosamine with detection limits between 1.2 and 11 nM with intra-day repeatability of 2–9% and inter-day repeatability of 3–9%. The optimized method has the same level of detection limit with a widely used anion exchange chromatography method. Besides the preliminary results reported in this study, it may be possible to achieve higher sensitivity and to detect more monosaccharides by the use of shorter and narrow-bore columns at different polarities in further studies.

Analysis of chemical components of Chinese medicine Ligustici Radix by achiral-chiral liquid chromatography-predictive multiple reaction monitoring

中药毛前胡为伞形科植物短片藁本Ligusticum brachylobum Franch.的干燥根,主要用于治疗风热咳嗽痰多、痰热喘满、咯痰黄稠等证,富含香豆素类化学成分,含有多组对映异构体和非对映异构体。为了深入研究毛前胡的化学成分组成,特别是对映异构体的组成,研究建立了非手性-手性色谱-预测多反应监测法(achiral-chiral-LC predictive MRM),同步实现毛前胡化学成分的化学选择性和立体选择性分离,以及高灵敏度定性分析。非手性色谱和手性液相色谱-串联质谱系统结合了RP-C18色谱柱的高效化学选择性分离能力以及手性色谱柱的立体选择性优势,有效避免了中心切割非手性-手性二维液相色谱构造复杂、重现性难以满足定量要求等缺陷。采用小内径核-壳型RP-C18色谱柱作为前端化学分离柱,实现结构类似香豆素的高效化学选择性分离;采用反相大内径AD-RH手性色谱柱,实现对映异构体的手性拆分;采用预测多反应监测模式,实现化学成分的高灵敏度检出;利用增强子离子扫描模式(EPI)采集各色谱峰的二级质谱信息,鉴定化学结构。通过定量离子对、定性离子对及两者的比值,判定是否为对映异构体。利用所构建的非手性-手性色谱耦联系统从毛前胡中共鉴定出60个化学成分,其中8对香豆素对映异构体得到了良好分离。本研究为毛前胡以及含有对映异构体中药的深入定性、定量分析提供可靠的方法。

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.

Vapor-phase chromatography in glass-capillary column of high polar and involatile hydrocarbon-containing ether derivatives of disulpho-benzoic acid

The paper deals with the results of capillary columns implementation in vapor-phase chromatography. Glass and narrow-bore columns covered with liquid stationary phase and polyphenyl ether were used, the mixtures of aliphatic alcohols, acids, amines and ether derivatives of disulpho-benzoic acid, which are semi-products of additive synthesis analyzed. The usage of water vapor as a moving carrier of nitrogen enabled to decrease the analysis time and improve division efficiency. The retention time, height values of equivalent theoretic plates and coefficient of analyzed components asymmetry were calculated. Obtained results justify the practicability of glass capillary application for the analysis of disulpho-benzoic acid derivatives via vapor-phase chromatography method.

Ionic liquids as water-compatible GC stationary phases for the analysis of fragrances and essential oils: Quantitative GC–MS analysis of officially-regulated allergens in perfumes

Qualitative and quantitative determination of volatile markers in aqueous based fragrances assumes ever-increasing importance, because of both the need for quality control and the safety-regulatory limitations introduced for several compounds. This study reports and critically discusses the results of applying new water-compatible ionic-liquid (IL) GC stationary phases, based on phosphonium and imidazolium derivative cations combined with trifluoromethanesulphonate (Watercol™) to the direct quantitative analysis of aqueous samples in the perfume field with GC–MS. Narrow-bore columns of different lengths, especially prepared for this study, were adopted to minimize the amount of water reaching the MS detector after GC separation. All GC–MS analysis steps were investigated, to achieve results compatible with quality control requirements for the volatiles of interest in this field, in terms of LODs, LOQs, and repeatability. Reliability of the GC–MS results was demonstrated by determining volatile allergens in two commercial perfumes, as per EU regulations concerning no-declaration limits for leave-on (0.001%) and rinse-off (0.01%) cosmetic products.

Slow injector-to-column sample transport to maximize resolution in liquid chromatography: Theory versus practice

A simple chromatographic model including extra-column sample bandspreading is built and validated experimentally. It is used to quantify the advantage of slowly transporting under isocratic elution of the injected sample band along the pre-column volume of the chromatographic system compared to the conventional injection method at constant flow rate. For fast analyses (<30 s), the model predicts a maximum relative gain of 20% for the isocratic peak capacity of weakly retained compounds (k < 1) at the price of a 60% increase in retention time (+20 s) for short (3.0 cm long) narrow-bore (2.1 mm i.d.) columns packed with sub-2 μm particles and for low-dispersion (∼2 μL2 extra-column volume variance) vHPLC systems. These predictions were confirmed experimentally using a 2.1 mm × 30 mm column packed with 1.9 μm XBridge-C18 for the rapid separation of small molecules in 25 s at 40 °C using a mixture of methanol and water (75/25, v/v) as the eluent. For longer analyses (>30 s), the model allows for the determination of the optimum and slow injection speed that maximizes the trade-off between the analysis time and the isocratic peak capacity. For 2.1 mm × 20 mm columns packed with 2 μm particles and a highest retention factor of 10, the optimum injection speed is about 10–15% of the operating column flow rate for a maximum relative gain of 5% in overall isocratic peak capacity at constant elution time. Overall, the proposed injection method is directly applicable for enhanced resolution of weakly retained polar compounds in RPLC without the need of changing separation conditions, which may strongly increase the retention times of the late apolar eluters.

Simultaneous Determination of Substance P and CGRP in Rat Brainstem Tissue Using LC-ESI-MS/MS Method

Background: Substance P and CGRP are neuropeptides that belong to the thachykinin and calcitonin family, respectively. They play an important role in neuropathic pain development and regulation. Methods: In this study, simultaneous analysis of Substance P and CGRP in rat brainstem tissue was conducted by using LC-ESI-MS/MS method. A gradient mobile phase was used with a Zorbax C8 150x2.1 mm, a particle size of 3.5 µm narrow-bore column. Mobile phases consist of 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (v/v) (B). Firstly, mass spectrometry parameters were optimized. After achieving chromatographic separation, the method has been validated and applied to rat brainstem samples. Results: SP and CGRP were eluted at 8.7 min and 10.1 min, respectively. Mass spectrum of neuropeptides was obtained in positive polarity and ion transitions of Substance P and CGRP were monitored at m/z 674.4→[600+254.4] and 952.2→1214.200, respectively. Linearity range for quantification was 5.85-438.52 ng/mL for Substance P and 0.21-155.28 ng/mL for CGRP. The limit of quantification was 5.85 ng/mL for Substance P and 0.21 ng/mL for CGRP. According to tissue samples analysis (n=6), basal endogen concentrations of Substance P and CGRP in brainstem tissue were calculated as 260.57±11.24 ng/g and 22.53±0.75 ng/g respectively. Conclusion: A novel LC-ESI-MS/MS method was successfully developed and validated according to the bioanalytical validation guideline published by the EMA including precision, accuracy, specificity and linearity. Acceptable results were achieved. Basal endogen concentrations of Substance P and CGRP in brainstem tissue were determined. Keywords: Substance P, CGRP, LC-ESI-MS/MS, rat brainstem tissue, neuropeptide, determination.

A Fast Quantitative Multi-analyte Method for Growth Promoters in Bovine Meat Using Bead-Disruption, 96-well SPE Clean-up and Narrow-Bore UHPLC-MS/MS Analysis

A new method for detecting low levels of growth promoters in bovine meat was developed with the following goal: easy, fast and sensitive analysis of a wide range of compounds, with reduced consumption of chemicals and disposables. Several classes of growth promoters were included, i.e. resorcylic acid lactones (RALs) and steroids, the latter including corticosteroids and gestagens. For sample treatment, 0.5 g of homogenised bovine meat was simultaneously disrupted and extracted in a bead-ruptor machine. The organic extraction solvent was further processed by solid-phase extraction (SPE) clean-up using 96-Well Oasis® HLB Plates. Six SPE washing steps were applied to remove matrix compounds after which the growth promoters were eluted and analysed using UHPLC-MS/MS. To achieve lower detection levels and to reduce LC-solvent consumption, a narrow-bore column with an internal diameter of 1 mm was used, instead of the conventional 2.1 mm. During analysis, the mass spectrometer was operated in negative and positive ionisation mode (ion switching). The newly developed method was validated according to the Commission Decision 2002/657. The results demonstrate that the method meets the criteria as established in this Commission Decision. The precision of the method for exogenous steroids varies between 85 and 115%, the CCα for the compounds ranges from 0.1–0.9 μg kg−1 and the expanded measurement uncertainty was lower than 36%. Compared to our current in-house methods with analysis times of 2 days for a maximum of 24 samples, the new method offers improved sample throughput (96 samples in less than 24 h) and lower detection limits.

Ultrafast Chiral Chromatography as the Second Dimension in Two-Dimensional Liquid Chromatography Experiments.

Chromatographic separation and analysis of complex mixtures of closely related species is one of the most challenging tasks in modern pharmaceutical analysis. In recent years, two-dimensional liquid chromatography (2D-LC) has become a valuable tool for improving peak capacity and selectivity. However, the relatively slow speed of chiral separations has limited the use of chiral stationary phases (CSPs) as the second dimension in 2D-LC, especially in the comprehensive mode. Realizing that the recent revolution in the field of ultrafast enantioselective chromatography could now provide significantly faster separations, we herein report an investigation into the use of ultrafast chiral chromatography as a second dimension for 2D chromatographic separations. In this study, excellent selectivity, peak shape, and repeatability were achieved by combining achiral and chiral narrow-bore columns (2.1 mm × 100 mm and 2.1 mm × 150 mm, sub-2 and 3 μm) in the first dimension with 4.6 mm × 30 mm and 4.6 mm × 50 mm columns packed with highly efficient chiral selectors (sub-2 μm fully porous and 2.7 μm fused-core particles) in the second dimension, together with the use of 0.1% phosphoric acid/acetonitrile eluents in both dimensions. Multiple achiral × chiral and chiral × chiral 2D-LC examples (single and multiple heart-cutting, high-resolution sampling, and comprehensive) using ultrafast chiral chromatography in the second dimension are successfully applied to the separation and analysis of complex mixtures of closely related pharmaceuticals and synthetic intermediates, including chiral and achiral drugs and metabolites, constitutional isomers, stereoisomers, and organohalogenated species.

On the feasibility to conduct gradient liquid chromatography separations in narrow-bore columns at pressures up to 2000 bar

The limits in operating pressures are extended for narrow-bore columns in gradient elution up to 2000bar. As the required pumps for these pressures are incompatible with common chromatographic solvents and are not suitable to apply a mobile phase composition gradient, a mobile phase delivery and injection system is described and experimentally validated which allows to use any possible chromatographic solvent in isocratic and gradient elution. The mobile phase delivery and injection system also allows to perform multiple separations without the need to depressurize the column. This system consists out of 5 dual on/off valves and two large volume loops in which the gradient and equilibration volume of initial mobile phase are loaded by a commercial liquid chromatography pump. The loops are then flushed toward the column at extreme pressures. The mobile phase delivery and injection system is first evaluated in isocratic elution and shows a comparable performance to a state-of-the-art commercial flow-through-needle injector but with twice the pressure rating. Distortion of the loaded gradient by dispersion in the gradient storage loop is studied. The effect of the most important parameters (such as flow rate, pressure and gradient steepness) is experimentally investigated. Different gradient steepnesses and volumes can be applied at different flow rates and operating pressures with a good repeatability. Due to the isobaric operation of the pumps, the gradient is monitored in real-time by a mass flow meter installed at the detector outlet. The chromatograms are then converted from time to volume-base. A separation of a 19-compound sample is performed on a 300×2.1mm column at 1000bar and on a 600×2.1mm column at 2000bar. The peak capacity was found to increase from 141 to 199 and thus scales with L as is predicted by theory. This allows to conclude that the inlet pressure for narrow-bore columns in gradient elution can be increased up to 2000bar without fundamental pressure-induced limitations.

An integrated electrophoretic mobility control device with split design for signal improvement in liquid chromatography–electrospray ionization mass spectrometry analysis of aminoglycosides using a heptafluorobutyric acid containing mobile phase

Electrophoretic mobility control (EMC) was used to alleviate the adverse effect of the ion-pairing agent heptafluorobutyric acid (HFBA) in the liquid chromatography–electrospray ionization mass spectrometry (LC-ESI-MS) analysis of aminoglycosides. Aminoglycosides separated by LC were directed to a connecting column before their detection via ESI. Applying an electric field across the connecting column caused the positively charged aminoglycosides to migrate toward the mass spectrometer whereas the HFBA anions remained in the junction reservoir, thus alleviating the ion suppression caused by HFBA. To accommodate the flow rate of a narrow-bore column, minimize the effect of electrophoretic mobility on separation, and facilitate the operation, an integrated EMC device with a split design was fabricated. With the proposed EMC device, the signals of aminoglycosides were enhanced by a factor of 5–85 without affecting the separation efficiency or elution order. For the analysis of aminoglycosides in bovine milk, the proposed approach demonstrates a sensitivity that is at least 10 times below the maximum residue limits set by most countries.

Intrinsic advantages of packed capillaries over narrow-bore columns in very high-pressure gradient liquid chromatography

250μm×100mm fused silica glass capillaries were packed with 1.8μm high-strength silica (HSS) fully porous particles. They were prepared without bulky stainless steel endfittings and metal frits, which both generate significant sample dispersion. The isocratic efficiencies and gradient peak capacities of these prototype capillary columns were measured for small molecules (n-alkanophenones) using a home-made ultra-low dispersive micro-HPLC instrument. Their resolution power was compared to that of standard 2.1mm×100mm very high-pressure liquid chromatography (vHPLC) narrow-bore columns packed with the same particles. The results show that, for the same column efficiency (25000 plates) and gradient steepness (0.04min−1), the peak capacity of the 250μm i.d. capillary columns is systematically 15–20% higher than that of the 2.1mm i.d. narrow-bore columns. A validated model of gradient chromatography enabled one to predict accurately the observed peak capacities of the capillary columns for non-linear solvation strength retention behavior and under isothermal conditions. Thermodynamics applied to the eluent quantified the temperature difference for the thermal gradients in both capillary and narrow-bore columns. Experimental data revealed that the gradient peak capacity is more affected by viscous heating than the column efficiency. Unlike across 2.1mm i.d. columns, the changes in eluent composition across the 250μm i.d. columns during the gradient is rapidly relaxed by transverse dispersion. The combination of (1) the absence of viscous heating and (2) the high uniformity of the eluent composition across the diameter of capillary columns explains the intrinsic advantage of capillary over narrow-bore columns in gradient vHPLC.

Effect of pre- and post-column band broadening on the performance of high-speed chromatography columns under isocratic and gradient conditions

We report on the results of an experimental and theoretical study of the effect of the extra-column band broadening (ECBB) on the performance of narrow-bore columns filled with the smallest particles that are currently commercially available. Emphasis is on the difference between the effect of ECBB under gradient and isocratic conditions, as well as on the ability to model and predict the ECBB effects using well-established band broadening expressions available from the theory of chromatography. The fine details and assumptions that need to be taken into account when using these expressions are discussed. The experiments showed that, the steeper the gradient, the more pronounced the extra-column band broadening losses become. Whereas the pre-column band broadening can in both isocratic and gradient elution be avoided by playing on the possibilities to focus the analytes on top of the column (e.g. by using the POISe injection method when running isocratic separations), the post-column extra-column band broadening is inescapable in both cases. Inducing extra-column band broadening by changing the inner diameter of the post-column tubing from 65 to 250μm, we found that all peaks in the chromatogram are strongly affected (around a factor of 1.9 increase in relative peak width) when running steep gradients, while usually only the first eluting peak was affected in the isocratic mode or when running shallow gradients (factor 1.6–1.8 increase in relative peak width for the first eluting analyte).

Conventional and enantioselective gas chromatography with microfabricated planar columns for analysis of real-world samples of plant volatile fraction

Within a project exploring the application of lab-on-chip GC to in-field analysis of the plant volatile fraction, this study evaluated the performance of a set of planar columns (also known as microchannels, MEMS columns, or microfabricated columns) of different dimensions installed in a conventional GC unit. Circular double-spiral-shaped-channel planar columns with different square/rectangular sections up to 2m long were applied to the analysis of both essential oils and headspace samples of a group of medicinal and aromatic plants (chamomile, peppermint, sage, rosemary, lavender and bergamot) and of standard mixtures of related compounds; the results were compared to those obtained with reference narrow-bore columns (l:5m, dc:0.1mm, df:0.1μm). The above essential oils and headspaces were first analyzed quali-and quantitatively with planar columns statically coated with conventional stationary phases (5%-phenyl-polymethylsiloxane and auto-bondable nitroterephthalic-acid-modified polyethylene glycol), and then submitted to chiral recognition of their diagnostic markers, by enantioselective GC with a planar columns coated with a cyclodextrin derivative (30% 6I–VII-O-TBDMS-3I–VII-O-ethyl-2I–VII-O-ethyl-β-cyclodextrin in PS-086). Column characteristics and analysis conditions were first optimized to obtain suitable retention and efficiency for the samples investigated. The planar columns tested showed performances close to the reference conventional narrow-bore columns, with theoretical plate numbers per meter (N/m) ranging from 6100 to 7200 for those coated with the conventional stationary phases, and above 5600 for those with the chiral selector.

Identification of isobaric lyso-phosphatidylcholines in lipid extracts of gilthead sea bream (Sparus aurata) fillets by hydrophilic interaction liquid chromatography coupled to high-resolution Fourier-transform mass spectrometry.

The numerous and varied biological roles of phosphatidylcholines (PC) and lysophosphatidylcholines (LPC) have fueled a great demand for technologies that enable rapid, in-depth structural examination of these lipids in foodstuffs. Here, we describe the capabilities of a newly configured combination of high-efficiency liquid chromatography and high-resolution/accuracy Fourier-transform mass spectrometry with electrospray ionization (LC-ESI-FTMS), designed for lipidomics applications that require the identification of PC in their lyso forms. The devised strategy, involving a separation by hydrophilic interaction liquid chromatography (HILIC) on spherical, fused-core ultrapure silica particles (2.7μm) of a narrow-bore column (2.1mm i.d.), enabled the identification of as many as 71 LPC species in the lipid extracts of gilthead sea bream (Sparus aurata) fillets. In this way, LPC as proton (43) and sodium (28) adducts, i.e., [M + H](+) and [M + Na](+) ions (with M representing the zwitterionic form), were identified. In several cases, the extremely high (sub-ppm) mass accuracy and the high chromatographic efficiency available with the adopted instrumentation enabled the distinction of isobaric and closely eluting LPC species. Informative tandem mass spectra, based on high-energy collision induced dissociation (HCD), were also obtained, thus distinguishing regioisomeric LPC species (i.e., LPC differing only for the location of the residual side chain on the glycerol backbone) and between proton and sodium adducts. Graphical Abstract Extracted Ion Current chromatogram (XIC) obtained for the m/z value 568.339, showing the presence of two regioisomeric Lysophosphatidylcholines. The corresponding high collisional energy tandem MS spectra, obtained using a HCD cell, are shown as insets.

Exploring the speed-resolution limits of supercritical fluid chromatography at ultra-high pressures

The limits of supercritical fluid chromatography have been experimentally explored using inlet pressures at the limits of the current commercial instrumentation (400–600 bar), as well as pressures significantly surpassing this (up to 1050 bar). It was found that efficiencies in the range of 200,000 theoretical plates can be achieved for a void time t0 of roughly 6min using superficially porous particles (2.7 and 4.6μm) while remaining within the pressure limits of current commercial instrumentation and columns. If lower efficiencies are sufficient (<100, 000 plates), smaller particles (e.g. 1.8μm) provide the best trade-off between analysis time and efficiency. Apparent efficiencies of 83,000 (k′=2.2) to 76,000 (k′=6.6) plates could be achieved for void times around 1min by pushing the pressure limits up to 1050 bar on a column length of 500mm. As the optimal mobile phase velocity for these small particle columns is even higher, it is required to use narrow-bore columns (2.1mm ID) to remain within the instrument limits of flow rate. The smaller column volume however puts a strain on the separation efficiency due to extra-column band broadening, resulting in losses up to 50% for weakly retained compounds for column lengths below 250mm. It is also illustrated that when using sub-2μm particles, especially for separations where a significant amount of organic modifier is required, the current pressure limits of state-of-the-art instrumentation can sometimes be insufficient. For a gradient running from 4 to 40 v% methanol on a 300mm column at the optimal flow rate the pressure increases from 420 to 810 bar. Operating SFC-columns with a large pressure gradient induces several (undesired) side effects which have been investigated as well. It has been found that, since the viscosity increases strongly with pressure in SFC, the optimal flow rate and the minimal plate height can significantly change when the column length is changed. Whereas e.g. a 3×150mm column (2.7μm particles) has an optimal flow rate of 1.5ml/min and minimal plate height of 5.66μm, a 3×1050mm column has an optimal flow rate of 1.2ml/min and a minimal plate height of 6.25μm. Nevertheless, an increase in operating pressure drop in SFC results in a significant gain in kinetic performance.

Characterization and kinetic performance of 2.1 × 100 mm production columns packed with new 1.6 μm superficially porous particles.

The overall kinetic performance of three production columns (2.1 mm × 100 mm format) packed with 1.6 μm superficially porous CORTECS-C18 + particles was assessed on a low-dispersive I-class ACQUITY instrument. The values of their minimum intrinsic reduced plate heights (h(min) = 1.42, 1.57, and 1.75) were measured at room temperature (295 K) for a small molecule (naphthalene) with an acetonitrile/water eluent mixture (75:25, v/v). These narrow-bore columns provide an average intrinsic efficiency of 395,000 plates per meter. The gradient separation of 14 small molecules shows that these columns have a peak capacity about 25% larger than similar ones packed with fully porous BEH-C18 particles (1.7 μm) or shorter (50 mm) columns packed with smaller core-shell particles (1.3 μm) operated under very high pressure (>1000 bar) for steep gradient elution (analysis time 80 s). In contrast, because their permeabilities are lower than those of columns packed with larger core-shell particles, their peak capacities are 25% smaller than those of narrow-bore columns packed with standard 2.7 μm core-shell particles.