Two-dimensional Supercritical Fluid Research Articles

Development of a Two-Dimensional Supercritical Fluid Chromatography System in Multiple Heart-Cutting Modes.

In this study, we present the development of a loop-based two-dimensional supercritical fluid system in multiple heart-cutting modes (mSFC-SFC), with diode-array and mass spectrometric detection. The instrument design was developed to be as simple as possible, based on a single SFC instrument, with the sole addition of three external 2-port 6-position valves. The objective was to achieve the most complete transfer of a peak from the first to the second dimension, whatever the composition of the mobile phase, i.e., whatever the proportion of carbon dioxide and methanol cosolvent along a wide gradient elution. Thanks to fine adjustment of the valve switching times, the first-dimension peaks were parked in 50 μL or 100 μL loops and later discharged to the second dimension. The interest of this instrument was then demonstrated with a sample application on a natural product: an extract of Citrus aurantium L. bark was analyzed, with a particular focus on chiral flavonoids, neohesperidin, and naringin. In this system, the first dimension was an achiral separation of the flavonoids, based on a polar diethylamine-bonded silica stationary phase (ACQUITY Torus DEA), while the second dimension used a stereoselective polysaccharide stationary phase (CHIRALPAK IB-3) to resolve flavonoid diastereomers. Excellent repeatability was demonstrated, with relative standard deviation values on retention times and peak areas all below 2%, together with excellent peak capacity and peak shapes (no distortion observed), making it possible to quantify diastereomers in the second-dimension separation. This good repeatability was also shown for the transfer rate between the two dimensions, which reached a value of 83%. Finally, transferring a compressible sample from the first to the second dimension is demonstrated to yield excellent performance, despite the large loop volume.

Determination of vitamin D3 in daily oily supplements by a two-dimensional supercritical fluid chromatography-liquid chromatography-mass spectrometry system.

A two-dimensional system composed of supercritical fluid chromatography (SFC) and reverse phase liquid chromatography (RPLC) coupled a tandem mass spectrometry (MS) was developed for the quantitative analysis of vitamin D in daily oily supplements. Two six-port switching valves are configured, allowing four different valve positions. When the valve positions were fixed at Position A, this system worked at SFC-MS mode. When the valve positions switched between Position B and C, this system worked at a SFC-LC-MS switching mode. Vitamin D3 in two kinds of oily drops, Baby Ddrops and Vitamin AD drops, was determined at both SFC-MS and SFC-LC-MS switching modes by using the same system. The linearity, repeatability and recovery were investigated using the internal and external standard methods for the two modes. The results obtained from the internal standard method are better than those of the external standard method at either mode. The coefficient of determination (r2) for the internal standard method is more than 0.999, with a linear range of 20-1000μg/L. Both Baby Ddrops and Vitamin AD drops were analyzed with good repeatability (<3.21%) and recovery (94.1%-116.1%) using internal standard method. When calculated by the external standard method, as compared to SFC-MS mode, SFC-LC-MS switching mode has better linearity (r2>0.999), repeatability (Baby Ddrops: 4.45%, Vitamin AD drops: 1.12%) and recovery (86.3%-107.1%). The results indicate that the two-dimensional SFC-MS/SFC-LC-MS system is useful for determination of vitamin D in oily drops. If it works at SFC-MS mode, the internal standard is required. When SFC-LC-MS switching mode is used, external standard method can also obtain the accurate results with good precision.

An offline two-dimensional supercritical fluid chromatography × reversed phase liquid chromatography tandem quadrupole time-of-flight mass spectrometry system for comprehensive gangliosides profiling in swine brain extract

Gangliosides, widely distributed in tissues and body fluid, have been connected to the therapy of cancer and brain related diseases. The complexity of the gangliosides structures with different polar moieties coexisting, a carbohydrate moiety and a ceramide chain, make it a great challenge in separation and analysis science. This study aimed to develop a strategy on the basis of high-accuracy data collected by an offline two-dimensional (2D) supercritical fluid chromatography (SFC) × reversed phase liquid chromatography (RPLC)/quadrupole time-of-flight (Q-ToF) system, and to integrate an in-house library with self-developed software for fast screening and identification of gangliosides from a complex sample (swine brain extract). Subsequent positive-mode MS/MS was used to validate the identified gangliosides. Finally, 153 gangliosides were separated and 79 of them were identified by the in-house library and self-developed software, 4-fold more than those by manual identification (18 gangliosides). Among the identified ones, 20 were detected in swine brain for the first time. This study established an offline 2D SFC × RPLC system and provided a new method for fast screening and automatic identification of gangliosides in complex mixtures. It will be conducive to further study of biological functions of gangliosides.

Development of a heart-cutting supercritical fluid chromatography-high-performance liquid chromatography coupled to tandem mass spectrometry for the determination of four tobacco-specific nitrosamines in mainstream smoke

This paper proposed a newly developed heart-cutting two-dimensional supercritical fluid chromatography-high-performance liquid chromatography system coupled to tandem mass spectrometry (SFC-HPLC-MS/MS) for the determination of four tobacco-specific nitrosamines (TSNAs) in cigarette mainstream smoke. The orthogonality of five SFC columns and two HPLC columns was evaluated. The 1-AA column was applied for the first dimensional (1D) SFC separation to isolate the target compounds from the complex cigarette smoke matrices, and a trapping column in conjunction with an isocratic pump was employed to capture the 1D elutes. Then, the trapped 1D elutes were transferred into the C18 column through a two-position/six-port valve for the second dimensional (2D) analysis. The ion suppression was significantly reduced by the established SFC-HPLC system; meanwhile, the matrix interferences were eliminated as the results demonstrated. A dynamic range between 0.1 and 20ng/mL was achieved with LOQs of 0.72μg/cig for N-nitrosonornicotine (NNN), 0.66μg/cig for nicotine-derived nitrosamine ketone (NNK), 0.81μg/cig for N-nitrosoanatabine (NAT), and 0.39μg/cig for N-nitrosoanabasine (NAB). All the results revealed that the presented method exhibited good repeatabilities and recoveries and could be used as a rapid and reliable approach for routine analysis of TSNAs in mainstream smoke.

Oxidative Depolymerisation of Lignosulphonate Lignin into Low-Molecular-Weight Products with Cu\u2013Mn/\u03b4-Al2O3

Lignin depolymerisation receives great attention due to the pressing need to find sustainable alternatives to fossil sources for production of fuels and chemicals. In this study, alumina-supported Cu–Mn and Ni–Mo catalysts were tested for oxidative depolymerisation of a technical lignin stream—sodium lignosulphonates—to produce valuable low-molecular-weight aromatics that may be considered for applications in the fuels and chemicals sector. The reactions were performed at elevated temperature and oxygen pressure, and the product mixtures were analysed by size exclusion chromatography, two-dimensional nuclear magnetic resonance spectroscopy and supercritical fluid chromatography mass spectrometry. The best performance was obtained with Cu–Mn/δ-Al2O3, which was thoroughly characterised before and after use by nitrogen physisorption, scanning electron microscopy, energy dispersive spectroscopy, powder X-ray diffraction, thermal gravimetric analysis, inductively coupled plasma optical emission spectrometry and X-ray photoelectron spectroscopy. Major products identified were vanillin, p-hydroxybenzaldehyde, vanillic acid and p-hydroxybenzoic acid as well as smaller aliphatic aldehydes, acids and lactones.

Study on the separation of triterpene saponins from Panax notoginseng by off-line two-dimensional supercritical fluid chromatography–ultrahigh-performance liquid chromatography

The off-line two-dimensional supercritical fluid chromatography (SFC)–ultrahigh-performance liquid chromatography (UHPLC) was selected to separate the triterpene saponins from Panax notoginseng. The separation by SFC was performed on an Atlantis® HILIC silica column. Methanol was selected as a modifier, and the most time-saving gradient was developed. The decrease of the column temperature and the increase of the back pressure could shorten the retention time but had no effect on the separation selectivity. Then, the back pressure, column temperature, and flow rate were set as 131 bar, 45 °C, and 4.0 mL min−1, respectively. The retention behavior of the saponins from P. notoginseng was different between SFC and reversed-phase liquid chromatography (RPLC), which facilitated to construct an off-line SFC/RPLC–mass spectrometry (MS) system. In first dimension, a total of eight fractions were collected under SFC and further analyzed by RPLC–MS in second dimension. The result indicated that the retention behavior of triterpene saponins was mainly controlled by the hydrogen bonding interactions which were affected by the number and types of sugars, as well as the aglycone in the structure of triterpene saponins. Thus, the presence of “clustering effect” under SFC was observed, namely, one SFC peak always contained several saponins with same number of sugars and similar structure of aglycone. The clustering effect of triterpene saponins promised SFC to be used as first dimension to complete the preliminary crude separation in the two-dimensional liquid chromatography.

Active modulation in neat carbon dioxide packed column comprehensive two-dimensional supercritical fluid chromatography

After demonstrating in a first paper the feasibility of SFCxSFC without decompression of the mobile phase, a modified interface has been developed in order to perform active modulation between the two SFC dimensions. In this paper, it is shown that the new interface enabled independent control of modulation parameters in SFCxSFC and performed a band compression effect of solutes between the two SFC dimensions. The effectiveness of this new modulation process was studied using a Design of Experiments. The SFCxSFC prototype was applied to the analysis of a real oil sample to demonstrate the benefits of the active modulator; in comparison to our previous results obtained without active modulation, better separation was obtained with the new interface owing to the peak compression occurring in the modulator.

Purification of lignans from Fructus Arctii using off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography.

As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high-purity compounds. In total, 12 high-purity compounds were isolated from Fructus Arctii. Their structures were identified by using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method for the purification of lignans from Fructus Arctii. The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products.

Alkaloids analysis using off-line two-dimensional supercritical fluid chromatography × ultra-high performance liquid chromatography.

In this study, an off-line two-dimensional (2-D) supercritical fluid chromatography (SFC) × ultra-high performance liquid chromatography (UHPLC) method with high orthogonality was developed for the analysis of the practical amide alkaloids fraction from P. longum L. The effects of SFC parameters such as column type, organic modifier, temperature and back-pressure on separation were systematically evaluated. Different selectivity was observed for different columns (BEH, BEH 2-EP, XAmide and CSH FP). An investigation was then carried out of the orthogonality of different columns and systems following a geometric approach with a set of amide alkaloid samples. The orthogonality between a CSH FP column and a BEH column reached 50.79%, which was much higher than that for the other columns. While the orthogonality between SFC and UHPLC based on an XAmide column and an HSS T3 column reached 69.84%, which was the highest of all the combinations. At last, the practical amide alkaloids fraction was analyzed with an off-line 2-D chromatography SFC × UHPLC system. In total, at least 340 peaks were detected by this method. Rapid separation in these two dimensions and easy post treatment of SFC facilitated this 2-D system for the separation of complex samples.

Two-dimensional supercritical fluid chromatography/mass spectrometry for the enantiomeric analysis and purification of pharmaceutical samples

A new analytical two-dimensional supercritical fluid chromatography/mass spectrometry system (2D SFC/SFC/MS) has been designed and implemented to enhance the efficiency and quality of analytical support in drug discovery. The system consists of a Berger analytical SFC pump and a modifier pump, a Waters ZQ 2000 mass spectrometer, a set of switching valves, and a custom software program. The system integrates achiral and chiral separations into a single run to perform enantiomeric analysis and separation of a racemic compound from a complex mixture without prior clean up. The achiral chromatography in the first dimension separates the racemate from all other impurities, such as un-reacted starting materials and by-products. Mass-triggered fractionation is used to selectively fractionate the targeted racemic compound based on its molecular weight. The purified racemate from the achiral chromatography in the first dimension is then transferred to the chiral column in the second dimension to conduct the enantiomeric separation and analysis. A control software program, we coined SFC2D, was developed and integrated with MassLynx to retrieve acquisition status, current sample information, and real time mass spectrometric data as they are acquired. The SFC2D program also monitors the target ion signal to carry out mass-triggered fractionation by switching the valve to fractionate the desired peak. The 2D SFC/SFC/MS system uses one CO 2 pump and one modifier pump for both first and second dimension chromatographic separations using either gradient or isocratic elution. Similarly, a preparative 2D SFC/SFC/MS system has been constructed by modifying an existing Waters preparative LC/MS system. All components except the back pressure regulator are from the original LC/MS system. Applications of the 2D SFC/SFC/MS methods to the separation and the analysis of racemic pharmaceutical samples in complex mixtures demonstrated that an achiral separation (in first dimension) and a chiral separation (in second dimension) can be successfully combined into a single, streamlined process both in analytical and preparative scale.

Supercritical fluid chromatography and two-dimensional supercritical fluid chromatography of polar car lubricant additives with neat CO 2 as mobile phase

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. Their total amount in the commercial lubricant varies from a few percents to 20–25%. 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 as it should involve sample treatment and preparative scale separations in order to simplify the sample. The aim of this work is to determine the feasibility of the separation of low molecular weight lubricant additives using various packed columns with pure CO 2 as a mobile phase to enable implementation of flame ionisation detection as universal detector. This is part of a hypernated system including more sophisticated specific detectors, such as AED, FTIR or MS to obtain detailed structural information of compounds. This paper is devoted to the comparison of some stationary phases supposed to provide hydrocarbon group type separation (silica and normal phase) or separations on alkyl-bonded silica in non-aqueous mode of some selected classes of additives in test mixtures or in base stocks. Adsorption chromatography allows partial separation of additives from the base stocks while the direct elution of test additives can only be obtained on reversed phase supports having a very efficient silanol group protection so the interaction of the more polar compounds is much reduced. A two-dimensional scheme of analysis is also described. It combines adsorption chromatography to separate most of the polar additives from the base stock and alkyl-bonded silica for more detailed separation of the additives. However, overlapping between groups of compounds and the lack of resolution between some additives and the base stock should be addressed by the implementing of selective detectors.

Development of Supercritical Fluid Extraction Coupled to Comprehensive Two-dimensional Supercritical Fluid Chromatography (SFE-SFC¥SFC)

Supercritical fluid extraction was coupled to comprehensive two-dimensional supercritical fluid chromatography with conventional packed columns. With this system, three modes of operations were feasible, such as SFE-SFC, SFCxSFC, and SFE-SFCxSFC. The operations for valve switching and data acquisition were automated using in-house software. In order to evaluate the present system, SFE-SFC for polymer additives in polypropylene, SFCxSFC for a mixture of benzene derivatives with different polarities, and SFE-SFCxSFC for styrene oligomers in polystyrene were performed. With SFE-SFCxSFC, diastereomers were resolved for linear tetramers and pentamers as well as trimers with the tetraline moiety.

Group-Type Analysis of Oxygenated Compounds with a Silica Gel Porous Layer Open Tubular Column and Comprehensive Two-Dimensional Supercritical Fluid and Gas Chromatography

A porous layer open tubular (PLOT) silica gel column was used together with subcritical CO2 as the mobile phase to effect the group separation of polar oxygenated compounds. Aliphatic and aromatic compounds were shown to elute together. This group was followed by ethers and aldehydes, which were separated from compounds containing an alcohol functional group. Compounds with a carboxylic acid moiety could also be eluted from the silica gel. The group separation obtained when a silica gel PLOT column is used together with subcritical CO2 was also demonstrated to be valuable as the first dimension of a comprehensive two-dimensional SFCxGC analysis where the GC analysis in the second dimension is performed with a fast and independently heated temperature programmed gas chromatograph. With this combination of SFC and GC, many of the oxygenated compounds, routinely found in petroleum samples, could successfully be separated and identified.

Comprehensive two-dimensional supercritical fluid and gas chromatography with independent fast programmed heating of the gas chromatographic column.

With comprehensive two-dimensional supercritical fluid and fast, independent temperature-programmed gas chromatography (SFCxGC), a polar column was used in the first dimension to achieve group-type analysis. The eluent of this separation was repetitively sampled and transferred to a fast, resistively heated gas chromatograph to obtain the boiling point distribution over the entire polarity separation. The SFC was operated isothermally with stopped flow to provide a sufficient time span for the GC analysis. The GC analysis had a typical cycle time of 1 min for the system demonstrated here. During this time, the GC column was independently heated at a rate of 450 degrees C/min to 250 degrees C and actively cooled again to -50 degrees C before the next GC injection took place. The analysis of petrochemical samples is presented to illustrate the technique.

Separation of fatty acid methyl esters by comprehensive two-dimensional supercritical fluid chromatography with packed columns and programming of sampling duration.

Fatty acid methyl esters from various fats and oils were separated by comprehensive two-dimensional supercritical fluid chromatography with conventional packed columns and FID detection. The first dimension was a silica gel column and the second dimension was an ODS column. This combination was largely orthogonal for the separation of fatty acid methyl esters. The first dimension separations were primarily based on the number of double bonds while the second dimension separations were based on the chain length. The highly-ordered chromatograms and improved resolution allowed the easy detection and identification of minor components. Although the first dimension separations were performed under isobaric conditions where the peak width increased in proportion to the retention, the programming of the sampling duration allowed us to maintain the optimum re-injection frequency (3-4 times) per peak into the second dimension and so to minimize the total analysis time without deteriorating the resolution.

Two-dimensional supercritical fluid mercury formed on a sapphire substrate

Under high temperature and pressure near the critical point of fluid mercury, a thin film of fluid mercury is formed on a sapphire substrate. The coverage of the mercury wetting film jumps discontinuously when a prewetting transition line is crossed. In the present work, we report our recent studies on the properties of the mercury wetting film on sapphire by using optical reflectivity measurements and optical emissivity measurements. In the prewetting supercritical region, we observed two types of anomalies, denoted by ‘EPW1’ and ‘EPW2’. ‘EPW1’ is characterized by a sharp dip in the emissivity, and is attributed to the fluctuations in the wetting film caused by the two-dimensional critical phenomena. ‘EPW2’ is characterized by a steep increase in the reflectivity, and is attributed to the cross-over from the complete wetting to the critical adsorption.

Correlation between Local and Long-Range Structure in Compressible Supercritical Lennard-Jones Fluids: State-Point Dependence

Evaluation of local-density-specific radial distribution functions in a two-dimensional supercritical Lennard-Jones fluid has enabled us to elucidate how the correlations between a solvent particle's local density and the extended surrounding solvent structure change as a function of the thermodynamic condition. We find that the standard correlation length provides only a very crude measure of these correlations and that, for example, such inhomogeneity-induced correlations remain present at bulk densities surprisingly far from the critical density in this two-dimensional system. Additionally, these local-density-specific radial distribution functions provide insight into the state-point dependence of the origin of mean local-density enhancements, showing that the potential-induced and the correlation-induced contributions both die out rapidly with increasing temperature, whereas they are maximally important at different bulk densities.

Domain-Based Characterization of Density Inhomogeneities in Compressible Supercritical Fluids

We develop a location-based coarse-graining method for characterizing solvent density inhomogeneities and use it to investigate a two-dimensional supercritical Lennard-Jones fluid. The strength of this method is that it treats any high- and low-density domains present in the fluid equivalently, rather than focusing on the high-density “clusters” alone. As the critical region is approached, we find that the distribution of local densities found in this fluid becomes quite broad, while, more globally, the high- and low-density domain structure changes from singly continuous to bicontinuous. Concurrently, these domains grow in size, as expected from the divergence of the correlation length; yet, we find that the correlation length is a very poor indicator of this change, greatly underestimating the length of large domains. This result is in part due to the very asymmetric topology of the domains, which, we find, is relatively invariant to changes in thermodynamic conditions or to the proximity of the critica...

What causes the vibrational lifetime plateau in supercritical fluids?

Using molecular dynamics simulation, we show that the vibrational relaxation lifetime of a diatomic in a two-dimensional supercritical rare-gas fluid exhibits the same unexpected plateau behavior as was observed experimentally for W(CO)6 in supercritical CO2 by Fayer and co-workers, and we link this behavior to average local solvent density enhancements arising from the solvent’s near-critical density fluctuations.

Quantitative hydrocarbon group analysis of gasoline and diesel fuel by supercritical fluid chromatography

A two-dimensional supercritical fluid chromatographic system is described for the determination of saturates, alkenes and mono-, di- and tri-aromatics in gasoline and diesel fuel. Aromatics were isolated from saturates/alkenes and group separated according to the number of aromatic rings on a capillary column packed with silica of particle size 4 μm. The alkenes were separated from saturates on a 38-mm capillary column packed with a silica-based cation exchanger which had been modified in situ with silver nitrate. Elution of alkenes was facilitated by the use of a relatively high column temperature, 65°C, and operation in the back-flush mode. Carbon dioxide was used as the mobile phase. Relative standard deviations were generally in the range 0.4–20.0%. At a low content of alkenes, 0.5%, a relative standard deviation of 8.4% was achieved. The analysis time was under 8 min. The utility of the method is demonstrated by the analysis of feedstock before and after hydration.