Supercritical Fluid Chromatography Research Articles

Novel Pinhole Emitter Chip for Micro Supercritical Fluid Chromatography-Mass Spectrometry with Integrated Dilution-Free Fluidic Back-Pressure Regulation.

We present a novel chip-based device featuring a pinhole emitter for mass spectrometry (MS) coupling with integrated fluidic back-pressure regulation for supercritical mobile phases. This design enables facile coupling of packed capillary columns used for supercritical fluid chromatography (SFC) with atmospheric pressure ionization mass spectrometry. The monolithic microfluidic chips were fabricated using selective laser-induced etching, seamlessly integrating multiple functions, including comb-shaped particle retention structures for column packing and ports for zero-clearance connection with standard fused silica capillaries. The integrated restrictive pinhole MS emitter generated by dielectric breakdown is a key innovation of the micro SFC-MS platform. It enables a controlled decompression of the supercritical CO2-based mobile phase within few micrometers to efficiently transfer the analytes from the compressed supercritical fluid into the ambient gas phase in front of the MS orifice. The inclusion of an arrowhead-shaped fluidic element further enables precise, dilution-free back-pressure regulation. With a minimal postcolumn volume of just 3 nL, the system shows excellent MS coupling performance, as demonstrated by rapid SFC-MS analysis of pharmaceuticals and natural products.

A non-target evaluation of drinking water contaminants in pilot scale activated carbon and anion exchange resin treatments

This study evaluates the effectiveness of five types of Granular Activated Carbon (GAC) and one anion exchange resin in a pilot plant for treating groundwater for drinking water production, specifically targeting the removal of persistent compounds like PFAS. Using liquid chromatography and supercritical fluid chromatography coupled with high-resolution mass spectrometry, hundreds of features (i.e. peak at specific mass and retention time) were detected in the groundwater by non-target analysis. Initially, after treating <3200 bed volumes (BV), the GAC filter materials showed < 6 % breakthrough for all features from the groundwater, with decreasing efficiency down to 79 % breakthrough after seven month (69,000 treated BV for µGAC). Using resin as a lag filter after GAC did not improve the removal of compounds detected in positive electrospray ionization mode. However, it enhanced removal by up to 35% for compounds detected in negative electrospray ionization mode, indicating higher selectivity of resin for acidic compounds like PFAS. The shortest detected PFAS (PFBA and PFPeA) broke through completely for all GAC and the resin material except the proprietary blended GAC (at 15,700 treated BV), which had only 19% breakthrough for PFPeA. The so far rarely detected perfluoro(4-ethylcyclohexane)sulfonic acid (PFECHS) was well adsorbed by GAC coupled to resin and by the proprietary blended GAC. Pesticides were effectively removed by GACs, but not by the resin filter. Contaminants not previously detected in groundwater, 2,4,5-trichlorobenzenesulfonic acid (TCBS) and 2-amino-4-chloro-5-methylbenzenesulfonic acid (ACMBS), were effectively removed (>92 %), but high ACMBS concentrations (360 ng/L) in groundwater are of concern. The drinking water after the resin filter revealed 20 new contaminants, such as tributylamine derivatives and monobutyl phthalate, indicating resin filters contribution to drinking water contamination. Accelerated migration experiments of the resin revealed additional contaminants, such as NDBA and further phthalates, highlighting the need for continued monitoring and evaluation of resin materials in water treatment systems.

Four-Dimensional Lipidomic Analysis Using Comprehensive Online UHPLC × UHPSFC/Tandem Mass Spectrometry.

Multidimensional chromatography offers enhanced chromatographic resolution and peak capacity, which are crucial for analyzing complex samples. This study presents a novel comprehensive online multidimensional chromatography method for the lipidomic analysis of biological samples, combining lipid class and lipid species separation approaches. The method combines optimized reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) in the first dimension, utilizing a 150 mm long C18 column, with ultrahigh-performance supercritical fluid chromatography (UHPSFC) in the second dimension, using a 10 mm long silica column, both with sub-2 μm particles. A key advantage of employing UHPSFC in the second dimension is its ability to perform ultrafast analysis using gradient elution with a sampling time of 0.55 min. This approach offers a significant increase in the peak capacity. Compared to our routinely used 1D methods, the peak capacity of the 4D system is 10 times higher than RP-UHPLC and 18 times higher than UHPSFC. The entire chromatographic system is coupled with a high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) in both full-scan and tandem mass spectrometry (MS/MS) and with positive- and negative-ion polarities, enabling the detailed characterization of the lipidome. The confident identification of lipid species is achieved through characteristic ions in both polarity modes, information from MS elevated energy (MSE) and fast data-dependent analysis scans, and mass accuracy below 5 ppm. This analytical method has been used to characterize the lipidomic profile of the total lipid extract from human plasma, which has led to the identification of 298 lipid species from 16 lipid subclasses.

Separation of phenylpropionic acids both by strong anion exchange stationary phase and strong cation exchange stationary phase in supercritical fluid chromatography, using the same additive

This study demonstrates the expanded application of ion-exchange stationary phases (including strong cation exchange, SCX and strong anion exchange, SAX) in supercritical fluid chromatography (SFC), and more importantly, provides a deeper understanding of the retention mechanisms of these two stationary phases when using the same acidic additive. Phenylpropionic acid compounds (belonging to phenolic acids) were selected as probes. On the SCX column, the π-π and polar interactions originating from the bonded benzenesulfonic acid groups were important foundations for prolonging the retention time of solutes, but they were also the main reason for solutes’ tailing profiles. It was found that adding 0.1% phosphoric acid can generate sufficient strength of electrostatic repulsion to obtain satisfactory peak shapes. Here, phosphoric acid can be adsorbed on the surface of the stationary phase to accumulate more negative charges, and at the same time, the phosphate anion and the phenylpropionic acid in mobile phase might combine into the contact ion pair (CIP)- like through the polar interactions to form the apparently negatively charged solute. On the SAX column, phosphate anions generated by ionization of phosphoric acid, were considered as counterions to effectively mask the electrostatic attraction of quaternary ammonium groups, to significantly reduce the retention of phenylpropionic acids, but improve their resolution. Finally, utilizing two developed SFC methods, i.e., SCX with MeOH + 0.1% phosphoric acid in CO2, or SAX with MeOH + 0.3% phosphoric acid in CO2, the baseline separation of the extract of Lonicerae Japonicae Flos was achieved within 6 min and 10 min, respectively.

Finding the ideal solvent for the analysis of polar analytes using supercritical fluid chromatography.

The analysis of polar analytes with the help of hydrophilic interaction liquid chromatography (HILIC) using classic methods of high-performance liquid chromatography is not without its downsides. In these applications, acetonitrile is prevalent as main eluent and sample diluent. This results not only in slow diffusion processes during the separation, but also in often unstable sample solutions where polar analytes are concerned. Furthermore, there are ecological concerns. With the use of supercritical fluid chromatography (SFC) which uses supercritical carbon dioxide as eluent, and other green solvents as alternative for the sample preparation, the separation of polar analytes could be vastly improved with this technique. Fast diffusion within carbon dioxide led to shorter analysis times and higher plate numbers. Regarding sample diluents, small alcohols such as ethanol and 2-propanol, as well as acetone, yielded promising results while analytes showed higher solubility and stability within these solvents compared to acetonitrile. Other green solvents such as dihydrolevoglucosenone (Cyrene) and dimethyl carbonate were found to be unsuitable sample diluents for applications in SFC.

An orthogonal approach for analysis of underivatized steroid hormones using ultrahigh performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS).

The crucial role of steroid hormones in health and diseases merits their high-throughput, accurate and affordable measurements in biological specimens. Despite advances in analytical methods, sensing and quantifying steroid hormones remains challenging. Immunoassays offer excellent sensitivity but are inherently labour-intensive, costly, and prone to false positives. Mass spectrometry (MS) has been increasingly utilised, with the main hurdle being the isobaric tendencies of similar analytes, which complicates their separation and accurate quantification. This study compares ultrahigh-performance supercritical fluid chromatography separation (UHPSFC) and ultra-high-performance liquid chromatography (UHPLC) for MS detection. It optimises the column chemistry, temperature, and pressure to provide an operational protocol for the resolution and quantification of analytes. It presents the systematic characterisation of UHPSFC-MS performance by investigating spiked blood samples using Solid-Phase Extraction (SPE) and describes the matrix effects associated with MS measurements. Although both separation methods showed adequate resolution, specificity, and retention time, UHPSFC-MS was superior for five out of seven columns tested. With added high-throughput capacities, UHPSFC-MS, thus, offers an optimal solution for the analysis of steroid hormones for research, medical chemistry, and clinical diagnostics.

Optimizing conditions in online RPLC × SFC for the analysis of complex samples containing neutral compounds: Solving injection issues

The online combination of reversed-phase liquid chromatography and supercritical fluid chromatography (online RPLC × SFC) is an attractive technique for the characterization of complex samples containing neutral compounds as the two techniques are highly complementary, especially with a polar stationary phase in supercritical fluid chromatography (SFC). However, the setup is challenging due to the presence of hydro-organic solvents in RPLC, which become injection solvent in SFC. In this study, numerous key experimental parameters were identified and found to have a major effect on peak shape under RPLC × SFC conditions. These parameters included the organic modifier in reversed-phase liquid chromatography (RPLC), the co-solvent in SFC, the gradient conditions and the column ID in SFC, the configuration of the valve and finally, the injection volume in SFC that should be maximized. Acetonitrile (ACN) in RPLC, a mixture of ACN and methanol (MeOH) (50/50, v/v) in SFC, a minimum initial composition of 5% B in SFC, column IDs of 1.0 mm and 2.1 mm in RPLC and SFC respectively and flushing the interface loops with pure CO2 while adding the co-solvent after the valve, are all conditions that have been identified as perfectly suitable for online RPLC × SFC. They were successfully applied to the online RPLC × SFC separations of microalgae bio-oil samples. Despite unusual injection conditions, the peaks were symmetrical over the entire chromatogram, leading to a high separation power.

Supercritical fluid chromatography for milligram preparation of enantiomers.

Preparative chromatographic enantioseparation is now the preferred technique for obtaining milligram quantities of pure enantiomers in the initial phase of development of a therapeutic compound. Supercritical fluid chromatography offers several advantages over liquid chromatography and was therefore selected for the preparative enantioseparation of a new potential anti-inflammatory molecule. Approximately 10mg of each of the two enantiomers was successfully prepared using a Chiralpak AD-H (tris-3,5-dimethylphenylcarbamate of amylose) polysaccharide-based stationary phase with 40% of ethanol as a co-solvent, using a stacked injection mode. A peak distortion was observed during volume overloading, which may be due to the mixed-stream injection method.

A Dual Column pH Switchable Water Stationary Phase System for Separation Control in Supercritical Fluid Chromatography.

A dual column system comprised of a pH switchable water stationary phase column and a conventional non-polar capillary column is introduced for use in Supercritical Fluid Chromatography (SFC). By removing or adding NH4OH to the system hydration source, the water stationary phase pH can be rapidly switched between acidic (measured at pH∼3) and basic (measured at pH∼9) in seconds, while the operating character of the conventional column is unchanged. This switch modulates the velocity of ionizable analytes about 20-fold in the system, whereas non-ionizable analytes are not affected. In this way, the retention time of acids and/or bases can be reproducibly altered (<1% RSD; n = 3) in SFC separations. As a result, analyte selectivity and resolution can be readily controlled during analyses. For example, a selectivity reversal (alpha from 0.4 to 1.6) and a resolution increase (from 0 to 13) are demonstrated. Rapid stationary phase pH switching also allows multiple acids, bases, and/or neutral analytes to be determined simultaneously. Applications demonstrate that this method can greatly simplify complex mixture analysis in SFC by helping to separate target analytes from interfering matrix components.

Supercritical Fluid Chromatography in Bioanalysis-A Review.

In the last decade, the instrumentation improvements in supercritical fluid chromatography (SFC) and the hyphenation to mass spectrometry (MS), have increased the SFC acceptance between scientists, becoming today a valuable tool in analytical chemistry. The unique selectivity, short analysis times, low consumption of organic solvents, and the greener mobile phase, have contributed to expanding its applicability which has led to an increase in the number of publications especially in the bioanalysis area. This work reviews the advantages and main applications of SFC in bioanalysis during the last 5 years. Fundamental aspects concerning mobile phase composition, stationary phase, hyphenation to MS as well as matrix effect have been discussed. Finally, the most relevant applications have been summarized.

Fast Determination of Propofol in Human Plasma Using C18-Functionalized Magnetic Nanomaterials Followed by Supercritical Fluid Chromatography.

Novel C18-functionalized magnetic nanomaterials; i.e., C18@poly-styrene-divinylbenzene-glycidyl methacrylate-Fe3O4 (C18@PS-DVB-GMA-Fe3O4) have been synthesized by using N, N-dimethyloctadecylamine as modifying agent, which could be beneficial to remove the blood phospholipids. The C18@PS-DVB-GMA-Fe3O4 nanoparticles have been used and evaluated in the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) procedure for human plasma prior to the analysis of propofol by supercritical fluid chromatography (SFC). In the QuEChERS procedure, human plasma samples are directly mixed with extraction solvent and C18@PS-DVB-GMA-Fe3O4 nanoparticles, and the extraction and cleanup procedures have been accomplished simultaneously. The SFC separation was performed with a C18 column (Thermo Scientific™ Acclaim™ 120, 250 × 4. 6mm, 5μm) within 5min, using thymol as the internal standard. Supercritical carbon dioxide was used as the mobile phase with methanol as the cosolvent at the flow rate of 1.0mL/min. The column temperature was 38°C, and detection wavelength was 275nm. A good linearity was obtained among the propofol concentration range of 0.5-10mg/L (R2 = 0.9997) with the limit of detection of 0.17mg/L. Recoveries were in the range of 76.5-91.9%, with RSD less than 7.9%. These results suggested that method is convenient, rapid with high accuracy and little matrix effect, and suitable for rapid determination of propofol plasma concentration.

On the fractionation of lignin oligomers by stepwise gradient reversed-phase liquid chromatography

With the increased interest in lignin valorization, the analytical challenge to separate a complex mixture of a vast number of phenolics has made chromatography an indispensable step in lignin analysis. High-resolution separations, such as gas chromatography, reversed-phase liquid chromatography and supercritical fluid chromatography have typically been targeting low-molecular-weight compounds, while larger lignin oligomers have received less attention. These compounds have proven to be difficult to separate due to the inherent complexity of the high-molecular-weight fraction of lignins, in fact, even high-resolving linear reversed-phase gradients elute them as one wide zone. To tackle this, in this study we show that a crude fractionation of lignin oligomers can be achieved by applying stepwise reversed-phase gradients. A commonly employed reversed-phase system with water:acetonitrile mobile phase is evaluated for this task. Special attention was devoted to uncovering the molecular level explanation of the retention phenomenon. Our results indicate that separation is mainly governed by reversed-phase retention phenomena without any major exclusion or viscosity-related effects, shown by great fits to linear retention models (R2avg = 0.9599 for five different oligomers) and apparent differences in retentivity between different stationary phases. The influence of the gradient shape was demonstrated by the comparison of stepwise gradients with different number and frequency of steps, leading to the conclusion that gradients with a low number of steps yield fewer, but better resolved fractions, while finer multi-step gradients can be used to distinguish more fractions.

Ion Source Complementarity for Characterization of Complex Organic Mixtures Using Fourier Transform Mass Spectrometry: A Review.

Complex organic mixtures are found in many areas of research, such as energy, environment, health, planetology, and cultural heritage, to name but a few. However, due to their complex chemical composition, which holds an extensive potential of information at the molecular level, their molecular characterization is challenging. In mass spectrometry, the ionization step is the key step, as it determines which species will be detected. This review presents an overview of the main ionization sources employed to characterize these kinds of samples in Fourier transform mass spectrometry (FT-MS), namely electrospray (ESI), atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), atmospheric pressure laser ionization (APLI), and (matrix-assisted) laser desorption ionization ((MA)LDI), and their complementarity in the characterization of complex organic mixtures. First, the ionization techniques are examined in the common direct introduction (DI) usage. Second, these approaches are discussed in the context of coupling chromatographic techniques such as gas chromatography, liquid chromatography, and supercritical fluid chromatography.

Rapid Analysis for α-Tocopherol and Its Oxidative Products in the Pisum sativum L. Leaf Using Supercritical Fluid Chromatography-Medium Vacuum Chemical Ionization Tandem Mass Spectrometry.

A method for the rapid determination of α-tocopherol (α-T) and its oxidative products in plant tissue has been developed using supercritical fluid extraction (SFE) coupled with supercritical fluid chromatography (SFC) and medium vacuum chemical ionization (MVCI) with tandem mass spectrometry. The method is designed to study changes in levels for α-T and its oxidative products in plant cells during photosynthesis, aiming to observe the light response curves. α-T oxidation is a non-enzymatic self-defense mechanism in plant cells. Unlike enzyme-involved reactions, it cannot be stopped, so the oxidation continues in crude extracts even after extraction. Therefore, a real-time in-situ method is essential for tracking the light response curves. To optimize the selective reaction monitoring method, the reaction mixture of α-T and singlet oxygen (1O2), generated by rose Bengal under light illumination, was used as the source of oxidative products. The relative abundance changes in α-tocopherylquinone and 8a-hydroperoxy tocopherone in Pisum sativum L. (Pea) leaves under excessive light illumination have been preliminarily analyzed as part of the light response curve study. The method archives a throughput of 10-15 minutes for analyzing duplicate leaf samples. This process includes cutting off the leaf, sectioning it, placing the sample in a frozen SFE vessel, and conducting SFE/SFC analysis. Consequently, the average throughput is approximately 5-7 minutes per sample.

A novel application of hydrophilic interaction liquid chromatography for the identification of compounds with intramolecular hydrogen bonds

The incorporation of intramolecular hydrogen bonds (IMHB) into small molecules constitutes an interesting optimization strategy to afford potential drug candidates with enhanced solubility as well as permeability and consequently improved bioavailability (if metabolic stability is high). Common methods to assess IMHB rely on spectroscopic or diffraction techniques, which, however, have limited throughput when screening for hit compounds in early phases of drug discovery. Inspired by literature findings using supercritical fluid chromatography (SFC) as an indirect method for IMHB identification in a screening context, we aimed at developing a secondary chromatographic methodology taking advantage of commonly used HPLC-MS instrumentation. In this work, we explored hydrophilic interaction liquid chromatography (HILIC) and developed a method for discriminating compounds based on their hydrogen bonding features. By quantifying retention of different matched molecular pairs (MMP) and using information about their low energy conformations from quantum-mechanical calculations, we defined a hydrogen bonding-driven adsorption (kads) chromatographic parameter to assess a compound’s propensity to forming IMHB. In addition to the MMP analysis, we found that the kads parameter allows for the differentiation of analytes forming IMHB regardless of the comparison with control compounds.

RECENT ADVANCES IN GAS, LIQUID AND SUPERCRITICAL FLUID CHROMATOGRAPHY IN THE CONTEXT OF SUSTAINABLE/ GREEN CHEMISTRY

Background: Chromatographic techniques such as gas chromatography (GC), liquid chromatography (LC) and supercritical fluid chromatography (SFC) have long been employed in analytical chemistry. However, using or producing certain organic solvents and non-renewable gases is hazardous to the environment. Green chemistry is essential for addressing the environmental hazards associated with traditional procedures. Objectives: This review focuses on recent updates and profound insights into new, greener techniques introduced in GC, LC and SFC. Methodology: To compile this review, several electronic databases were searched, preferably from January 2019-December 2023. Results: This review highlights advancements in miniaturization, rapid methods, and eco-friendly solvents in chromatographic techniques. Integrating micro-electro-mechanical systems (MEMS) and nano-electro-mechanical systems (NEMS) in GC enhances sensitivity and resolution while reducing solvent consumption. Innovations like monolithic micro-GC chips and the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method further streamline analysis times. In LC, supercritical fluids are utilized as green mobile phases, optimizing techniques like electrochemical flow liquid chromatography (EFLC), high-performance liquid chromatography (HPLC), and ultra-high-performance liquid chromatography UHPLC. In contrast, SFC employs rapid methods, such as ultra-high-performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS). These developments collectively promote sustainability and efficiency in chromatographic practices. Conclusion: Green chromatography has become an essential tool in pharmaceutical analysis because it safeguards the operator’s health and the environment. It opens new horizons and generates new ideas for further experimentation while focusing on recent cutting-edge green technologies incorporated in GC, LC and SFC. These advancements significantly enhance safety and sustainability in analytical practices.

Using artificial neural networks to elucidate retention interactions on stationary phases with amine moieties dedicated to supercritical fluid chromatography

The retention mechanisms of small molecules on a wide range of stationary phases using different mobile phase compositions are one of the most discussed topics in current state-of-the-art supercritical fluid chromatography (SFC). Although various theories have been published on retention behavior, prediction, and stability over time, an in-depth insight into the mechanisms is still lacking. Our study focused on the description of the retention mechanisms on columns with amine moieties, i.e., 2-ethylpyridine, 2-picolylamine, 1-aminoanthracene, and diethylamine, dedicated to SFC, using three different compositions of organic modifier, i.e., methanol, methanol + 10 mmol/L NH3, and methanol + 2 % H2O, in CO2. An extensive set of analytes characterized by more than 200 molecular descriptors was used. The importance of the effect of each molecular descriptor on the retention behavior was determined by artificial neural networks. Thus, the descriptors increasing and/or decreasing retention on individual columns using different mobile phase compositions were defined. The data collected over one year of column use were statistically evaluated to describe the retention behavior of small molecules on the tested stationary phases using three organic modifiers and to describe the changes in retention behavior over time.As the development of the SFC method can be laborious and time-consuming, this publication offers a detailed description of the interactions taking place between the analyte and the SFC stationary phase. Understanding these fundamental processes will enable faster development of SFC methods using quality-by-design principles.

Discovery of the Alternaria mycotoxins alterperylenol and altertoxin I as novel immunosuppressive and antiestrogenic compounds in vitro.

Alternaria mycotoxins may pose significant challenges to food safety and public health due to the wide spectrum of reported adverse effects. Despite this, critical information on the immunomodulatory and antiestrogenic properties of most of these contaminants is still lacking. The present study aimed to identify the mycotoxins responsible for the immunosuppressive and antiestrogenic effects of a complex extract of Alternaria mycotoxins (CE) obtained by growing an Alternaria alternata strain on rice. Through a toxicity-guided fractionation procedure involving the production of CE-fractions by supercritical fluid chromatography and mycotoxin quantification by LC-MS/MS, the mycotoxins alternariol (AOH), tenuazonic acid (TeA), altertoxin I (ATX-I), and alterperylenol (ALTP) were identified as potential toxicologically relevant constituents contributing to the in vitro effects exerted by the extract. The assessment of the immunomodulatory effects, performed by applying the NF-κB reporter gene assay in THP1-Lucia™ monocytes, revealed the scarce contribution of AOH to the effects exerted by the CE. TeA showed no effect on the NF-κB pathway up to 250µM, whereas ATX-I and ALTP suppressed the LPS-mediated pathway activation at concentrations ≥ 1µM. The evaluation of antiestrogenic effects, performed in Ishikawa cells by applying the alkaline phosphatase assay, revealed the ability of ALTP (≥ 0.4µM) and ATX-I (≥ 2µM) to suppress the estrogen-dependent expression of enzyme activity. Given the risk of detrimental impacts stemming from alterations in endocrine and systemic immune responses by the investigated mycotoxins, further studies are needed to elucidate their underlying mechanisms of action and comprehensively evaluate the health risks posed by these toxins.

Complementarity of two-dimensional gas chromatography and two-dimensional liquid chromatography for the analysis of depolymerised lignin

Two-dimensional gas chromatography (GC × GC) and two-dimensional liquid chromatography (LC × LC) are nowadays widely used in academia and industry due to their high separation power. However, as far as we know, the complementarity of these two techniques has not yet been thoroughly studied based on the analysis of the same sample. Therefore, this was undertaken here by analysing the liquid fraction obtained after depolymerising a natural waste – lignin – with GC × GC and off-line comprehensive LC × SFC (SFC: supercritical fluid chromatography). Using complementary techniques is also important for lignin valorisation, as thorough structural characterisation of the depolymerised product can aid with developing and improving valorisation processes. For the tentative identification, NIST library was used for GC × GC–MS results and MS-DIAL together with SIRIUS for LC × SFC-MS/MS data. This allowed to study which compounds are detectable with the different 2D methods but also to discuss the limitations of the data analysis processes. The previous knowledge that LC techniques are more suitable than GC × GC for the analysis of larger oligomers and other low volatility compounds was confirmed; however, it was seen that GC × GC enabled the analysis of smaller compounds, such as aliphatic alcohols and saturated compounds. Overall, the study demonstrates the complementarity of the two techniques but also draws attention to the different detectable compound groups and classifications that the two techniques can provide.

Supercritical fluid chromatography- Nanospray ionization-mass spectrometry (SFC-nSI-MS)

A nanospray emitter coupled to a supercritical fluid chromatograph (SFC-nSI-MS) for mass spectrometric (MS) analysis of fatty acids (FA) positional isomers is introduced. The experimental setup uses conventional bore columns before the SF back-pressure regulator (pre-BPR). The flow is then split and nanosprayed using a short emitter post-BPR. A C18 column was used to resolve positional isomers of unsaturated FA with a 5 min gradient. Chromatographic resolution of the nSFC was compared to a LC-MS system with superior resolving power demonstrated in the nSFC MS system. This system has proven quantitative performance for analyzing pharmaceutical effects on FA composition in a complex biological matrix like E coli lysate.