Supercritical Fluid Chromatography Research Articles

Development of an ultra-high-performance supercritical fluid chromatography method for the analysis of phenols in the pyrolysis aqueous fraction.

The pyrolysis process consists of the thermal decomposition of biomass in an inert atmosphere, which produces a liquid (bio-oil) composed of a complex mixture of organic compounds, including an oil and water phase. The aqueous fraction can reach up to 45% w/w, and understanding its composition is of utmost importance in determining its intended destination, whether for the reuse of compounds in industrial applications or for treating the effluent for disposal. In this study, a fast, direct, and efficient method using ultra-high-performance supercritical fluid chromatography (UHPSFC) was developed and optimized for monitoring phenols in aqueous samples obtained from the pyrolysis processing of six different biomass sources. The following parameters were evaluated for method optimization: stationary phase type, mobile phase flow, organic modifier, sample diluent, temperature, pressure, and modifier gradient time. With a total analysis time of 26min, out of the fourteen (14) investigated phenolic compounds, eleven (11) were successfully separated after method optimization, and among them, five (5) were quantified in all six aqueous fractions. The aqueous fractions of residue from cowpea pod (1.89mg.mL-1), sugar apple (3.09mg.mL-1), and acerola (4.79mg.mL-1) presented lower concentrations compared to grape (8.16mg.mL-1), pine nuts (6.68mg.mL-1), and guava (6.05mg.mL-1) fractions. However, even at lower concentrations, all biomasses showed promising results regarding the phenolic compound content, analytes that have high added value for the chemical industry.

Applicability of supercritical fluid chromatography for oligonucleotide analysis: A proof-of-concept study

We evaluated the suitability of supercritical fluid chromatography (SFC) for oligonucleotide analysis using 4-mer oligonucleotides with various phosphorothioate (PS) contents as model compounds. Column screening showed that the diol-modified column was able to separate sequences with different PS contents. Optimization of the column body and additives allowed us to analyze polar oligonucleotides using SFC. Various sequences were also analyzed using the optimized method. A good peak shape was obtained when the guanine plus cytosine content of the analyte was two or less in the 4-mer oligonucleotides. Furthermore, we found that the retention times of the selected sequences were positively correlated with polar surface areas, indicating that oligonucleotides interact with polar stationary phases. In contrast, more hydrophobic full PS sequences were retained more strongly in the diol column than the full phosphodiester (PO) sequences. This suggests that the diol column has unique selectivity for PO and PS linkages. These results indicate that SFC is potentially applicable to oligonucleotide analysis with a separation mechanism that is different from that of ion-pair reversed-phase liquid chromatography.

Improving the position specificity of Themomyces lanuginosus lipase based on semi-rational design

Diacylglycerol (DAG) is an intermediate product in lipid metabolism and plays an important physiological role in human body. It is mainly prepared by hydrolyzing lipid with lipase. However, research on the detection method of 1, 2-diacylglycerol (1, 2-DAG) and 1, 3-diacylglycerol (1, 3-DAG) and catalytic specificity of lipase was not enough, which limits its wide application. To address these challenges, an efficient quantitative detection method was first established for 1, 2-DAG (0.025-0.200 g/L) and 1, 3-DAG (0.025-0.150 g/L) by combining supercritical fluid chromatography with evaporative light scattering detector and optimizing the detection and analysis parameters. Based on the molecular docking between Thermomyces lanuginosus lipase (TLL) and triolein, five potential substrate binding sites were selected for site-specific saturation mutation to construct a mutation library for enzyme activity and position specificity screening. The specificity of sn-1, 3 of the I202V mutant was the highest in the library, which was 11.7% higher than the specificity of the wild type TLL. In summary, the position specificity of TLL was modified based on a semi-rational design, and an efficient separation and detection method of DAG isomers was also established, which provided a reference for the study of the catalytic specificity of lipase.

Controlling the positive ion electrospray ionization of poly(ethylene glycols) when using ultra-high-performance supercritical fluid chromatography-mass spectrometry.

Poly(ethylene glycols) are complex polymers often added to pharmaceutical formulations to improve drug solubility and delivery. One of the main challenges when using chromatographic techniques coupled to mass spectrometry is the unselective ionization of poly(ethylene glycols) oligomers. Additionally, when the chain length is large enough, multiple charged species are formed, further complicating the mass spectra and processing. This study uses the advanced oligomer separation provided by supercritical fluid chromatography with a mass spectrometry approach that selectively ionizes poly(ethylene glycols) as ammoniated molecules to simplify data analysis and facilitate batch-to-batch comparisons. Several visual representations of the response of the ionization events based on the polymer molecular weight and the repeating unit were used to elucidate trends in ionization. Evaluation of the influence of the oligomer length and end-group on the electrospray ionization of the polymer allowed the development of a process to enable selective ionization for these complex polymers.

Use of N-(4-aminophenyl)piperidine derivatization to improve organic acid detection with supercritical fluid chromatography-mass spectrometry.

The analysis of organic acids in complex mixtures by LC-MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native (i.e., underivatized or untagged) form. These compounds have also been difficult to measure using supercritical fluid chromatography (SFC)-MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N-(4-aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC-MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N-(4-aminophenyl)piperidine to achieve detection limits down to 0.5 ppb, with overall improvements in detection limit ranging from 25-to-2100-fold. The effect of the derivatization group on sensitivity, which increased by at least 200-fold for compounds that were detectable in their native form, and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC-MS, especially for those that are undetectable in their native form.

Analytical and Preparative Separation of Softwood Lignans by Supercritical Fluid Chromatography

Lignans are widespread polyphenolic secondary plant metabolites possessing high biological activity. One of the most promising industrial-scale sources of such compounds is coniferous knotwood, containing a large number of polyphenolic compounds. Their use in pharmaceutical and other industries is limited by the difficulty in obtaining high-purity preparations from plant material and the requirement of advanced separation techniques. In this study, supercritical fluid chromatography on polar stationary phases was proposed for the efficient separation and identification of spruce, pine, fir, and larch knotwood extractives. Among the six tested sorbents, the best results were shown by silica with grafted diol and 2-ethylpyridine groups under conditions of gradient elution with a carbon dioxide–methanol mobile phase, which ensured the efficient retention and separation of analytes due to donor–acceptor interactions. Scaling up the method on a DIOL stationary phase provided a semi-preparative separation of extractives within 30 min to obtain 14 individual compounds with a purity of 90–99% and yields from 0.3 to 51% of the dry extract. These included eight lignans (nortrachelogenin, matairesinol, oxomatairesinol, α-conidendrin, 5-hydroxymatairesinol and its isomer, lariciresinol, and secoisolariciresinol), two oligolignans, three stilbenes (pinosylvin and its methyl ester, pterostilbene), and flavonoid taxifolin. The developed approach is distinguished with low operational costs, low consumption of organic solvents, environmental safety, and it is fully consistent with the principles of green chemistry.

Columns in analytical-scale supercritical fluid chromatography: From traditional to unconventional chemistries.

Within this review, we thoroughly explored supercritical fluid chromatography (SFC) columns used across>3000 papers published from the first study carried out under SFC conditions in 1962 to the end of 2022. We focused on the open tubular capillary, packed capillary, and packed columns, their chemistries, dimensions, and trends in used stationary phases with correlation to their specific interactions, advantages, drawbacks, used instrumentation, and application field. Since the 1990s, packed columns with liquid chromatography and SFC-dedicated stationary phases for chiral and achiral separation are predominantly used. These stationary phases are based on silica support modified with a wide range of chemical moieties. Moreover, numerous unconventional stationary phases were evaluated, including porous graphitic carbon, titania, zirconia, alumina, liquid crystals, and ionic liquids. The applications of unconventional stationary phases are described in detail as they bring essential findings required for further development of the supercritical fluid chromatography technique.

A novel switchable water stationary phase for supercritical fluid chromatography

A novel pH switchable water stationary phase is presented for use in supercritical fluid chromatography (SFC). By adding NH4OH to the water coating and system hydration, changes in CO2 pressure and temperature allow a wide range of stationary phase pH conditions (∼3–9) to be achieved, which impact analyte retention properties. For example, 100 atm and 50 °C produces an acidic water stationary phase (pH near 4.0) where octanoic acid readily elutes while the base caffeine does not. Conversely, at 80 atm and 120 °C a basic water stationary phase (pH near 8.0) is obtained and the opposite occurs. Further, under constant pressure and temperature conditions, simply adding or removing NH4OH from the system is also found to readily allow switching between the basic and acidic water stationary phase modes and demonstrates control over ionizable analyte elution. For instance, hexanoic acid elution is near 40 times more delayed on a basic water stationary phase and, as such, it can be eluted at later points in time as desired by removing the NH4OH and switching to an acidic stationary phase. Experiments indicate that stationary phase pH switching occurs uniformly across the 15 m column length within about 18 s and that analyte retention times are very reproducible upon performing a switch (1.4% RSD; n = 3). Results demonstrate the selectivity factor between acidic and neutral analytes can be reversed and increased about 35 times, while in other trials resolution also similarly increased near 40-fold. By rapidly switching the stationary phase pH back and forth between acidic and basic modes, the selectivity between ionizable analytes could also be increased as desired. Various applications with the system show that it can vastly increase the separation between target analytes and matrix components as required by the dynamics of a particular separation.

Microstructure characterization and mechanistic insight into polyether polyols and their associated polyurethanes.

This article reviews the analytical tool chest used for characterizing alkoxylates and their associated copolymer mixtures. Specific emphasis will be placed upon the use of mass spectrometry-based techniques as rapid characterization tools for optimizing reaction processes in an industrial R&D setting. An initial tutorial will cover the use of matrix-assisted laser desorption/ionization-mass spectrometry and tandem mass spectrometry fragmentation for detailed component analysis (e.g., polyol and isocyanate) of a model polyurethane-based foam. Next, this critical feedback information will be used with the guidance of mass spectrometry to initiate the development of a new, more efficient, tris(pentafluorophenyl)borane (FAB) catalyst-based alkoxylation process for generating the next generation of glycerin-initiated poly(propylene oxide)-co-poly(ethylene oxide)copolymers. Examples will be provided for each step in the FAB-based optimization process that were required to generate the final product. Following this example, two-dimensional liquid chromatography, supercritical fluid chromatography, and ion mobility separations, along with their coupling to mass spectrometry, will be reviewed for their efficiency in characterizing and quantitating the components within these complex polyether polyol mixtures.

Detection of walnut oil adulterated with high-linoleic acid vegetable oils using triacylglycerol pseudotargeted method based on SFC-QTOF-MS

Authentication of walnut oil (WO) is challenging due to the adulteration of high-linoleic acid vegetable oils (HLOs) with similar fatty acid composition. To allow the discrimination of WO adulteration, a rapid, sensitive and stable scanning method based on supercritical fluid chromatography quadrupole time-of-flight mass spectrometry (SFC-QTOF-MS) was established to profile 59 potential triacylglycerol (TAGs) in HLOs samples within 10min. Limit of quantitation of the proposed method is 0.002µgmL-1 and the relative standard deviations range from 0.7% to 12.0%. TAGs profiles of WO samples from various varieties, geography origins, ripeness, and processing methods were used to construct orthogonal partial least squares-discriminant analysis (OPLS-DA) and OPLS models that were highly accurate in both qualitative and quantitative prediction at adulteration levels as low as 5% (w/w). This study advances the TAGs analysis to characterize vegetable oils and holds promise as an efficient method for oil authentication.

Quantification of coumarins, furocoumarins and polymethoxyflavones in hydroalcoholic fragrances by supercritical fluid chromatography‐tandem mass spectrometry

ABSTRACT Citrus essential oils, thanks to their pleasant aroma, are certainly the most used ingredients in the formulation of hydroalcoholic fragrances. The non-volatile fraction of Citrus essential oil is composed for 10–20% of coumarins, furocoumarins and polymethoxyflavones. It is well known that furocoumarins induce photosensitization and have potential carcinogenic and mutagenic effects. It follows that furocoumarins levels in cosmetics product are constantly monitored by opinions and regulations issued by the International Fragrance Association. The aim of this research article was to quantify coumarins, furocoumarins and polymethoxyflavones in thirty commercial hydroalcoholic fragrances using supercritical fluid chromatography in combination with triple quadrupole mass spectrometry technique (SFC-QqQ-MS). According to author’s knowledge, this is the first report on the determination of oxygen heterocyclic compounds in hydroalcoholic fragrances by means of SFC-QqQ-MS technique.

Employment of chiral columns with superficially porous particles in chiral separations of cobalt bis (dicarbollide) and nido-7,8-C2 B9 H12 (1-) derivatives.

Derivatives of the nido-7,8-C2 B9 H12 (1-) (dicarbollide ion) and [3,3'-Co-(1,2-C2 B9 H11 )2 ](1-) cobalt sandwich (COSAN) ion represent groups of extremely chemically and thermally stable abiotic compounds. They are being investigated in many research areas, that is, medicinal chemistry, material sciences, analytical chemistry, and electrochemistry. The chirality of these compounds remains still grossly overlooked, what is also reflected in limited number of reports on their chiral separations. Continued progress depends on reliable, fast, and cost-effective methods for such separations. Recently, chiral separations of COSAN derivatives were achieved in liquid chromatography and supercritical fluid chromatography. Only five anionic derivatives of nido-7,8-C2 B9 H12 (1-) were successfully enantioseparated in liquid chromatography. Efforts to separate anionic nido-7,8-C2 B9 H12 (1-) in supercritical chromatography have failed, and only a few dicarbollide ions were separated using liquid chromatography. Generally, all chiral separations in liquid chromatography took about 30 min. Herein, we identify a versatile column capable of separating both COSAN and nido-7,8-C2 B9 H12 (1-) derivatives and achieve faster analyses times employing commercially available superficially porous chiral stationary phases. The semisynthetic hydroxypropyl β-cyclodextrin-based column (CDShell-RSP) is identified as the column of choice from the tested columns by separating 19 of 27 compounds from each structural motifs tested mainly in less than 10min. The dihydroxyalkyl, oxygen-bridged hydroxyalkyl, and bisphenylene-bridged COSAN derivatives were baseline separated in less than 5min exceeding the results of supercritical fluid chromatography. Methods developed herein will aid synthetic chemists without the possession of a supercritical fluid chromatograph to achieve fast chiral separations of COSAN and derivatives of nido-7,8-C2 B9 H12 (1-) on a common liquid chromatograph without the need of dedicated instrumentation.

Qualitative and Quantitative Analyses of the Enantiomers of Nicotine and Nornicotine Employing Chiral Supercritical Fluid Chromatography.

An optimized method employing chiral supercritical fluid chromatography with diode array UV-VIS detection has been developed for the quantitative analysis of nicotine and nornicotine enantiomer distributions. The method parameters that were optimized included: column type (stationary phases, Chiralpak IG-3), column temperature (40°C), modifier types and concentration (isopropyl alcohol, 10%), additive types and concentrations (diethylamine, 0.2%), elution times (<6min, flow rate 3mL/min) and resolution factor (>1.2). These optimized conditions led to nicotine and nornicotine enantiomer detection limits of ~5ng/μL with accompanying %RSD values of <2% from the analyses of commercially available nicotine-containing formulations.

Evaluation of control of additive concentration in gradient analysis of supercritical fluid chromatography-coupled to tandem mass spectrometry

Mobile phase additives are used to improve retention behavior in chromatography. In supercritical fluid chromatography (SFC), for which supercritical fluid carbon dioxide (SF-CO2) is used as the main mobile phase, additives can only be added into the modifier. For that reason, when gradient analysis is performed by changing the modifier ratio to SF-CO2, the additive concentration in the mobile phase increases in parallel with the modifier ratio. In a preliminary study performed using the conventional SFC system, ammonium acetate was necessary to improve the peak shape of a polar steroid, dehydroepiandrosterone sulfate (DHEA-S), while the peak intensity of a non-polar steroid, progesterone, decreased by 78% compared to that in the absence of the additive in mobile phase when gradient elution was performed. Since ammonium acetate had both favorable and unfavorable effects on sensitive and simultaneous analysis of these two steroid compounds, a compromise between these effects had to be sought. A three-pump configuration of SFC was developed by adding a pump unit to SFC instrument, which enabled control of the additive concentration independently of the modifier ratio, for the purpose of investigating the additive effect in detail using both steroids as model compounds. The putative cause of the decrease in peak intensity of progesterone was excessively elevated additive concentration in gradient analysis. When the additive concentration in the mobile phase was controlled to ensure that it did not increase during gradient analysis, the peak intensities of progesterone, cortisol, corticosterone, and testosterone were 55%, 40%, 25%, and 17% higher than when the additive concentration was not controlled, respectively. On the other hand, the peak intensity of DHEA-S was almost identical between the conditions, with an increase of 2% with three-pump instrument. The three-pump configuration showed the potential to solve problems relating to the use of modifier additives by keeping their concentration constant in gradient SFC analysis.

Quantitative Determination of Related Substances in Propofol by Supercritical Fluid (CO2) Chromatography

Quantitative Determination of Related Substances in Propofol by Supercritical Fluid (CO2) Chromatography

Qualitative and Quantitative Analyses of the Enantiomers of Nicotine and Related Alkaloids Employing Chiral Supercritical Fluid Chromatography in Commercial Nicotine Samples and in E-Cigarette Products

Summary Several commercial sources of tobacco-derived nicotine (TDN) and synthetic nicotine (SyN) and a variety of e-cigarette liquids employing either TDN or SyN have been evaluated to determine the enantiomer distributions of R- and S-nicotine and R- and S-nornicotine by chiral supercritical fluid chromatography (chiral-SFC) with UV diode array detection (DAD-UV). The data generated are used to test the mismatched vs. matched hypothesis of Cheetham et al. as a means to distinguish products containing TDN from products with SyN. Two sets of experiments were conducted in this study. The first experiment was conducted on a series of 11 commercial nicotine samples (three characterized as tobacco-derived and eight characterized as synthetic nicotine). The commercial nicotine samples were either from a tobacco-derived nicotine (TDN) source or were synthetic nicotine (SyN). Some of the commercial nicotine samples were nicotine salts. The second experiment was conducted on e-liquids from a set of 11 e-cigarettes. The nicotine in the e-liquids was either from TDN or SyN. The e-liquid samples were differentiated based on the advertised information on the internet or from printed information on the e-cigarette packaging. None of the three commercial TDN samples in the first experiment could be unequivocally characterized as coming from a tobacco source. Five of the eight commercial SyN samples were correctly characterized as SyN based on the matched vs. mismatched nicotine and nornicotine hypothesis of Cheetham et al. In the second experiment, none of the e-liquids characterized as being from TDN sources could be unequivocally characterized as coming from a tobacco source. All of the e-liquids characterized as being from SyN sources were either characterized as equivocal or of uncertain origin based on the matched vs. mismatched nicotine and nornicotine hypothesis of Cheetham et al. These sets of experiments represent an excellent example of the difficulty that the United States Food and Drug Administration is having in trying to determine if TDN or SyN is being used in tobacco products. Even highly advanced chromatographic methods such as chiral-SFC were not able to unequivocally distinguish products with TDN from products with SyN 100% of the time. Other analytical methods such as 14C quantitation of nicotine samples by accelerator mass spectrometry offer a more reliable determinate of nicotine source (TDN vs. SyN) and can be used to identify misbranded products labelled as containing SyN, even though this methodology is more expensive and offered in limited locations.

Electrospray Ionization Efficiency Predictions and Analytical Standard Free Quantification for SFC/ESI/HRMS.

Supercritical fluid chromatography (SFC) is a promising, sustainable, and complementary alternative to liquid chromatography (LC) and has often been coupled with high resolution mass spectrometry (HRMS) for nontarget screening (NTS). Recent developments in predicting the ionization efficiency for LC/ESI/HRMS have enabled quantification of chemicals detected in NTS even if the analytical standards of the detected and tentatively identified chemicals are unavailable. This poses the question of whether analytical standard free quantification can also be applied in SFC/ES/HRMS. We evaluate both the possibility to transfer an ionization efficiency predictions model, previously trained on LC/ESI/HRMS data, to SFC/ESI/HRMS as well as training a new predictive model on SFC/ESI/HRMS data for 127 chemicals. The response factors of these chemicals ranged over 4 orders of magnitude in spite of a postcolumn makeup flow, expectedly enhancing the ionization of the analytes. The ionization efficiency values were predicted based on a random forest regression model from PaDEL descriptors and predicted values showed statistically significant correlation with the measured response factors (p < 0.05) with Spearman's rho of 0.584 and 0.669 for SFC and LC data, respectively. Moreover, the most significant descriptors showed similarities independent of the chromatography used for collecting the training data. We also investigated the possibility to quantify the detected chemicals based on predicted ionization efficiency values. The model trained on SFC data showed very high prediction accuracy with median prediction error of 2.20×, while the model pretrained on LC/ESI/HRMS data yielded median prediction error of 5.11×. This is expected, as the training and test data for SFC/ESI/HRMS have been collected on the same instrument with the same chromatography. Still, the correlation observed between response factors measured with SFC/ESI/HRMS and predicted with a model trained on LC data hints that more abundant LC/ESI/HRMS data prove useful in understanding and predicting the ionization behavior in SFC/ESI/HRMS.

Vacuum differential mobility spectrometry combined with column-switching liquid chromatography- mass spectrometry for the analysis of pyrrolizidine alkaloids in tea samples

The benefit of combining liquid chromatography (LC), supercritical fluid chromatography (SFC) and vacuum Differential Mobility Spectrometry – Mass Spectrometry (vDMS-MS) was investigated for the analysis of fourteen diastereomeric pyrrolizidine alkaloids (PA); intermedine, echinatine, lycopsamine, indicine, intermedine-N-oxide, echinatine-N-oxide, indicine-N-oxide, lycopsamine-N-oxide, senecivernine, senecionine, jacobine, senecivernine-N-oxide, senecionine-N-oxide, retrorsine. The mobile phase composition (15–100% MeOH and ACN), flow rate (8–100 µL/min), vDMS cell pressure, and F value showed an effect on the mobility behavior of the analytes. At 15% MeOH with a flow rate of 100 µL/min and 33 mbar vDMS pressure, 8 out 14 PA could be partially or totally separated by vDMS-MS. As well as providing an additional separation dimension vDMS improved the selectivity and a 5-minute assay method was developed for the quantification of 10 out of 14 single diastereomeric PA in tea samples, using a short LC column-switching and hyphenated to vDMS-MS in the selected ion monitoring mode. The performance of the method was found to be comparable with a 12-minute standard LC-MS/MS method using detection in the selected reaction monitoring mode. Additionally, the combination of vDMS and SFC-MS was investigated and suggests that the mixture of CO2/MeOH influences the CV shifting of the PA to more negative compensation voltage, and the signal-to-noise ratio is improved by a factor of three compared to SFC-MS without vDMS.

Facilitated on-line supercritical fluid extraction - supercritical fluid chromatography for nonpolar and polar compounds from milk thistle seeds

Plant seeds, as those from milk thistle (Silybum marianum), are a valuable source of nonpolar and polar compounds with potentially interesting biological activity. The main nonpolar compounds are triglycerides, which are also the main components of all vegetable oils. In addition, specific polar compounds – flavonolignans, called silymarin, have been found in large amounts in milk thistle seeds extract. These flavonoids derivatives have different biological activity, for instance hepatoprotective effects. In order to extract and analyze both nonpolar (triglycerides) and polar compounds (flavonolignans) from milk thistle seeds through a sequential methodology, an on-line supercritical fluid extraction - supercritical fluid chromatography (SFE-SFC) method was developed. Different ways of transferring the extracts from SFE to SFC (i.e. direct on-column transfer and loop transfer) were compared, and particularly for their effect on chromatographic quality. In this respect, nonpolar and polar compounds caused different issues, especially as polar compounds required a significant portion of co-solvent in the extraction step, favoring early elution in the chromatographic column. First, on-line SFE-SFC was used for triglycerides analysis and allowed the comparison of transfer modes. Then, on-line kinetics were performed to measure defatting time before polar molecules extraction. Finally, the eventual benefit of loop transfer was also investigated for the analysis of flavonolignans, polar molecules whose analysis can be difficult by on-line SFE-SFC. The aim of this paper is to discuss the versatility of on-line SFE-SFC and how challenging the coupling can be, especially when both non-polar and polar molecules must be analyzed independently in a single sample.

Supercritical fluid chromatography based on reversed-phase/ ion chromatography mixed-mode stationary phase for separation of spirooxindole alkaloids

The present paper illustrates the versatility of the supercritical fluid chromatography (SFC) since, for the first time, four spirooxindole alkaloids (SOAs) including two pairs of isomers were separated by using two types of reversed-phase/ ion chromatography (RP/IC) mixed-mode stationary phases. Two mixed-mode stationary phases (C8SAX and C8SCX) was simultaneously provided dispersive and electrostatic interactions, which were suitable for the separation of such alkaloids. This study tried to provide an in-depth understanding of the SFC separation mechanism of the mixed-mode stationary phase through investigation of the impact of changes in mobile phase composition on alkaloids’ retention behavior. On C8SAX, due to the strong electrostatic repulsion, there was a very narrow elution window of the alkaloids, of which behaviors were hardly affected by adding diethylamine in mobile phase. When adding formic acid or acidic ammonium formate, the prolonged retention time of alkaloids was presented because of the shielded effect of formate anions on the electrostatic repulsion. In particular, better peak shape and improved resolution were obtained by using acidic ammonium formate due to the deactivation of silanol groups by ammonium cations. On the other hand, both formic acid and acidic ammonium formate can strengthen the electrostatic attraction of C8SCX, causing difficult elution of the alkaloids. Ammonium cations from either the protonated diethylamine or the ionized ammonium formate, were considered as counter ions to effectively mask the electrostatic attraction of C8SCX, to significantly reduce the retention of alkaloids, but improve the resolution. Finally, utilizing two developed SFC methods, i.e., C8SAX with EtOH+ 10 mM acidic ammonium formate in CO2, or C8SCX with EtOH+0.1% diethylamine in CO2, the baseline separation of corynoxeine and isocorynoxeine, rhynchophylline and isorhynchophylline was achieved within 5 min.