Supercritical Fluid Chromatography Separation Research Articles

Investigation of robustness for supercritical fluid chromatography separation of peptides: Isocratic vs gradient mode

We investigated and compared the robustness of supercritical fluid chromatography (SFC) separations of the peptide gramicidin, using either isocratic or gradient elution. This was done using design of experiments in a design space of co-solvent fraction, water mass fraction in co-solvent, pressure, and temperature. The density of the eluent (CO2-MeOH-H2O) was experimentally determined using a Coriolis mass flow meter to calculate the volumetric flow rate required by the design. For both retention models, the most important factor was the total co-solvent fraction and water mass fraction in co-solvent. Comparing the elution modes, we found that gradient elution was more than three times more robust than isocratic elution. We also observed a relationship between the sensitivity to changes and the gradient steepness and used this to draw general conclusions beyond the studied experimental system.To test the robustness in a practical context, both the isocratic and gradient separations were transferred to another laboratory. The gradient elution was highly reproducible between laboratories, whereas the isocratic system was not. Using measurements of the actual operational conditions (not the set system conditions), the isocratic deviation was quantitatively explained using the retention model. The findings indicate the benefits of using gradient elution in SFC as well as the importance of measuring the actual operational conditions to be able to explain observed differences between laboratories when conducting method transfer.

Chiral Separations Using a Modified Water Stationary Phase in Supercritical Fluid Chromatography

A novel means of achieving chiral separations in supercritical fluid chromatography (SFC) using a water stationary phase is presented. By adding various chiral selectors to the phase, different chiral analytes can be readily separated using neat CO2 as a mobile phase. For example, by adding β-cyclodextrin, it is found that certain flavanone enantiomers can be separated, while using the antibiotic vancomycin as a selector provides separation of some chiral phenoxypropionic acids. Other additives such as sodium chloride and triethylamine are also explored and found to enhance certain separations when also present in the water phase. While column pressure has a moderate impact on chiral analyte retention and separation in this SFC method, column temperature has a comparatively larger influence. In particular, relatively cooler temperatures below about 5 °C are found to markedly increase resolution and selectivity. For instance, notably large resolution of 4.7 is achieved for a phenoxypropionic acid pair at 0 °C and 150 atm CO2. Since the method does not require modifier to elute such polar species, it is also readily compatible with FID detection and does not generate organic waste. Therefore, results indicate that this approach could be a potentially simple and flexible means of achieving chiral separations in SFC.

Supercritical fluid chromatographic separation on preparative scale and in continuous mode

Chromatography with supercritical fluids (SFC) is a powerful tool for carrying out separations of substances with very similar structure and volatility, such as isomers of various kinds. In this contribution, we discuss the separation process aiming at the recovery of products on a much larger scale than analytical. Nevertheless, for developing such a process, the separation is carried out first on analytical scale to determine optimum operating conditions and select stationary phase, mobile phase, and co-solvent (modifier), if necessary. Determination of isotherms is helpful for simulating the separation process. Verification of the separation process includes the recycling of the mobile phase. The chromatographic process can be a batch process in the elution mode, or a continuous process, approximated as simulated moving bed (SMB). The batch SFC process, although using a higher quantity of mobile phase is less cost intensive than the SFC-SMB process.

Supercritical fluid chromatography coupled with tandem mass spectrometry: A high-efficiency detection technique to quantify Taxane drugs in whole-blood samples.

We present a technique to rapid determine taxane in blood samples by supercritical fluid chromatography together with mass spectrometry. The aim of this study was to develop a supercritical fluid chromatography with mass spectrometry method for the analysis of paclitaxel, cabazitaxel, and docetaxel in whole-blood samples of rats. Liquid-dry matrix spot extraction was selected in sample preparation procedure. Supercritical fluid chromatography separation of paclitaxel, cabazitaxel, docetaxel, and glyburide (internal standard) was accomplished within 3min by using the gradient mobile phase consisted of methanol as the compensation solvent and carbon dioxide at a flow rate of 1.0mL/min. The method was validated regarding specificity, the lower limit of quantification, repeatability, and reproducibility of quantification, extraction recovery, and matrix effects. The lower limit of quantification was found to be 10ng/mL since it exhibited acceptable precision and accuracy at the corresponding level. All interday accuracies and precisions were within the accepted criteria of ±15% of the nominal value and within ±20% at the lower limit of quantification, implying that the method was reliable and reproducible. In conclusion, this method is a promising tool to support and improve preclinical or clinical pharmacokinetic studies with the taxanes anticancer drugs.

The Effect of Pressure and Solvent on the Supercritical Fluid Chromatography Separation of Tocol Analogs in Palm Oil.

There are six tocol analogs present in palm oil, namely α-tocopherol (α-T), α-tocomonoenol (α-T1), α-tocotrienol (α-T3), γ-tocotrienol (γ-T3), β-tocotrioenol (β-T3) and δ-tocotrienol (δ-T3). These analogs were difficult to separate chromatographically due to their similar structures, physical and chemical properties. This paper reports on the effect of pressure and injection solvent on the separation of the tocol analogs in palm oil. Supercritical CO2 modified with ethanol was used as the mobile phase. Both total elution time and resolution of the tocol analogs decreased with increased pressure. Ethanol as an injection solvent resulted in peak broadening of the analogs within the entire pressure range studied. Solvents with an eluent strength of 3.4 or less were more suitable for use as injecting solvents.

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

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

Is supercritical fluid chromatography hyphenated to mass spectrometry suitable for the quality control of vitamin D3 oily formulations?

Nowadays, many efforts are devoted to improve analytical methods regarding efficiency, analysis time and greenness. In this context, Supercritical Fluid Chromatography (SFC) is often regarded as a good alternative over Normal Phase Liquid Chromatography (NPLC). Indeed, modern SFC separations are fast, efficient with suitable quantitative performances. Moreover, the hyphenation of SFC to mass spectrometry (MS) provides additional gains in specificity and sensitivity. The present work aims at the determination of vitamin D3 by SFC-MS for routine Quality Control (QC) of medicines specifically. Based on the chromatographic parameters previously defined in SFC-UV by Design of Experiments (DoE) and Design Space methodology, the method was adapted to work under isopycnic conditions ensuring a baseline separation of the compounds. Afterwards, the response provided by the MS detector was optimized by means of DoE methodology associated to desirability functions. Using these optimal MS parameters, quantitative performances of the SFC-MS method were challenged by means of total error approach method validation. The resulting accuracy profile demonstrated the full validity of the SFC-MS method. It was indeed possible to meet the specification established by the European Medicines Agency (EMA) (i.e. 95.0 − 105.0% of the API content) for a dosing range corresponding to at least 70.0-130.0% of the API content. These results highlight the possibility to use SFC-MS for the QC of medicine and obviously support the switch to greener analytical methods.

Early Development Scale-Up of a Structurally-Challenging 5-Lipoxygenase Activating Protein (FLAP) Inhibitor

A practical and efficient synthesis of the FLAP inhibitor 1 was developed addressing multiple scale-up and safety concerns posed by the established synthesis and utilized a resolution strategy (replacing supercritical fluid chromatography (SFC) separation) for expedient access to the key structural component of 1: the challenging chiral quaternary center. Also highlighted are in situ IR monitoring, condensation to form the 1,2,4-oxadiazole ring, and an efficient Suzuki-Miyaura coupling.

The effect of high concentration additive on chiral separations in supercritical fluid chromatography

Supercritical Fluid Chromatography is frequently used to efficiently handle separations of enantiomers. The separation of basic analytes usually requires the addition of a basic additive in the mobile phase to improve the peak shape or even to elute the compounds. The effect of increasing the concentration of 2-propylamine as additive on the elution of a series of basic compounds on a Chiralpak-AD stationary phase was studied. In this study, unusual additive concentrations ranging from 0.3% to 10% of 2-propylamine 2-propylaminein the modifier were explored and the effect on retention, peak shape, selectivity and resolution was evaluated. The addition of a large quantity of additive allowed to drastically improve the selectivity and the resolution, and even enantiomers elution order reversal was observed by changing the concentration of basic additive. The role of the ratio additive/modifier appeared a key to tune the enantioselectivity. Finally, the impact of these drastic conditions on the column material was evaluated.

Development of a supercritical fluid chromatography method with ultraviolet and mass spectrometry detection for the characterization of biomass fast pyrolysis bio oils

The characterization of complex mixtures is a challenging issue for the development of innovative processes dedicated to biofuels and bio-products production. The huge number of compounds present in biomass fast pyrolysis oils combined with the large diversity of chemical functions represent a bottleneck as regards analytical technique development. For the extensive characterization of complex samples, supercritical fluid chromatography (SFC) can be alternative to usual separation techniques such as gas (GC) or liquid chromatography (LC). In this study, an approach is proposed to define the best conditions for the SFC separation of a fast pyrolysis bio-oil. This approach was based on SFC data obtained directly from the bio-oil itself instead of selecting model compounds as usually done. SFC conditions were optimized by using three specific, easy-to-use and quantitative criteria aiming at maximizing the separation power. Polar stationary phases (ethylpyridine bonded silica) associated to a mix of acetonitrile and water as polarity modifier provided the best results, with more than 120 peaks detected in SFC-UV.

Separation and purification of six components from the roots of Rheum officinale Baill. by supercritical fluid chromatography

ABSTRACTThe dried roots of Rheum officinale Baill., named Dahuang in Chinese, has many pharmacological effects and has been widely used for the treatment of many diseases. To develop a harmless and eco-friendly method for the separation of components in Dahuang is of great interest for quality control and pharmacological study of Dahuang. A method for separation and purification of components in Dahuang using supercritical fluid chromatography (SFC) is established in this work. Samples were prepared by extraction of Dahuang powders with 20% H2SO4 and benzene under reflux. The extracts were obtained by evaporating benzene extracts and separating by SFC on YMC-Diol column using supercritical CO2 with CH3OH 4% (v/v) as the modifier. The flow rate of the mobile phase was 15 mL/min, the column pressure was 13 MPa, and the column temperature was 318 K. Cinnamic acid and five kinds of hydroxyanthraquinones including rhein, emodin, aloe emodin, chrysophanol, and physcion were obtained by SFC. The purities of the obtained compounds were all above 97% as determined by high performance liquid chromatography and their chemical structures were identified by nuclear magnetic resonance and mass spectrum. The thermodynamics of chromatographic process was also studied and it revealed that the SFC separation process of these compounds on YMC-Diol column was controlled by enthalpy.

General screening and optimization strategy for fast chiral separations in modern supercritical fluid chromatography

High throughput general chiral screening method using supercritical fluid chromatography was developed. This method takes an advantage of very fast gradient screening (3 min + 1 min isocratic hold) and generic enantioselectivity of the combined additive formed by 0.1% trifluoroacetic (TFA) acid and 0.1% diethylamine (DEA). The TFA/DEA combined additive was systematically added to organic modifiers methanol and isopropanol. Among five tested polysaccharide-based chiral stationary phases, amylose tris(3,5-dimethylphenylcarbamate) and cellulose tris(3,5-dimethylphenylcarbamate) provided the best enantioseparation success rate. Therefore, the proposed initial first-line screening includes four experiments using these two stationary phases and the above mentioned two combinations: CO2/methanol and CO2/isopropanol + the combined additive. If these stationary phases fail in the screening step, cellulose tris(3-chloro-4-methylphenylcarbamate) and cellulose tris(3,5-dichlorophenylcarbamate) can be proposed for the screening in the second line.For further optimization in case of insufficient resolution obtained in the screening phase fine tuning of temperature, BPR pressure and gradient slope was tested with unsuccessful results. An improvement of enantioselectivity was obtained only when gradient elution was replaced by isocratic elution with substantially lower amount of organic modifier, when changing the concentration of the additive or when using combined organic modifier, such as methanol/acetonitrile (1:1). Finally, to enable the MS compatibility, also volatile additives including ammonium formate and ammonium acetate were tested. The results were more encouraging than expected. Volatile buffers thus make an interesting option in chiral SFC screening methods, however, at the cost of somewhat lower enantioselectivity.

Enantioselective Analytical- and Preparative-Scale Separation of Hexabromocyclododecane Stereoisomers Using Packed Column Supercritical Fluid Chromatography.

Hexabromocyclododecane (HBCDD) is an additive brominated flame retardant which has been listed in Annex A of the Stockholm Convention for elimination of production and use. It has been reported to persist in the environment and has the potential for enantiomer-specific degradation, accumulation, or both, making enantioselective analyses increasingly important. The six main stereoisomers of technical HBCDD (i.e., the (+) and (−) enantiomers of α-, β-, and γ-HBCDD) were separated and isolated for the first time using enantioselective packed column supercritical fluid chromatography (pSFC) separation methods on a preparative scale. Characterization was completed using published chiral liquid chromatography (LC) methods and elution profiles, as well as X-ray crystallography, and the isolated fractions were definitively identified. Additionally, the resolution of the enantiomers, along with two minor components of the technical product (δ- and ε-HBCDD), was investigated on an analytical scale using both LC and pSFC separation techniques, and changes in elution order were highlighted. Baseline separation of all HBCDD enantiomers was achieved by pSFC on an analytical scale using a cellulose-based column. The described method emphasizes the potential associated with pSFC as a green method of isolating and analyzing environmental contaminants of concern.

Supercritical fluid chromatography for separation and preparation of tautomeric 7-epimeric spiro oxindole alkaloids from Uncaria macrophylla

Increasing challenge arising from configurational interconversion in aqueous solvent renders it rather difficult to isolate high-purity tautomeric reference standards and thus largely hinders the holistic quality control of traditional Chinese medicine (TCM). Spiro oxindole alkaloids (SOAs), as the markers for the medicinal Uncaria herbs, can easily isomerize in polar or aqueous solvent via a retro-Mannich reaction. In the present study, supercritical fluid chromatography (SFC) is utilized to separate and isolate two pairs of 7-epimeric SOAs, including rhynchophylline (R) and isorhynchophylline (IR), corynoxine (C) and corynoxine B (CB), from Uncaria macrophylla. Initially, the solvent that can stabilize SOA epimers was systematically screened, and acetonitrile was used to dissolve and as the modifier in SFC. Then, key parameters of ultra-high performance SFC (ultra-performance convergence chromatography, UPC2), comprising stationary phase, additive in modifier, column temperature, ABPR pressure, and flow rate, were optimized in sequence. Two isocratic UPC2 methods were developed on the achiral Torus 1-AA and Torus Diol columns, suitable for UV and MS detection, respectively. MCI gel column chromatography fractionated the U. macrophylla extract into two mixtures (R/IR and C/CB). Preparative SFC, using a Viridis Prep Silica 2-EP OBD column and acetonitrile-0.2% diethylamine in CO2 as the mobile phase, was finally employed for compound purification. As a result, the purity of four SOA compounds was all higher than 95%. Different from reversed-phase HPLC, SFC, by use of water-free mobile phase (inert CO2 and aprotic modifier), provides a solution to rapid analysis and isolation of tautomeric reference standards for quality control of TCM.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

Additive free preparative chiral SFC separations of 2,2-dimethyl-3-aryl-propanoic acids

A series of racemic 2,2-dimethyl-3-aryl-propanoic acids were resolved by chiral supercritical fluid chromatography (SFC) without the use of an acidic additive, trifluoroacetic acid (TFA). The use of additive-free protic methanol as co-solvent in CO2 was expanded to successfully resolve other series of carboxylic acid containing racemates. Large-scale SFC of racemic acid 4, 3-(1-(4-fluorophenyl)-1H-indazol-5-yl)-2,2-dimethyl-3-phenylpropanoic acid, in methanol without TFA as additive on both Chiralpak AD-H and Chiralcel OJ-H will be discussed, along with impact on throughput and solvent consumption. Investigation of co-solvent effect on peak sharpening of acid racemate 20, 2-(2-chloro-9-fluoro-5H-chromeno[2,3-b]pyridin-5-yl)-2-methylpropanoic acid, without TFA further indicated that methanol in CO2 provided improved peak shape compared with isopropanol (IPA) and acetonitrile. Finally, we discuss the resolution of basic aromatic chiral amines without the addition of basic additives such as diethylamine (DEA) and application of this protocol for the large-scale SFC separation of weakly basic indazole-containing racemate 14, methyl 3-(1H-indazol-5-yl)-2,2-dimethyl-3-phenylpropanoate, in methanol without DEA.

Development of a Practical Synthesis of Functionalized Azaxanthene-Derived Nonsteroidal Glucocorticoid Receptor Modulators

An efficient route to two functionalized 2-aryl-5H-chromeno[2,3-b]pyridines (azaxanthenes) is reported. The addition of lithiated 2,6-dichloropyridine to salicylaldehyde followed by cyclization was a key process improvement identified for the formation of the azaxanthene core. Further elaboration of 2-chloro-5H-chromeno[2,3-b]pyridin-5-ol at the 5 position was accomplished via Lewis acid-catalyzed coupling with commercially available ((1-methoxy-2-methylprop-1-en-1-yl)oxy)trimethylsilane. A partial classical resolution coupled with a preparative chiral supercritical fluid chromatography (SFC) separation was used to isolate the desired enantiomer of the azaxanthene carboxylic acid that is a common intermediate for both compounds 1 and 2. Suzuki–Miyaura cross-coupling with appropriately substituted boronic acids, followed by condensation with 2-amino-1,3,4-thiadiazole, provided the target compounds with an overall yield of approximately 10%. The use of stable, amorphous materials to support clinical compari...

A Partial Classical Resolution/Preparative Chiral Supercritical Fluid Chromatography Method for the Rapid Preparation of the Pivotal Intermediate in the Synthesis of Two Nonsteroidal Glucocorticoid Receptor Modulators

Preparative chiral supercritical fluid chromatography (SFC) employing an enantioenriched feedstock obtained via partial classical resolution enabled rapid access to kilogram quantities of a key intermediate common to two glucocorticoid (GC) receptor modulator candidates for which neither chemical nor enzymatic resolutions provided the desired chiral purity (>99.0 HPLC area percent). We developed a partial classical resolution of the racemate that afforded 85:15 enantioenriched mixtures with 90% recovery of the desired enantiomer and reduced the SFC processing time by 54%. The SFC method used a 5 cm × 28 cm column packed in-house with 20 μm Chiralpak AD, a total column flow rate of 150 g/min, a cosolvent composition of 12% methanol, and a column back pressure of 75 bar. The feed processing rate was up to 80 mL/h, which translated to 67 g/day of the enantioenriched mixture or 57 g/day of the desired enantiomer. This combined classical resolution/SFC process was employed to obtain a total of 1.0 kg of the desired enantiomer with an enantiomeric excess greater than 99.5% in 79% isolated yield by running the SFC separation over 23 days, 24 h/day, and 5 days/week. Stable column pressure drops of 7–11 bar were measured throughout the manufacturing campaign. This separation approach enabled the first large-scale preparation of the two GC compounds and permitted their evaluation in toxicological and first-in-human studies within a few months of entering development.

Supercritical fluid chromatography coupled with in-source atmospheric pressure ionization hydrogen/deuterium exchange mass spectrometry for compound speciation

An experimental setup for the speciation of compounds by hydrogen/deuterium exchange (HDX) with atmospheric pressure ionization while performing chromatographic separation is presented. The proposed experimental setup combines the high performance supercritical fluid chromatography (SFC) system that can be readily used as an inlet for mass spectrometry (MS) and atmospheric pressure photo ionization (APPI) or atmospheric pressure chemical ionization (APCI) HDX. This combination overcomes the limitation of an approach using conventional liquid chromatography (LC) by minimizing the amount of deuterium solvents used for separation. In the SFC separation, supercritical CO2 was used as a major component of the mobile phase, and methanol was used as a minor co-solvent. By using deuterated methanol (CH3OD), AP HDX was achieved during SFC separation. To prove the concept, thirty one nitrogen- and/or oxygen-containing standard compounds were analyzed by SFC-AP HDX MS. The compounds were successfully speciated from the obtained SFC-MS spectra. The exchange ions were observed with as low as 1% of CH3OD in the mobile phase, and separation could be performed within approximately 20min using approximately 0.24mL of CH3OD. The results showed that SFC separation and APPI/APCI HDX could be successfully performed using the suggested method.

An improved classification of stationary phases for ultra-high performance supercritical fluid chromatography

Supercritical fluid chromatography (SFC) has recently benefited of new instrumentation, together with the availability of many ultra-high performance columns (sub −2μm fully porous particles or sub −3μm superficially porous particles), rendering it more attractive than ever. Most of these columns commonly used in SFC were initially developed for HPLC use, with an increasing number of stationary phases specifically designed for SFC. While the availability of different stationary phase chemistries is an advantage to achieve successful SFC separations, selecting a column for method development remains difficult. For this reason, we have previously developed a classification of stationary phases dedicated to SFC use. It is based on linear solvation energy relationships (LSER) with Abraham descriptors (for neutral species). While current interest in SFC is strong in the pharmaceutical industry, the need to take account of interactions occurring with ionisable species is pressing. We have previously shown how a modified version of the solvation parameter model, adapted to take account of ionic and ionizable species, could be applied to the characterization of SFC systems. In the present paper, based on this modified LSER model, and on the analysis of 109 neutral and ionisable species, we propose an improved classification of 31 ultra-high performance stationary phases to facilitate method development with SFC.