Supercritical Fluid Chromatography Method Research Articles

Application of sub-/supercritical fluid chromatography for the fingerprinting of a complex therapeutic peptide.

The application area of supercritical fluid chromatography expanded tremendously over the last years and more polar analytes such as biomolecules have become accessible. The growing interest in biopharmaceuticals and associated regulatory requirements demand alternative analytical tools. The orthogonal nature of supercritical fluid chromatography compared to reversed-phase liquid chromatography meets these needs and makes it a useful option during research and development. In this study, we present a systematic approach for the development of a supercritical fluid chromatography method for fingerprinting of tyrothricin, a complex therapeutic peptide covering a mass range from 1200 to 1900Da. The substance was chosen due to the presence of cyclic and linear peptides and isomeric or highly similar amino acid sequences. Different column chemistries covering neutral, basic, and zwitterionic functionalities in combination with acidic, basic, and neutral additives were screened. Subsequently, Design-of-Experiments principles were utilized to perform optimization of the chromatographic parameters. The final mass spectrometry-compatible gradient method using a diol stationary phase, carbon dioxide, and a modifier consisting of methanol/water/methanesulfonic acid (100:2:0.1, v:v:v) was found to provide orthogonality and superior resolution to other methods published. Isomeric peptide compounds coeluting in reversed-phase liquid chromatography were resolved by applying the final method.

Advanced Development of Supercritical Fluid Chromatography in Herbal Medicine Analysis.

The greatest challenge in the analysis of herbal components lies in their variety and complexity. Therefore, efficient analytical tools for the separation and qualitative and quantitative analysis of multi-components are essential. In recent years, various emerging analytical techniques have offered significant support for complicated component analysis, with breakthroughs in selectivity, sensitivity, and rapid analysis. Among these techniques, supercritical fluid chromatography (SFC) has attracted much attention because of its high column efficiency and environmental protection. SFC can be used to analyze a wide range of compounds, including non-polar and polar compounds, making it a prominent analytical platform. The applicability of SFC for the separation and determination of natural products in herbal medicines is overviewed in this article. The range of applications was expanded through the selection and optimization of stationary phases and mobile phases. We also focus on the two-dimensional SFC analysis. This paper provides new insight into SFC method development for herbal medicine analysis.

Liquid crystals purity assay using nonaqueous capillary electrokinetic chromatography.

A method for purity control of newly synthesized lactic acid-based liquid crystals has been developed. The electrokinetic chromatography proved to be suitable for the separation of these electroneutral substances from their impurities. The separations were performed in an acidic acetonitrile-based background electrolyte (BGE) with a pseudostationary phase formed by a cationic surfactant. During the optimization step, appropriate concentrations of cetyltrimethylammonium bromide, acetic acid, and water were seeked. In the optimized method, separations were carried out in acetonitrile with 1-mol/L acetic acid, 80-mmol/L cetyltrimethylammonium bromide, and 6% (v/v) water. Interesting positive effects of a small water content in the BGE on electroosmotic flow and resolution of liquid crystal substances from their impurities were observed and discussed. Samples of five liquid crystal substances, both pure and containing impurities from synthesis, were analyzed. The identification of analytes was based on a comparison of relative migration times related to the migration time of mesityl oxide. For all five samples, impurities were separated from the liquid crystals and the method thus showed its viability. To the best of our knowledge, this method is used for the first time for the purity control of newly synthesized liquid crystals. This method can be used to confirm or complement the results obtained by commonly used high-performance liquid chromatography and supercritical fluid chromatography methods. Furthermore, the electrokinetic chromatography method requires very small amounts of sample, solvents, and buffer constituents. Overall, its operational costs are significantly lower.

Rapid fingerprint analysis based on supercritical fluid chromatography for quality evaluation of Hedyotis diffusa

In this study, quality evaluations of Hedyotis diffusa (H. diffusa) batches by rapid fingerprint analysis based on supercritical fluid chromatography (SFC) were accomplished. Abundant chemical components of H. diffusa were effectively extracted by optimal supercritical fluid extraction conditions (20 % MeOH as modifier, 45 °C, 300 bar and 60 min). Then, the extract was separated by SFC on a Torus 1-AA column (100 × 3.0 mm i.d., 1.7 µm) within 10 min by gradient elution increasing from 5 % to 45 % modifier (MeOH containing 0.05 % TFA) at 1.2 mL/min, 30 °C and 2000 psi. The SFC approach exhibited short analysis time, while maintaining good peak shape and resolution. Seven major compounds were further identified by SFC coupled with tandem mass spectrometer to be phenylpropanoid, iridoids and anthraquinones. Finally, fingerprint analysis of 10 batches of H. diffusa by the developed SFC method was accomplished. The similarity values were between 0.894 and 0.968, indicating quality differences of H. diffusa from depending on origin and harvest year exist. The result demonstrates the feasibility of the SFC in batch quality evaluation of H. diffusa.

Ultrahigh-Performance Supercritical Fluid Chromatography and Detection of Multiple Biogenic Amines in Gentamicin Sulfate: Method Development Using Computer-Assisted Modeling.

In order to solve the problem of difficult separation of various biogenic amines (BAs), which have similar structures or very different polarities, in gentamicin, by conventional liquid chromatography, a new ultrahigh-performance supercritical fluid chromatography (UHPSFC) method was developed. In this method, 10 BAs were derivatized precolumn using dansyl chloride and separated using a UHPSFC system. By computational simulation, complete separation of 10 BAs was successfully achieved. Detection was performed using a photodiode array (PDA) and single-quadrupole mass spectrometry (MS) together with electrospray ionization (ESI). A wide linear range (10-2500 ng/mL) was achieved, with the limits of detection (LODs) between 1.2 and 10.0 ng/mL and the limits of quantification (LOQs) between 5.0 and 25.0 ng/mL. Apart from high sensitivity, this UHPSFC-PDA/ESI-MS detection method also displayed high accuracy, the matrix effect was reduced by an appreciable extent, and the recovery rates of the 10 BAs were between 84.1 and 117.1%. For comparison, high-performance liquid chromatography-tandem mass spectrometry (MS/MS) was also used for the detection of underivatized BAs in gentamicin, showing good linearity and high sensitivity (LODs from 0.05 to 1.00 ng/mL and LOQs from 1.00 to 12.50 ng/mL) for all BAs except for spermine and spermidine. Although single-quadrupole MS is inferior to MS/MS in terms of sensitivity, the UHPSFC method could detect more BAs. It also achieved the quantification limits required for impurity determination, demonstrating a potential strategy to offer a map overview of possible BA presence in fermentation antibiotics.

Chiral separation of β-blockers by supercritical fluid chromatography using Chiralpak-IG and Chiralpak IBN-5 columns.

Chiral separation of β-blockers is performed by utilizing the supercritical fluid chromatographic method. The chiral columns utilized were Chiralpak IG and Chiralpak IBN-5. The finest mobile phase was CO2 -0.2% TEA in methanol (60:40). The values atenolol enantiomers retention factors were 6.39 and 8.98. These values for propranolol enantiomers were 3.39 and 4.06. These values for betaxolol enantiomers were 4.08 and 4.68. The separation and resolution factor values for atenolol, propranolol, and betaxolol were 1.41 and 3.33, 1.19 and 2.23, and 1.15 and 1.87, separately and respectively. By comparison, it was observed that Chiralpak IG column is better than Chiralpak IBN-5 column. Supercritical fluid chromatography has been found as the best analytical technique due to its high speed, being eco-friendly, and being economic. The various most probable interactions responsible for the chiral resolution are hydrogen bonding, dipole-dipole interactions, steric effect, and π-π interactions. The reported methods are effective, efficient, and reproducible and may be used to separate and identify atenolol, propranolol, and betaxolol in any unknown samples.

Development of Supercritical Fluid Chromatographic Methods for Separation of Conazole Pesticide Enantiomers

Development of Supercritical Fluid Chromatographic Methods for Separation of Conazole Pesticide Enantiomers

Isolation of achiral aliphatic acid derivatives from Piper kadsura using preparative two-dimensional chiral supercritical fluid chromatography

The separation of structural analogues in natural products has always been one of the challenges in separation science, where supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an unconventional but potential solution. In this study, a preparative two-dimensional chiral SFC (2D cSFC) method that was established with two kinds of CSPs was applied in the isolation of the aliphatic acid derivatives in Piper kadsura (P. kadsura). The RPLC unseparated peaks of two samples A and B of P. kadsura were evenly scattered on the CSP-1 column while they clustered into two groups on the CSP-2 column by SFC. There was impressively complementary selectivity between CSP-1 and CSP-2, which were used for construction of 2D cSFC. The first dimension (1D) separation with CSP-1 fractionated the sample A into six parts by a heart-cutting method and the sample B into nine parts for a comprehensive 2D analysis; then 29 and 71 peaks were respectively found in these parts in the second dimension (2D) separation with CSP-2. Further through 2D preparative separation, 19 high purity components were obtained, and the chemical structures of two of them were confirmed, including a novel unsaturated aliphatic acid compound (8Z,10Z)-12-methoxyheptadeca-8,10-dienoic acid and a known octadecadienoic acid lactone Lactariolide. The 2D cSFC method presented the superiority of separating the achiral compounds of complex samples.

Ion pair-based mobile phase additives to improve the separation of alkaloids in supercritical fluid chromatography

In this study, a supercritical fluid chromatography (SFC) method based on ion pair reagents was used to separate alkaloids. The chromatographic parameters, including the stationary phase, additive type, additive concentration, outlet pressure, temperature and flow rate, were optimized. Baseline separation was completed in 20 min on an Agilent Pursuit 5 PFP column (4.6 × 150 mm) using carbon dioxide as the mobile phase and 7.5 mM sodium 1-pentanesulfonate as an additive with gradient elution at 140 bar, 60 °C, and a flow rate of 1.5 mL/min. The retention rate and resolution of the analytes were satisfactory. The limits of detection were 27.04–298.03 ng/mL, and the limits of quantification were 90.15–993.42 ng/mL. The recoveries of low and high concentrations were 77.46–111.86% and 83.84–111.00%, respectively. This ion pair additive greatly improved the separation efficiency of alkaloids. Consequently, this SFC method was successfully applied to the separation of alkaloids from Rhizoma corydalis.

Separation of chiral and achiral impurities in paroxetine hydrochloride in a single run using supercritical fluid chromatography with a polysaccharide stationary phase

Separating paroxetine hydrochloride and its impurities using conventional reversed-phase liquid chromatography (RPLC) is challenging due to their highly similar structures. In the present study, a rapid, simple, sensitive and environmentally friendly method was developed for the determination of chiral and achiral impurities in raw materials of paroxetine hydrochloride using chiral supercritical fluid chromatography (SFC). The impacts of chiral stationary phases (CSPs), mobile phases, column temperature and back pressure on the retention and separation of analytes were comprehensively evaluated. After method optimization, a satisfying result was obtained on a cellulose tris-(3-chloro-4-methylphenylcarbamate) stationary phase in 4.0 min using 70% CO2 and 20 mM ammonium acetate in 30% methanol as the mobile phase. Molecular docking was further performed to understand the interactions between the analytes and CSP. The results suggested that hydrogen bonding and π–π interactions were the dominant interactions. The affinity given by the software was in good agreement with the elution order and free energy (△G) values obtained from van’t Hoff equations. The results of molecular docking also provide insights into the different retentions of N-methylparoxetine at different temperatures. The results of method validation revealed that the method was sensitive with a limit of detection of approximately 0.05 μg·mL−1 (corresponding to approximately 0.005% paroxetine hydrochloride in the sample solution). The relative standard deviations (RSDs) of precision and intra-assay precision were all less than 2.0%, and the recoveries of the method were 93.8~105.3% with RSDs less than 3.0%. The chiral and achiral RPLC methods included in the Chinese pharmacopoeia and the SFC method proposed in this study were simultaneously used to determine the impurity content in the raw materials of paroxetine hydrochloride. The results showed that impurities that cannot be detected by the reference method can be accurately quantified using the SFC method. In addition, the SFC method has advantages in terms of throughput, analysis cost and simplicity. This study can provide a reference for further research of impurities in paroxetine hydrochloride and promote the application of chiral SFC in the rapid separation of structurally similar compounds.

Supercritical fluid chromatography for the analysis of natural dyes: From carotenoids to flavonoids.

Plant-derived natural dyes are used in a variety of formulated products, from food to cosmetics and pharmaceutics. In addition to their color, they also provide some bioactivity. While they are mostly analyzed with high-performance liquid chromatography, supercritical fluid chromatography was also employed for several dye families, mostly for carotenoids and chlorophylls, and more recently for anthraquinones and flavonoids. These supercritical fluid chromatography methods are described in this review. Because the dyes have different structures and structural variations (polarity, isomers, etc.), the best chromatographic system to achieve their separation is not always the same. Hydrophobic stationary phases are preferred for the most hydrophobic dyes (chlorophylls and carotenoids) while polar stationary phases are preferred for the polar dyes (anthraquinones and flavonoids). Regarding the mobile phase composition, chlorophylls and carotenoids are best eluted with moderate proportions of co-solvent in CO2 (about 40%), while the most polar glycosylated flavonoids require higher proportions of co-solvent and acidic additives. Because dyes are colorful, ultraviolet-visible detection is often sufficient, while mass spectrometry offers additional structural information. Furthermore, fundamental information can also be gained through chromatographic analysis of dyes: either solubility in supercritical fluids, in view of their extraction, or retention behavior providing an understanding of stationary phase properties.

Separation and characterization of phenylamides from Piper kadsura using preparative supercritical fluid chromatography and ultra-high-performance supercritical fluid chromatography-tandem mass spectrometry.

A preparative supercritical fluid chromatography method for the separation of Piper kadsura obtained five phenylamide compounds, which had the same structural skeleton, but changed in the number and position of methoxyl substituents. To improve the separation selectivity of these structural analogues, silica, phenyl, and chiral stationary phases were screened. Only through the combination of Chiral C and phenyl columns could the separation of the five phenylamides be solved. The two-step strategy using preparative supercritical fluid chromatography presented good orthogonality that ensured the purity of the phenylamides. Then, an ultra-high-performance supercritical fluid chromatography hyphened tandem mass spectrometry method was developed, and the fragmentation pattern of phenylamides was summarized. It mainly cleaved in the amide bond to produce the fragment ion, which could help to judge the substituent positions. Twenty-eight possible molecular weights of hydroxyl and methoxyl substituted phenylamides were calculated and screened. Nine compounds were extracted in three [M + H]+ ions at m/z 284.13, 314.13, and 344.13, including five purified compounds and the other four positional or trans-cis phenylamide isomers in low content. The methods developed in this research were useful in the separation and characterization of phenylamide analogues.

Review of Determination of Chlortalidone Levels in Pharmaceutical Preparations and Biological Matrix

Chlortalidone is a compound that is often used in the pharmaceutical world as an oral drug to treat hypertension. This review article discusses the summary of methods for determining the concentration of chlorthalidone in pharmaceutical preparations and biological matrices. We took the data collected in this review article was taken through trusted sites such as Google Scholar with the search keywords "chlorthalidone determination," "pharmaceutical preparations," and "biological matrices," with a period of the last twenty years (2001-2021). This review article aims to provide an overview of the assay used to determine the concentration of chlorthalidone either in the form of a single substance or pharmaceutical dosage forms. Overall, the determination of chlorthalidone levels has been carried out using various analytical methods. Including spectrophotometry, high-performance liquid chromatography (HPLC), chemometrics and thin-layer chromatography (TLC) - densitometric, capillary zone electrophoresis (CZE) methods, spectrofluorimetric methods, liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) methods, ultrafast liquid chromatography (UFLC), supercritical fluid chromatography method and high-performance thin-layer chromatography (HPTLC)-densitometric method. Analysis with the HPLC technique is widely used in research because it can detect samples with the lowest concentrations up to the nanogram level.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of six compounds in Guizhi Fuling capsule and tablet samples.

A fast and simple ultra-high performance supercritical fluid chromatography method has been developed for the determination of six analytes, namely (paeonol, coumarin, cinnamic alcohol, cinnamic acid, paeoniflorin, and amygdalin) in Guizhi Fuling capsule and tablet samples. The influence of the key chromatographic parameters for the separation purposes was evaluated. The optimal column was Trefoil CEL1 column. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol at flow rate of 1.0mL/min. The back pressure of the system was set to 1.38 × 107 Pa and the temperature to 45°C. The six compounds were separated within 11min by the proposed ultra-high performance supercritical fluid chromatography method with satisfactory resolution. Method validation confirmed that the procedure is accurate with the recovery rates from 87.04 to 104.30%, intraday precision values less than 4.81% and interday precision less than 5.22%, and linear with R2 higher than 0.9967. Therefore, this work provides a simple and novel method for the simultaneous analysis of six compounds in Guizhi Fuling capsule and tablet samples.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an ‘instrument type’ effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Large-scale supercritical fluid chromatography purification of unstable STING agonist intermediates

A regioisomeric mixture of the nucleoside derivative, Intermediate 1, required resolution by preparative supercritical fluid chromatography (SFC) in order to obtain the desired regioisomer as a key intermediate in a STING agonist program. Various chiral columns and solvents including methanol, acetonitrile, isopropanol, and the mixture of acetonitrile and isopropanol as organic modifiers in carbon dioxide at different temperatures were screened to obtain the best regioisomeric resolution. A key issue associated with interconversion between the regioisomers via silyl migration during purification was investigated in methanol, acetonitrile, and the mixture of acetonitrile and isopropanol, and the optimal organic modifier in CO2 was established to mitigate the interconversion to an acceptable level (<5%). Taking into account peak resolution, throughput, interconversion and operation robustness, an efficient SFC method for large-scale purification was successfully developed and scaled up onto a 5 cm I. D. Chiralcel OJ-H column using 25% acetonitrile: isopropanol [1:1 (v/v)] with 0.1% ammonium hydroxide as the modifier in CO2 at a total flow rate of 270 mL/min and a temperature of 30°C. In addition, continual evaporation (i.e. every hour) of the desired isomer fraction stream post-separation ensured minimal further interconversion. A total of 258 grams were separated at a high throughput of 8.6 g/h. Regioisomeric purity of the desired isomer of Intermediate 1 was ≥98.2% and the recovery was ≥90.2%. A similar purification strategy was applied to the regioisomeric resolution of Intermediate 2, an analog of Intermediate 1. In total, 1028 grams of Intermediate 2 were processed at a high throughput of 12.5 g/h on a Viridis BEH 2-EP column. The regioisomeric purity of the desired isomer was ≥96.8% and the recovery was ≥90.7%.

The effect of water on the large-scale supercritical fluid chromatography purification of two factor XIa active pharmaceutical ingredients

BMS-962212, a parenteral Factor XIa inhibitor, was scaled-up for toxicity studies. Two steps of supercritical fluid chromatography (SFC) were developed for the chiral resolution of the penultimate and achiral purification of final active pharmaceutical ingredient (API), BMS-962212. A robust SFC process using Chiralcel OD-H with methanol-acetonitrile as modifier in CO2 was established to achieve a stable and uninterrupted operation with reduced mobile phase viscosity and system pressure drop. More than 230 g of the racemic penultimate was chirally resolved to reach >99% chiral purity, ready for final tert-butyl ester deprotection to provide the API. There were a significant number of impurities in BMS-962212 generated from the final step that needed to be removed. In contrast to conventional SFC conditions, an SFC method exploiting water and ammonia as additives in both the mobile phase and sample solution was developed to accomplish purification and desalting (i.e. removing TFA) of the zwitterionic API in one step. Water as an additive eliminated salt precipitation and improved the resolution while ammonia contributed to the desalting, details of which will be discussed in this article. A throughput of 2 g/h was achieved, and >80 g of the crude API was purified. The same strategy was applied to another Factor XIa API (compound A) and its penultimate.

Effect of the chemical composition of six hydrotreated light cycle oils for benzene, toluene, ethylbenzene, and xylene production by a hydrocracking process

The effect of the chemical composition of the hydrotreated light cycle oil (HDT LCO) on the benzene, toluene, ethylbenzene, and xylene (BTEX) production by a hydrocracking (HCK) procedure, is presented. Six different types of HDT LCOs were obtained by submitting two types of LCOs to hydrotreating (HDT) with different catalysts and experimental conditions. The products were analyzed as mono-, di- and tri-aromatic compounds using the supercritical fluid chromatography (SFC) method (ASTM D5186). The HDT LCOs were subjected to HCK with a 50/50 in weight mixture of nickel-molybdenum on alumina (NiMo/Al2O3) and H-ZSM5 (NiMo/H-ZSM5, 50/50) at 375 °C, 7.5 MPa, 1.2 h−1, and 750 m3/m3 H2/Oil. The HCK products were analyzed by gas chromatography with a flame ionization detector (GC-FID) and divided into five groups: gas, light hydrocarbons (LHCs), BTEX, middle hydrocarbons (MHCs), and heavy hydrocarbons (HHCs).The results showed that the BTEX formation ranged from 27.0 to 29.8 wt.% and it did not show a significant dependence on the mono-aromatic (59.9 and 75.6 wt.%), total aromatic (61.1–84.2 wt.%) contents or MHCs conversion (58.3–64.3 wt.%) from the departing HDT LCO feedstock. This result implies that, contrary to previous expectations, the BTEX formation does not directly depend on the amounts of total or mono-aromatic compounds when departing from real feedstocks. A GC-PIONA (paraffin, isoparaffin, olefin, naphthene, aromatic) characterization method (ASTM D6623) for mechanism understanding purpose was also carried out.

Rapid Purification of Drug Enantiomers using Supercritical Fluid Chromatographic Method: Ibuprofen as a Model Compound

Rapid Purification of Drug Enantiomers using Supercritical Fluid Chromatographic Method: Ibuprofen as a Model Compound

A Retention-Matching Strategy for Method Transfer in Supercritical Fluid Chromatography: Introducing the Isomolar Plot Approach

A strategy to match any retention shifts due to increased or decreased pressure drop during supercritical fluid chromatography (SFC) method transfer is presented. The strategy relies on adjusting the co-solvent molarity without the need to adjust the back-pressure regulator. Exact matching can be obtained with minimal changes in separation selectivity. To accomplish this, we introduce the isomolar plot approach, which shows the variation in molar co-solvent concentration depending on the mass fraction of co-solvent, pressure, and temperature, here exemplified by CO2–methanol. This plot allowed us to unify the effects of the co-solvent mass fraction and density on retention in SFC. The approach, which was verified on 12 known empirical retention models for each enantiomer of six basic pharmaceuticals, allowed us to numerically calculate the apparent retention factor for any column pressure drop. The strategy can be implemented either using a mechanistic approach if retention models are known or empirically by iteratively adjusting the co-solvent mass fraction. As a rule of thumb for the empirical approach, we found that the relative mass fraction adjustment needed is proportional to the relative change in the retention factor caused by a change in the pressure drop. Different proportionality constants were required to match retention in the case of increasing or decreasing pressure drops.