Fluid Chromatography Research Articles

Advantages of dimethyl carbonate as organic modifier for enantioseparation of novel psychoactive substances in sub/supercritical fluid chromatography

BackgroundSub/supercritical fluid chromatography is regarded as a greener separation technique due to the use of carbon dioxide as the main component of the mobile phase compared to conventional liquid chromatography techniques. Organic co-solvents are usually added to carbon dioxide to increase elution strength of the mobile phase. Therefore, it is of great importance to test applicability of green co-solvents in separation methods and to include them among commonly used mobile phase components. ResultsA comprehensive study of the suitability of green solvent dimethyl carbonate as a co-solvent for enantioseparation in sub/supercritical fluid chromatography was conducted with a set of novel psychoactive substances from various groups. The experiments were performed on polysaccharide-based columns. For successful enantioseparation of these compounds, the presence of basic or mixed mobile phase additives was essential. The obtained results clearly show that dimethyl carbonate is a suitable co-solvent for enantioseparation on polysaccharide-based columns in sub/supercritical fluid chromatography and in some cases surpasses commonly used co-solvents as methanol and propan-2-ol. SignificanceThe use of more sustainable co-solvents, such as dimethyl carbonate, instead of conventional ones to carbon dioxide presents a greener approach to analytical applications and reduces the overall environmental impact of analytical processes.

On the greenness of separation modes containing compressed fluids

BackgroundIn the past three decades, liquid chromatography (LC) has been recognized as a significant environmental, health, and safety burden due to its heavy reliance on toxic organic solvents. Various chromatographic modes are in vogue today for complex analyses, such as sub/supercritical fluid chromatography (SFC) and enhanced fluidity liquid chromatography (EFLC). These modes are often advertised as "universally green" compared to the traditional allliquid reversed (RPLC) and normal phases (NPLC). Quantitative greenness evaluations must be done to validate or invalidate this assumption and allow separation scientists to make educated choices when deciding on what mode to use. ResultsIn this work, we modify the Analytical Method Greenness Score (AMGS) to include the cycle time of the instrument, and with the help of the first-order optimality condition (by setting the AMGS gradient = 0), we show that SFC and EFLC are not always the greenest option as they are often thought to be. Most of the greenness metrics have ignored the cycle time of instruments, yet this key component changes the entire AMGS response to flow rate. The complex case of separating tobacco alkaloid enantiomers (nicotine, nornicotine, anabasine, and anatabine) was selected as an illustrative example for comparing and contrasting separation modes using the modified greenness metric. These enantiomers have been selected due to their notorious difficulty in separation over the past 30 years. Using this family of molecules, four unique retention patterns were observed covering a wide variety of retention phenomena seen in small molecule enantioseparations. SignificanceThe modified AMGS metric will assist practicing analytical chemists in assessing the environmental impact of their separation methods from a single run in a given chromatographic mode. The proposed methodology identifies the minimum AMGS score corresponding to the greenest separation for routine chemical analysis.

Expanding cyclodextrin use in normal phase and super/subcritical fluid chromatographic modes for the chiral separation of 1,4-dihydropyridines

Cyclodextrin-based stationary phases are important chiral selectors in liquid chromatography. These chiral selectors are most commonly used in the reversed-phase mode because native cyclodextrin assumes a torus conformation with a hydrophobic cavity, facilitating inclusion complexation in aqueous environments. However, the value of native and aliphatic-derivatized cyclodextrins in other modes, such as the normal phase liquid chromatography (NPLC) or super/subcritical fluid chromatography (SFC), remains unexplored. In this work, we report chiral separations of pharmaceutically relevant compounds with the 1,4-dihydropyridine (DHP) scaffold on a 2-hydroxypropyl-β-cyclodextrin (CD-RSP) stationary phase in NPLC and SFC modes. Although CD-RSP is conventionally considered only effective in the reversed-phase mode, we show that these compounds tend to separate better in other modes. This is particularly apparent for analytes with hydrogen-bonding moieties. We propose that the separation mechanism primarily depends on external adsorption rather than inclusion complexation. The negligible impact of a complexation-competitive additive on retention in non-aqueous modes further supports this claim. Additionally, van Deemter analysis demonstrated the efficiency and environmental benefit of using this stationary phase in the SFC mode, further highlighting the promise of aliphatic derivatized cyclodextrin stationary phases for greener separations.

The enantioselective potential of amylose tris(3‑chloro-5-methylphenyl carbamate)-based chiral stationary phase in sub/supercritical fluid and normal phase liquid chromatography

The enantioselective potential of amylose tris (3‑chloro-5-methylphenylcarbamate) based chiral stationary phase in sub/supercritical fluid chromatography and normal phase liquid chromatography was evaluated. To test its enantioselective potential the enantiomeric resolution of a set of nineteen important chiral biologically active compounds was explored. The influence of the cosolvent and different mobile phase additives on the retention, enantioselectivity, and enantioresolution was investigated. The type, as well as the amount of cosolvent used in the mobile phase, are critical for enantioseparation efficiency in both techniques. Using basic or mixed additives in the mobile phases significantly improved enantioseparation and peak shape of different compounds, especially β-blockers. Results showed the high enantioselective potential of the chlorinated amylose-based chiral stationary phase in both chromatographic techniques with overall higher enantiorecognition ability in sub/supercritical fluid chromatography.

The Enantioselective Potential of NicoShell and TeicoShell Columns for Basic Pharmaceuticals and Forensic Drugs in Sub/Supercritical Fluid Chromatography.

The enantioselective potential of two macrocyclic glycopeptide-based chiral stationary phases for analysis of 28 structurally diverse biologically active compounds such as derivatives of pyrovalerone, ketamine, cathinone, and other representatives of psychostimulants and antidepressants was evaluated in sub/supercritical fluid chromatography. The chiral selectors immobilized on 2.7 μm superficially porous particles were teicoplanin (TeicoShell column) and modified macrocyclic glycopeptide (NicoShell column). The influence of the organic modifier and different mobile phase additives on the retention and enantioresolution were investigated. The obtained results confirmed that the mobile phase additives, especially water as a single additive or in combination with basic and acidic additives, improve peak shape and enhance enantioresolution. In addition, the effect of temperature was evaluated to optimize the enantioseparation process. Both columns exhibited comparable enantioselectivity, approximately 90% of the compounds tested were enantioseparated, and 30% out of them were baseline enantioresolved under the tested conditions. The complementary enantioselectivity of the macrocyclic glycopeptide-based chiral stationary phases was emphasized. This work can be useful for the method development for the enantioseparation of basic biologically active compounds of interest.

Insights into enantioselective separations of ionic metal complexes by sub/supercritical fluid chromatography

Sub/supercritical fluid chromatography (SFC) is a green separation technique that has been used to separate a wide variety of compounds and is proven to be immensely useful for chiral separations. However, SFC is currently not thought to be applicable for ionic compounds due to their low solubility in CO2, even with additives and organic modifiers. Recently, a large amount of research has been centered on octahedral complexes of Ru(II) and Os(II) with bidentate polypyridyl ligands due to their ability to serve in cancer treatment and other biological activities. These compounds exist as the delta (Δ) and lambda (Λ) enantiomers. Previously, similar compounds have been enantiomerically separated using HPLC and capillary electrophoresis, but never with SFC. Cyclofructan-6 (CF6) derivatized with (R)-naphthyl ethyl (RN) groups has been proven to be an effective chiral stationary phase for these separations in HPLC. This column chemistry was expanded to SFC to provide the first chiral separation of a wide variety (23 complexes in total) of ionic octahedral polypyridyl complexes. Unexpected behavior for mixing methanol and acetonitrile as the organic modifier will be discussed, along with the effects of additives. Enantioselectivity on CF6-RN chemistry is shown to be dependent on the conjugation level and rigidity of the metal complexes. Mass transfer kinetic behavior is also shown, and high-efficiency baseline resolved rapid separations are shown for fast screening or quantitation of representative coordination complexes.

Additives in chiral packed column super/subcritical fluid chromatography: A little goes a long way

The introduction of additives has revolutionized super/subcritical fluid chromatography (SFC) by expanding the range of compounds that could be analyzed by the technique. From being considered a mere extension of gas chromatography, incorporation of a modifier, and subsequently an additive has made SFC a workhorse for chiral separations. Additives are by far the smallest component of the SFC mobile phase but can result in changing the polarity and acidity of the mobile phase, suppression of ionization, deactivation of the stationary phase, and act as an ion pairing agent. A wide variety of compounds have been tested as additives including but not limited to diethylamine, trifluoroacetic acid, ammonium acetate, and water. This review summarizes the different role played by additives in the SFC mobile phase. Further this work aims to help the reader by critically evaluating different additives used in enantiomeric separations with packed column SFC.

Sub/supercritical Fluid Chromatography for Chiral Compounds Analysis

Chirality is an essential feature of nature as it is common for many biologically active compounds. The different biological effects of individual enantiomers in a chiral environment are generally known. Therefore, there is a need for fast, efficient, and robust methods for their separation, quantification, and purification, too. The easiest way is to use chromatographic methods utilizing chiral stationary phases. Sub/supercritical fluid chromatography has become popular in the field of enantioselective separations in various scopes and, in some cases, has become a method of the first choice.
 Therefore, this review article covers actual trends and possibilities of sub/supercritical fluid chromatography in enantioseparations. Ways to influence enantioselectivity of the separation system by column coupling, screening approaches, and processes of methodical development for fast and efficient analyses are discussed.
 Sub/supercritical fluid chromatography under suitable experimental conditions provides fast and highly efficient separation of chiral compounds.

Exploring the utility of natural deep eutectic solvents as additives in super/subcritical fluid chromatography- insights into chiral recognition mechanism

A push towards sustainability has led to the widespread adoption of not only green synthetic methodologies but also green analytical techniques. Supercritical fluid chromatography (SFC) is considered a greener technique compared to HPLC. In this work we investigate the utility of natural deep eutectic solvents (NADES) as green additives in chiral SFC to replace toxic additives like triethylamine and trifluoroacetic acid. A total of 33 compounds belonging to different classes were screened with four immobilized polysaccharide stationary phases, using 0.2% NADES additives in methanol modifier. A total of 25 compounds had some separation with the tested NADES. Novel selectivities were obtained using choline chloride-D-glucose when compared to commonly used additives. Other NADES were also synthesized and tested in order to shed light on chiral recognition mechanism with the new additives.

High efficiency functionalized hydrophilic cyclofructans as stationary phases in sub/supercritical fluid chromatography

Packed column SFC has become very popular for preparative and analytical separations due to the low cost of CO2, its accessible critical temperature, and pressure, with the additional benefit of a low environmental burden. Currently, there is a shortage of new polar stationary phase chemistries for SFC. In this work, two new functionalized cyclofructan columns are introduced and evaluated for their performance in achiral SFC separations for the first time. Cyclofructan (CF6), a macrocyclic oligosaccharide, was covalently linked with benzoic acid (BCF6) and propyl sulfonic acid (SCF6) groups by ether bonds. Superficially porous particles (2.7 μm) bonded with modified CF6 showed markedly different selectivity than native CF6. In SFC, peak shapes of amines and basic compounds are often compromised. We show that small quantities (~5.7% v/v) of water added to the methanol modifier in CO2 improves peak symmetries of primary, secondary, and tertiary amines. Efficiencies as high as 200,000 plates/m (reduced plate height ~ 1.8) were observed for benzamide and amitriptyline on the BCF6 column. The relative standard deviations (RSDs) of retention times on BCF6 were about 1.4%, and on SCF6 were less than 1%. Amines on the SCF6 column showed plate counts as high as 170,000 plates/m. Tetramethylammonium acetate is examined as an alternative to water in MeOH. A run time of 36 min with methanol, trifluoroacetic acid, triethylamine mobile phase was reduced to <5 min with complete baseline resolution for a set of amines. The new stationary phases allow greener approaches towards solving separation problems.

Expanding the range of sub/supercritical fluid chromatography: Advantageous use of methanesulfonic acid in water-rich modifiers for peptide analysis

The aim of this work was to expand the applicability range of UHPSFC to series of synthetic and commercialized peptides. Initially, a screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points. Secondly, methanesulfonic acid (MSA) was evaluated as a potential replacement for TFA. Due to its stronger acidity, MSA gave better performance than TFA at the same concentration level. Furthermore, the use of reduced percentages of MSA, such as 8 mM, yielded similar results to those observed with 15 mM of MSA. The optimized UHPSFC method was, then, used to compare the performance of UHPSFC against RP-UHPLC for peptides with different pI and with increasing peptide chain length. UHPSFC was found to give a slightly better separation of the peptides according to their pI values, in few cases orthogonal to that observed in UHPLC. On the other hand, UHPSFC produced a much better separation of peptides with an increased amino acidic chain compared to UHPLC. Subsequently, UHPSFC-MS was systematically compared to UHPLC-MS using a set of linear and cyclic peptides commercially available. The optimized UHPSFC method was able to generate at least similar, and in some cases even better performance to UHPLC with the advantage of providing complementary information to that given by UHPLC analysis. Finally, the analytical UHPSFC method was transferred to a semipreparative scale using a proprietary cyclic peptide, demonstrating excellent purity and high yield in less than 15 min.

Macrocyclic glycopeptide-based chiral selectors for enantioseparation in sub/supercritical fluid chromatography.

Increasing number of reported works dealing with macrocyclic glycopeptide-based columns in sub/supercritical fluid chromatography (SFC) points to the growing interest in this area. With the development and production of sub 2µm fully porous particles and superficially porous particles with bonded macrocyclic glycopeptides, significant improvements have been made in ultrafast high efficiency chiral SFC. This review article gives an overview of macrocyclic glycopeptide-based chiral selectors that were used in theoretical studies and/or applications in SFC. The review covers the period from 1997 when macrocyclic glycopeptides were first used in SFC till the end of July 2020 according to Web of Science. This work can also be helpful to analysts searching for an appropriate method for the separation/determination of enantiomers of their interest.

Review Article: Bioseparation

The Bio separation is stringent to the area of engineering for biochemistry. All bio separation operation should be suits to detach, purifying, or regain the coveted product. The bio separation depends on, essential property involved thermo constancy, solubility, diffusion, shipment, isoelectric pH, and other things. The purifying of biological products is get by a bio separations operations include a lengthy series of procedures, and all procedure needs the employ single or more unity operations, like filtrate, extraction, chromatography and desiccation. The Filtering isolate a components depending to volume while a liquid solute flux during a pored midst down a compressing differential. Super filtration of a protein solute at fixed size is completed with the addendum of H2O or buffer to the feed in an process named diafiltration. Fluid chromatography is a form of absorption operation, which is, depends on the affinity of different solution for specified form of solid. The generality efficiency chromatography process employ each the adsorption sites ready. While chromatography to purifying a protein is scaled up from the laboratory level, knowing of the laboratory elution profile is useful in interpreting the great level elution profile.

Advanced reaction monitoring of pharmaceutical processes enabled with sub/supercritical fluid chromatography

Reaction monitoring is crucial in order to guide pharmaceutical chemical processes towards successful outcomes. Many drug substances require the application of water/additive-free solvents to guide process efforts which are intrinsically incompatible with reversed phase liquid chromatography (RPLC) conditions. Sub/supercritical fluid chromatography (SFC) helps to overcome many of these challenges delivering unmatched speed, with low cost, high-efficiency and an additive-free environment ideal for reaction monitoring of species chemically susceptible to water. Herein, we provide several examples where traditional RPLC fails or delivers sub-optimal performance in monitoring processes. These examples include using additive-free SFC-UV and SFC-MS methodologies to overcome difficulties associated with: 1) on-column RPLC hydrolysis, 2) improving normal phase liquid chromatography, replacing capillary zone electrophoresis methods, and 3) avoiding the use of tedious pre-column derivatization procedures.

Separation of achiral anti-diabetic drugs using sub/supercritical fluid chromatography with a polysaccharide stationary phase: Thermodynamic considerations and molecular docking study

A simple, fast and environmental friendly supercritical fluid chromatography-photodiode array (SFC-PDA) method was developed for the simultaneous determination of metformin and three sodium-glucose co-transporter 2 (SGLT-2) inhibitors (canagliflozin, dapagliflozin and empagliflozin). The impact of different stationary phases, co-solvents, column temperature and outlet pressure was extensively evaluated for selective separation and quantitation of the drugs. Amongst several achiral and chiral stationary phases tested, the best results were obtained on amylose based Chiralpak IG column using CO2 and 0.1 % diethylamine in methanol and isopropanol 50:50 (v/v) in 60:40 ratios as the mobile phase. The separation efficiency was mainly dependent on the nature of stationary phase and the mobile phase composition. The limit of detection (LOD) and quantitation (LOQ) of the method were 0.155 and 0.469, 0.062 and 0.187, 0.015 and 0.045, 0.028 and 0.084 μg/mL for metformin, canagliflozin, dapagliflozin and empagliflozin, respectively. The chromatographic resolution between metformin-canagliflozin, metformin-dapagliflozin and metformin-empagliflozin pairs was 6.87, 3.44 and 6.47, respectively. The intra-batch and inter-batch precision of the method was ≤1.64 % while the accuracy was in the range of 96.2–103.3 %. The method was used to analyze metformin and SGLT-2 inhibitors in their binary fixed-dose formulations with acceptable accuracy (recovery) and precision. To support the experimental results, molecular docking was performed on Schrodinger software to provide a deeper insight into the interactions between the analytes and the chiral stationary phase and to understand their elution orders under optimized conditions.

Pesticide Identification through PCA-ANN Classification of Cyclic Voltammetry using Screen-Printed Electrodes

Introduction Pollution caused by the use of pesticides is a growing concern, strongly linked to the expansion of agricultural operations based on fulfilling the food requirement for humans [1]. Therefore, over the previous decade, there has been a huge increase in research within this area, through both the development of new, innovative detection approaches and the improvement of existing techniques.Several analytical methods were used to analyze pesticides, including fluorimetry, capillary electrophoresis, spectrophotometry, mass spectroscopy, and gas or fluid chromatography [2]. Most of these devices present a number of disadvantages; rather bulky, expensive, and require time-consuming procedures. Research interest has increased on the application of electrochemical instruments for pesticide sensing, since electrochemistry takes benefit of its sensitivity and relative selectivity [3]. The response of electrochemical detectors is given straight with electrical signals, without the need to convert to other types of responses. It results in a less complex instrumentation and consequently low measurement cost, but also in portability, allowing the measurements to be carried out in the field. A potentiostat is used for current-voltage measurements, which forms the basis for electro-analysis. While potentiostats play a significant role in current electrochemical research, they are mostly used in the laboratory courses for little application. These devices have a low penetration due to the price, as even the least expensive commercially accessible laboratory potentiostats are selling for more than USD1000 [4]. The main proposal of this study is the development of a convenient, sensitive, selective and low cost electrochemical sensor that could be used for identification of pesticides in a solution. Cyclic Voltammetry Voltammetric techniques are useful tools in analytical sciences, especially in aqueous media research or in the solid-state of electro-active substances. In a Cyclic Voltammetry (CV) system, by sweeping the potential over time and recording the current, a voltammogram curve is achieved that is equal to the current as a function of the potential applied [5]. The application for voltammetric methods for analytical purposes is due to the fact that the current recorded at the electrode of the indicator is directly proportional to the analyte concentration. With regard to pesticide electro-analysis, most of which are organic compounds with electro-active functional groups, CV and pulse voltammetry are often used. CV is quite common among voltammetric techniques and is usually used to explore the electrochemical behavior of new redox systems in the first place, and CV can also achieve micromolar detection limits. Method The initial electrochemical measurements of cyclic voltammetry (CV) and chronoamperometry were performed with an Autolab potentiostat (Metrohm, model PGSTAT302) with Nova software version 1.7, and the data set for each pesticide will be obtained from this device. To voltammetrically detect the pesticides; two kinds of commercial Screen-Printed Carbon Electros (SPCE purchased from Quasense Co., Ltd) and one homemade electrode (using carbon paste Loctite Edag PR406 E&C) are utilized. To meet the objective of developing a low cost pesticide detector, a homemade potentiostat responsible of carrying out the CV is built by following an open source reference [4], and manually soldering the components to the PCB of the referenced potentiostat schematic. The CV measurements are recorded from the developed device using a Python program that linearly sweeps the potentiostat’s reference electrode voltage from -1V to 1V, while capturing the corresponding current at the working electrode. Results and Conclusions Tables 1 shows 5 types and concentrations of pesticides under investigation.Table 1: 5 types and concentrations of pesticides under investigation Pesticide Concentrations (ng mL-1) Chlopyrifos (organophosphte) 100 1200 Dicofol (organochlorine) 100 1200 Cabaryl (carbarmate) 100 1200 Cypermethrin (pyrethrum) 100 1200 Pirimiphos-methyl 100 1200 Similar to spectroscopy methods, patterns discovered in CV information can be considered as a fingerprint of the experimental system being studied, which is unique and coherent [6]. The use of chemometric tools such as Principal Component Analysis (PCA) or Artificial Neural Networks (ANN) can help to identify and process the electrochemical fingerprint shown by the pesticide sample. The proposed approach is based on coupling voltammetric responses obtained from an SPCE on the five kinds of pesticides, using PCA to visualize the dissimilarities between samples, and finally using ANN to classify the obtained results of the PCA score plot in order to identify each pesticide separately.

Generic Enhanced Sub/Supercritical Fluid Chromatography: Blueprint for Highly Productive and Sustainable Separation of Primary Hindered Amines

α,α-Diaryl primary amines are highly important chemical building blocks in agrochemical and pharmaceutical active ingredients. One of the limitations of current chromatographic methodologies for th...

Ramifications and Insights on the Role of Water in Chiral Sub/Supercritical Fluid Chromatography.

More than 40 cosolvents have been used with carbon dioxide to alter its solvation strength. Among the most interesting systems is the subcritical/supercritical CO2/alkanol eluents. Using small amounts of water in CO2/MeOH is known to be beneficial in chiral subcritical/supercritical chromatography. However, the ramifications of introducing water as a cosolvent component is not entirely understood. In this work, we demonstrate important aspects of the CO2/MeOH/H2O system on nine chiral stationary phases with very different surface chemistries, encompassing derivatized polysaccharides, macrocyclic glycopeptides, iso-butylmercaptoquinine, isopropyl macrocyclic oligosaccharides, and π-electron acceptor/π-electron donor phases. A hydrophilicity scale has been shown to be useful in predicting if a given chiral column chemistry would show a significant enhancement in separation efficiency in the presence of water in the CO2/MeOH system. We demonstrate up to 8-fold enhancements in plate counts of chiral separations with a concomitant decrease in retention times, as predicted by the qualitative test. The same chiral analysis can now be completed in almost a third of the time with the addition of small amounts of water, thereby decreasing organic solvent consumption by a considerable amount. Hydrophobic stationary phases show a minimal increase in efficiency and decrease in analysis times and optimized separations show much larger reduced plate heights, compared to more hydrophilic stationary phases. Furthermore, the presence of water can alter the nature of the adsorption isotherm under nonlinear conditions. Small amounts of water can be used to tune nonlinear tailing peaks into fronting ones, significantly improving preparative enantiomeric separations.

Chaotropic Effects in Sub/Supercritical Fluid Chromatography via Ammonium Hydroxide in Water-Rich Modifiers: Enabling Separation of Peptides and Highly Polar Pharmaceuticals at the Preparative Scale.

Chromatographic separation, analysis and characterization of complex highly polar analyte mixtures can often be very challenging using conventional separation approaches. Analysis and purification of hydrophilic compounds have been dominated by liquid chromatography (LC) and ion-exchange chromatography (IC), with sub/supercritical fluid chromatography (SFC) moving toward these new applications beyond traditional chiral separations. However, the low polarity of supercritical carbon dioxide (CO2) has limited the use of SFC for separation and purification in the bioanalytical space, especially at the preparative scale. Reaction mixtures of highly polar species are strongly retained even using polar additives in alcohol modifier/CO2 based eluents. Herein, we overcome these problems by introducing chaotropic effects in SFC separations using a nontraditional mobile phase mixture consisting of ammonium hydroxide combined with high water concentration in the alcohol modifier and carbon dioxide. The separation mechanism was here elucidated based on extensive IC-CD (IC couple to conductivity detection) analysis of cyclic peptides subjected to the SFC conditions, indicating the in situ formation of a bicarbonate counterion (HCO3-). In contrast to other salts, HCO3- was found to play a crucial role acting as a chaotropic agent that disrupts undesired H-bonding interactions, which was demonstrated by size-exclusion chromatography coupled with differential hydrogen-deuterium exchange-mass spectrometry experiments (SEC-HDX-MS). In addition, the use of NH4OH in water-rich MeOH modifiers was compared to other commonly used basic additives (diethylamine, triethylamine, and isobutylamine) showing unmatched chromatographic and MS detection performance in terms of peak shape, retention, selectivity, and ionization as well as a completely different selectivity and retention behavior. Moreover, relative to ammonium formate and ammonium acetate in water-rich methanol modifier, the ammonium hydroxide in water additive showed better chromatographic performance with enhanced sensitivity. Further optimization of NH4OH and H2O levels in conjunction with MeOH/CO2 served to furnish a generic modifier (0.2% NH4OH, 5% H2O in MeOH) that enables the widespread transition of SFC to domains that were previously considered out of its scope. This approach is extensively applied to the separation, analysis, and purification of multicomponent reaction mixtures of closely related polar pharmaceuticals using readily available SFC instrumentation. The examples described here cover a broad spectrum of bioanalytical and pharmaceutical applications including analytical and preparative chromatography of organohalogenated species, nucleobases, nucleosides, nucleotides, sulfonamides, and cyclic peptides among other highly polar species.

Fast super/subcritical fluid chromatographic enantioseparations on superficially porous particles bonded with broad selectivity chiral selectors relative to fully porous particles

Superficially porous particles (SPPs) have shown advantages in enantiomeric separations in HPLC by conserving selectivity while providing higher efficiency separations with significantly reduced analysis times. The question arises as to whether the same advantages can be found to the same extent in super/subcritical fluid chromatography. In this work, the low viscosity advantage of carbon dioxide/MeOH mixtures is coupled with high-efficiency 2.7 μm superficially porous particles for enantiomeric separations. Given the fact that the viscosity of the mobile phase is typically ten times lower than liquid mobile phases it is possible to use flow rates as high as 14 mL/min on 5 cm packed columns. Superficially porous particles (SPPs) were grafted with teicoplanin (TeicoShell), a chemically modified macrocyclic glycopeptide (NicoShell), vancomycin (VancoShell), and isopropyl derivatized cyclofructan-6 (LarihcShell-P). One hundred chiral analytes were separated in a very short time frame, as little as 0.2 min (13 s). Even shorter separations can be obtained with advances in SFC instrumentation. The LarihcShell-P is the only chiral crown ether-based selector which showed high selectivity for primary amines. The Teicoshell column offered unique separations for acidic and neutral analytes. The NicoShell and the VancoShell were useful in separating amine (secondary and tertiary) containing pharmaceutical drugs and controlled substances. By chemically modifying a macrocyclic glycopeptide (NicoShell) we report the first enantiomeric separation of nicotine under SFC conditions within 3 min with a resolution of >3. Additionally, van Deemter plots are constructed comparing the fully porous particles and superficially porous particles bonded with the same chiral selectors. In toto the SPP advantages also were found for SFC. However instrumental shortcomings involving extra column effects and pressure limitations need to be addressed by instrument manufacturers to realize the full advantages of SPPs and other smaller particle supports.