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.

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.

The theory and practice of ultrafast liquid chromatography: A tutorial.

Modern high-throughput experimentation and challenging analytical problems of academic/industrial research have put the responsibility on separation scientists to develop new fast separation approaches. With the availability of high-pressure pumps, small particles with hydrolytically stable surface chemistries, reduced extra-column band broadening, and low volume detectors with fast signal processing, it is now feasible to do sub-minute to sub-second chromatography. Herein, the fundamental theoretical principles of ultrafast chromatography, along with practical solutions, are reviewed. Approaches for rapid separations in packed beds, narrow open tubular columns, and monoliths are demonstrated, along with the challenges that were faced. The instrumentation requirements (pumps, injection systems, detectors, column packing process) for using short columns ranging from 0.5 to 5 cm are examined, followed by real applications. One of the main problems in ultrafast chromatography is partial or complete peak overlap. As per Gidding's statistical overlap theory, peak overlap cannot be avoided for a completely random sample for a column with a given peak capacity. Signal processing techniques based on Fourier transform deconvolution of band broadening, power laws, derivatives, and iterative curve fitting are explained to help improve the chromatographic resolution. An example of ten peaks separated in under a second is shown and discussed. Other ultrafast separations in supercritical fluid chromatography or capillary electrophoresis are briefly mentioned to provide a complete understanding of this emerging field.

Enhancing supercritical fluid chromatographic efficiency: Predicting effects of small aqueous additives.

Supercritical fluid chromatography is becoming more prevalent, particularly in industry. This is due to the inexpensive, and more importantly, environmentally benign carbon dioxide that is used as the major component of the mobile phase. Water is minimally miscible with carbon dioxide at temperatures and pressures commonly used in SFC. However, the introduction of a polar alcohol modifier component increases the solubility of water in carbon dioxide. Previously, the addition of small amounts of water in the mobile phase was shown to provide significant gains in efficiency in chiral supercritical fluid chromatography, especially with polar stationary phases. In this work, we report the effect of the addition of small amounts of water on efficiency and retention factor with four different SFC stationary phases used for achiral analysis namely FructoShell-N (native cyclofructan-6), SilicaShell (bare silica), PoroShell 120 EC C18 (octadecyl silica) and Xselect C18 SB. This is the first reported use of FructoShell-N, a cyclofructan derivatized phase for SFC applications. We devised a predictive test to determine which analytes show an increase in efficiency using their known chemical constants (logKow, pKa, PSA and Hsum). We also use discriminant analysis to elucidate the most important analyte parameters that contribute to "water enhanced" efficiency gains.

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.

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.

Altered profiles and metabolism of l- and d-amino acids in cultured human breast cancer cells vs. non-tumorigenic human breast epithelial cells.

Herein we describe for the first time the endogenous levels of free l-and d-amino acids in cultured human breast cancer cells (MCF-7) and non-tumorigenic human breast epithelial cells (MCF-10A). d-Asp and d-Ser, which are co-agonists of the N-methyl-d-aspartate (NMDA) receptors, showed significantly elevated levels in MCF-7 cancer cells compared to MCF-10A cells. This may result from upregulated enzymatic racemases. Possible roles of these d-amino acids in promoting breast cancer proliferation by regulating NMDA receptors were indicated. d-Asn may also be able to serve as exchange currency, like specific l-amino acids, for the required uptake of essential amino acids and other low abundance nonessential amino acids which were elevated nearly 60 fold in cancer cells. The relative levels of specific l- and d-amino acids can be used as malignancy indicators (MIs) for the breast cancer cell line in this study. High MIs (>50) result from the increased demands of specific essential amino acids. Very low MIs (<1) result from the increased demands of specific d-amino acids (i.e., d-Ser, d-Asp) or the cellular release of amino acid exchange currency (i.e., l- and d-Asn) used in the upregulated amino acid antiporters to promote cancer cell proliferation.

Effective methodologies for enantiomeric separations of 150 pharmacology and toxicology related 1°, 2°, and 3° amines with core-shell chiral stationary phases.

Core-shell particles (superficially porous particles, SPPs) have been proven to provide high-throughput and effective separations of a variety of chiral molecules. However, due to their limited commercialization, many separations have not been reported with these stationary phases. In this study, four SPP chiral stationary phases (CSPs) were utilized for the enantiomeric separation of 150 chiral amines. These amines encompass a variety of structural and drug classes, which are particularly important to the pharmaceutical industry and in forensics. This comprehensive evaluation demonstrates the power of these CSPs and the ease of method development and optimization. The CSPs used in this study included the macrocyclic glycopeptide-based CSPs (VancoShell and NicoShell), the cyclodextrin-based CSP (CDShell-RSP), and the cyclofructan-based CSP (LarihcShell-P). These CSPs offered versatility for a variety of applications and worked in a complementary fashion to baseline separate all 150 amines. The LarihcShell-P was highly effective for separating primary amines. VancoShell, NicoShell, and CDShell-RSP were useful for separating all types of amines. These CSPs are multi-modal and can be utilized with mass spectrometry compatible solvents. Eighteen racemic controlled substances were simultaneously baseline separated in a single liquid chromatography-mass spectrometry (LC-MS) analysis. Details in high-performance liquid chromatography (HPLC) parameters will be discussed as well as the improved chromatographic performance afforded by the SPP CSPs.

A comprehensive methodology for the chiral separation of 40 tobacco alkaloids and their carcinogenic E/Z-(R,S)-tobacco-specific nitrosamine metabolites.

The predominant enantiomer of nicotine found in nature is (S)-nicotine and its pharmacology has been widely established. However, pharmacologic information concerning individual enantiomers of nicotine-related compounds is limited. Recently, a modified macrocyclic glycopeptide chiral selector was found to be highly stereoselective for most tobacco alkaloids and metabolites. This study examines the semi-synthetic and native known macrocyclic glycopeptides for chiral recognition, separation, and characterization of the largest group of nicotine-related compounds ever reported (tobacco alkaloids, nicotine metabolites and derivatives, and tobacco-specific nitrosamines). The enantioseparation of nicotine is accomplished in less than 20s for example. All liquid chromatography separations are mass spectrometry compatible for the tobacco alkaloids, as well as their metabolites. Ring-closed, cyclized structures were identified and separated from their ring-open, straight chain equilibrium structures. Also, E/Z-tobacco-specific nitrosamines and their enantiomers were directly separated. E/Z isomers also are known to have different physical and chemical properties and biological activities. This study provides optimal separation conditions for the analysis of nicotine-related isomers, which in the past have been reported to be ineffectively separated which can result in inaccurate results. The methodology of this study could be applied to cancer studies, and lead to more information about the role of these isomers in other diseases and as treatment for diseases.