M. Farooq Wahab

Advances in high-throughput and high-efficiency chiral liquid chromatographic separations

The need for improved liquid chromatographic chiral separations has led to the advancement of chiral screening techniques as well as the development of new, high efficiency chiral separation methods and stationary phases. This review covers these advancements, which primarily occurred over the last 15 years. High throughput techniques include multi-column screening units, multiple injection sequences, and fast gradient SFC screening. New separation methods and column technologies that aim at high efficiency chiral separations include the use of achiral UHPLC (i.e. sub-2μm) columns for separating derivatized chiral analytes or using chiral additives in the run buffer, UHPLC chiral stationary phases, and superficially porous particle based chiral stationary phases. Finally, the enhancement of chiral separations through these new technologies requires that certain instrumental considerations be made. Future directions in continuing to improve chiral separations are also discussed.

Salient Sub-Second Separations

Sub-second liquid chromatography in very short packed beds is demonstrated as a broad proof of concept for chiral, achiral, and HILIC separations of biologically important molecules. Superficially porous particles (SPP, 2.7 μm) of different surface chemistries, namely, teicoplanin, cyclofructan, silica, and quinine, were packed in 0.5-cm-long columns for separating different classes of compounds. Several issues must be addressed to obtain the maximum performance of 0.5 cm columns with reduced plate heights of 2.6 to 3.0. Modified UHPLC hardware can be used to obtain sub-second separations provided extra-column dispersion is minimized and sufficient data acquisition rates are used. Further, hardware improvements will be needed to take full advantage of faster separations. The utility of power transform, which is already employed in certain chromatography detectors, is shown to be advantageous for sub-second chromatography. This approach could prove to be beneficial in fast screening and two-dimensional liquid chromatography.

Carboxylate Modified Porous Graphitic Carbon: A New Class of Hydrophilic Interaction Liquid Chromatography Phases

Stationary phases for hydrophilic interaction liquid chromatography (HILIC) are predominantly based on silica and polymer supports. We present porous graphitic carbon particles with covalently attached carboxylic acid groups (carboxylate-PGC) as a new HILIC stationary phase. PGC particles were modified by adsorbing the diazonium salt of 4-aminobenzoic acid onto the PGC, followed by reduction of the adsorbed salt with sodium borohydride. The newly developed carboxylate-PGC phase exhibits different selectivity than that of 35 HPLC columns, including bare silica, zwitterionic, amine, reversed, and unmodified PGC phases. Carboxylate-PGC is stable from pH 2.0 to 12.6, yielding reproducible retention even at pH 12.6. Characterization of the new phase is presented by X-ray photoelectron spectroscopy, thermogravimetry, zeta potentials, and elemental analysis. The chromatographic performance of carboxylate-PGC as a HILIC phase is illustrated by separations of carboxylic acids, nucleotides, phenols, and amino acids.

Fundamental and Practical Insights on the Packing of Modern High-Efficiency Analytical and Capillary Columns

New stationary phases are continuously developed for achieving higher efficiencies and unique selectivities. The performance of any new phase can only be assessed when the columns are effectively packed under high pressure to achieve a stable bed. The science of packing columns with stationary phases is one of the most crucial steps to achieve consistent and reproducible high-resolution separations. A poorly packed column can produce non-Gaussian peak shapes and lower detection sensitivities. Given the ever larger number of stationary phases, it is impossible to arrive at a single successful approach. The column packing process can be treated as science whose unified principles remain true regardless of the stationary phase chemistry. Phenomenologically, the column packing process can be considered as a constant pressure or constant flow high-pressure filtration of a suspension inside a column with a frit at the end. This process is dependent on the non-Newtonian suspension rheology of the slurry in which the particles are dispersed. This perspective lays out the basic principles and presents examples for researchers engaged in stationary phase development. This perspective provides an extensive set of slurry solvents, hardware designs, and a flowchart, a logical approach to optimal column packing, thus eliminating the trial and error approach commonly practiced today. In general, nonaggregating but high slurry concentrations of stationary phases tend to produce well packed analytical columns with small particles. Conversely, C18 packed capillary columns are best packed using agglomerating solvents.

Peak Distortion Effects in Analytical Ion Chromatography

The elution profile of chromatographic peaks provides fundamental understanding of the processes that occur in the mobile phase and the stationary phase. Major advances have been made in the column chemistry and suppressor technology in ion chromatography (IC) to handle a variety of sample matrices and ions. However, if the samples contain high concentrations of matrix ions, the overloaded peak elution profile is distorted. Consequently, the trace peaks shift their positions in the chromatogram in a manner that depends on the peak shape of the overloading analyte. In this work, the peak shapes in IC are examined from a fundamental perspective. Three commercial IC columns AS16, AS18, and AS23 were studied with borate, hydroxide and carbonate as suppressible eluents. Monovalent ions (chloride, bromide, and nitrate) are used as model analytes under analytical (0.1 mM) to overload conditions (10-500 mM). Both peak fronting and tailing are observed. On the basis of competitive Langmuir isotherms, if the eluent anion is more strongly retained than the analyte ion on an ion exchanger, the analyte peak is fronting. If the eluent is more weakly retained on the stationary phase, the analyte peak always tails under overload conditions regardless of the stationary phase capacity. If the charge of the analyte and eluent anions are different (e.g., Br(-) vs CO3(2-)), the analyte peak shapes depend on the eluent concentration in a more complex pattern. It was shown that there are interesting similarities with peak distortions due to strongly retained mobile phase components in other modes of liquid chromatography.

Separations at the Speed of Sensors

The virtue of chemical sensors is speed and analyte specificity. The response time to generate an analytical signal typically varies from ∼1 to 20 s, and they are generally limited to a single analyte. Chemical sensors are significantly affected by multiple interferents, matrix effects, temperature, and can vary widely in sensitivity depending on the sensor format. Separation-based analyses remove matrix effects and interferents and are compatible with multiple analytes. However, the speed of such analyses has not been commensurate with traditional sensors until now. Beds of very small size with optimal geometry, containing core-shell particles of judicious immobilized selectors, can be used in an ultrahigh-flow regime, thereby providing subsecond separations of up to 10 analytes. Short polyether ether ketone lined stainless steel columns of various geometries were evaluated to determine the optimal bed geometry for subsecond analysis. Coupling these approaches provides subsecond-based detection and quantitation of multiple chiral and achiral species, including nucleotides, plant hormones, acids, amino acid derivatives, and sedatives among a variety of other compounds. The subsecond separations were reproducible with 0.9% RSD on retention times and showed consistent performance with 0.9% RSD on reduced plate height in van Deemter curves. A new powerful signal processing algorithm is proposed that can further enhance separation outputs and optical spectra without altering band areas on more complex separations such as 10 peaks under a second.

Total peak shape analysis: detection and quantitation of concurrent fronting, tailing, and their effect on asymmetry measurements

Most peak shapes obtained in separation science depart from linearity for various reasons such as thermodynamic, kinetic, or flow based effects. An indication of the nature of asymmetry often helps in problem solving e.g. in column overloading, slurry packing, buffer mismatch, and extra-column band broadening. However, existing tests for symmetry/asymmetry only indicate the skewness in excess (tail or front) and not the presence of both. Two simple graphical approaches are presented to analyze peak shapes typically observed in gas, liquid, and supercritical fluid chromatography as well as capillary electrophoresis. The derivative test relies on the symmetry of the inflection points and the maximum and minimum values of the derivative. The Gaussian test is a constrained curve fitting approach and determines the residuals. The residual pattern graphically allows the user to assess the problematic regions in a given peak, e.g., concurrent tailing or fronting, something which cannot be easily done with other current methods. The template provided in MS Excel automates this process. The total peak shape analysis extracts the peak parameters from the upper sections (>80% height) of the peak rather than the half height as is done conventionally. A number of situations are presented and the utility of this approach in solving practical problems is demonstrated.

Carboxylated cyclofructan 6 as a hydrolytically stable high efficiency stationary phase for hydrophilic interaction liquid chromatography and mixed mode separations

Stationary phases composed of native cyclofructan 6 (CF6) and benzoic acid modified CF6 were synthesized and evaluated for hydrophilic interaction liquid chromatography (HILIC). The ligands were bonded onto 2.7 μm core–shell silica using multipoint attachment technology. These cyclofructan 6 based columns exhibited excellent hydrolytic stability and efficiency (205000 N m−1). The new column chemistry was compared for stability with core–shell silica (the starting material) using neutral, positive and a negatively charged probes. Additionally, the advantage of the use of a pre-saturating column in HILIC mode is shown. The HILIC selectivity chart shows that the benzoic acid modified cyclofructan-6 column shows strong hydrophilicity as well as cation exchange property. A variety of hydrophilic/ionizable compounds were examined, and based on the selectivity chart, it was found that the new column chemistry is different from 33 commercial columns. The benzoic acid CF6 column can simultaneously separate acidic, neutral and basic drugs and produced considerably different retention and selectivity patterns for various classes of compounds including nucleic acid bases, β-blockers, salicylic acid and its analogues. The newly developed column chemistry also shows a potential to work in the reverse phase mode.

Mass spectrometry detection of basic drugs in fast chiral analyses with vancomycin stationary phases

Current trends in chiral analysis of pharmaceutical drugs are focused on faster separations and higher separation efficiencies. Core-shell or superficially porous particles (SPP) based chiral stationary phases (CSPs) provide reduced analysis times while maintaining high column efficiencies and sensitivity. In this study, mobile phase conditions suitable for chiral analyses with electrospray ionization LC-MS were systematically investigated using vancomycin as a representative CSP. The performance of a 2.7 µm SPP based vancomycin CSP (SPP-V) 10 cm × 0.21 cm column was compared to that of a corresponding 5 µm fully porous particles based analogue column. The results demonstrated that the SPP-V column provides higher efficiencies, 2–5 time greater sensitivity and shorter analysis time for a set of 22 basic pharmaceutical drugs. The SPP-V was successfully applied for the analysis of the degradation products of racemic citalopram whose enantiomers could be selectively identified by MS.

Improving Visualization of Trace Components for Quantification Using a Power Law Based Integration Approach

In some cases, trace component analysis only requires a sensitive and high-resolution mass spectrometer. However, enantiomers must be completely separated to be differentiated with a mass spectrometer, which is highly dependent on the stationary-mobile phase composition. In case of a challenging chiral separation, instead of trying new columns for screening purpose, resolution enhancement techniques could be used to resolve partially overlapping peaks. A well-known enhancement method is the power law, which increases the linear dynamic range of each analyte and reduces excessive noise. In many cases, the peak noise can decrease significantly by applying the power law. However, the main drawback is that this approach changes relative peak areas and heights of each peak in a non-linear fashion which limits its use for quantitative purposes. In this study, a normalized power law was utilized for extracting correct area information. It is a simple (5 step) protocol that only requires Microsoft Excel, and results in enhanced visualization of trace components, especially in low signal/noise environments, and makes integration convenient and reproducible. Several difficult chiral trace component analyses were investigated, including applications pertaining to ultrafast high-throughput chromatography, enantiopurity, and peak purity analysis. For complicated cases with multiple overlapped peaks of different resolutions, a segmented normalized power law was utilized. A trace component coeluting near a dead volume peak and a trace enantiomeric component in the tail of the corresponding enantiomeric peak were virtually enhanced. As an additional tool, first and second derivatives were utilized to identify if an enantiomeric impurity is coeluting with the dominant enantiomer under overload conditions. Idiosyncrasies of the derivative test are discussed. This study shows how these simple approaches can be used for accurate quantitation, specifically for trace enantiomeric components.