Dwight R. Stoll

Peer Reviewed: Zirconia Stationary Phases for Extreme Separations

Composite materials surpass standard silicon and carbon for stability and robustness in complicated reversed-phase column applications.

Smart Templates for Peak Pattern Matching with Comprehensive Two-Dimensional Liquid Chromatography

Comprehensive two-dimensional liquid chromatography (LCxLC) generates information-rich but complex peak patterns that require automated processing for rapid chemical identification and classification. This paper describes a powerful approach and specific methods for peak pattern matching to identify and classify constituent peaks in data from LCxLC and other multidimensional chemical separations. The approach records a prototypical pattern of peaks with retention times and associated metadata, such as chemical identities and classes, in a template. Then, the template pattern is matched to the detected peaks in subsequent data and the metadata are copied from the template to identify and classify the matched peaks. Smart Templates employ rule-based constraints (e.g., multispectral matching) to increase matching accuracy. Experimental results demonstrate Smart Templates, with the combination of retention-time pattern matching and multispectral constraints, are accurate and robust with respect to changes in peak patterns associated with variable chromatographic conditions.

Direct Identification of Rituximab Main Isoforms and Subunit Analysis by Online Selective Comprehensive Two-Dimensional Liquid Chromatography–Mass Spectrometry

In this proof-of-concept study, rituximab, which is a reference therapeutic monoclonal antibody (mAb), was characterized through the implementation of online, selective comprehensive two-dimensional liquid chromatography (sLC×LC) coupled with mass spectrometry (MS), using a middle-up approach. In this setup, cation exchange chromatography (CEX) and reverse-phase liquid chromatography (RPLC) were used as the first and second separation dimensions, respectively. As illustrated in this work, the combination of these two chromatographic modes allows a direct assignment of the identities of CEX peaks, using data from the TOF/MS detector, because RPLC is directly compatible with MS detection, whereas CEX is not. In addition, the resolving power of CEX is often considered to be limited; therefore, this 2D approach provides an improvement in peak capacity and resolution when high-performance second-dimension separations are used, instead of simply using the second-dimension separation as a desalting step. This was particularly relevant when separating rituximab fragments of medium size (25 kDa), whereas most of the resolution was provided by CEX in the case of intact rituximab samples. The analysis of a commercial rituximab sample shows that online sLC×LC-TOF-MS can be used to rapidly characterize mAb samples, yielding the identification of numerous variants, based on the analysis of intact, partially digested, and digested/reduced mAb samples.

High speed gradient elution reversed phase liquid chromatography of bases in buffered eluents. Part II. Full equilibrium

In this work we determined when the state of thermodynamic (full) equilibrium, i.e. time-invariate solute retention, was achieved in gradient elution reversed-phase chromatography. We investigated the effects of flow rate, temperature, organic modifier, buffer type/concentration, stationary phase type, n-butanol as eluent additive, and pore size. We also measured how selectivity varied with reequilibration time. Stationary phase wetting and the ability of the stationary phase to resist changes in pH strongly affect the time needed to reach full equilibrium. For example, full equilibrium is realized with many endcapped stationary phases after flushing with only two column volumes of acetonitrile-water containing 1% (v/v) n-butanol and 0.1% (v/v) trifluoroacetic acid. Trends in retention time (<0.010min) and selectivity become quite small after only five column volumes of reequilibration. We give practical guidelines that provide fast full equilibrium for basic compounds when chromatographed in buffered eluents.

Identifying orthogonal and similar reversed phase liquid chromatography stationary phases using the system selectivity cube and the hydrophobic subtraction model.

We have compared over 500 RPLC columns characterized by the hydrophobic subtraction model using the system selectivity cube (SSC). We have shown numerous differences in column selectivity even among columns in the same class (e.g., alkyl-silica, cyano, or embedded polar groups). We also illustrate the utility of our method for selecting alternative columns with different selectivities for problematic separations and for selecting orthogonal columns for use in two-dimensional separations. The system selectivity cube offers a visual way to easily compare many columns simultaneously and select those columns offering the desired selectivity.

High-speed gradient elution reversed-phase liquid chromatography of bases in buffered eluents: Part I. Retention repeatability and column re-equilibration

We studied the run-to-run repeatability of the retention times of both non-ionizable and basic compounds chromatographed using buffered eluents. The effect of flow rate, organic modifier and other additives, buffer type/concentration, stationary phase type, batch-to-batch preparation of the initial eluent, gradient time, sample type and intra-day changes on retention repeatability were examined. We also assessed the effect of column storage solvent conditions on the inter-day repeatability. Although retention repeatability is strongly influenced by many parameters (flow rate, solvent compressibility compensation, precision of temperature control, and buffer/stationary phase type), our primary finding is that with a reasonable size column (15cmx4.6mm (i.d.)) two column volumes of re-equilibration with initial eluent suffices to provide acceptable repeatability (no worse than 0.004min) for both non-ionizable and basic analytes under a wide variety of conditions. Under ideal conditions (e.g. the right buffer, flow rate, etc.) it is possible to obtain truly extraordinary repeatability often as good as 0.0004min. These absolute fluctuations in retention translate to worst case changes in resolution of 0.2 units and average changes of only 0.02 units.

Characterization of Fullerene-Modified Silica as a Complement to Graphite-like phases for Use in Two-Dimensional High Performance Liquid Chromatography

Carbonaceous sorbents have a long and rich history of development and application in all areas of separation science. Interest in these materials has been fueled by observations that solute-sorbent interactions are mainly adsorptive in nature and thus selectivities are frequently quite different from what is observed with small organic ligands bonded to porous substrates. However, despite over four decades of intense study and development of these materials for use in reversed-phase liquid chromatography, wide adoption continues to be hindered by a few significant, negative attributes of these materials, most notably irreversible adsorption and poor peak shape and separation efficiency for some classes of compounds. In this work we describe the results of a study aimed at characterization of C60 fullerene-modified silica (FMS) materials that we believe nicely complement existing graphite-like carbonaceous phases for use in liquid chromatography. Since their first synthesis about 20 years ago, FMS materials have received surprisingly little attention, which has been focused mainly on the separation of highly aromatic compounds. Here, we use retention data for well-established sets of both nonionizable and ionizable low molecular weight probe solutes to demonstrate that FMS both exhibits graphite-like characteristics (i.e., selectivity for structural isomers and enhanced retention of polar compounds) and has selectivity characteristics that are largely unique in comparison to over 600 other materials used for reversed-phase liquid chromatography. In addition, FMS exhibits much improved peak shape and separation efficiency for compounds that are known to be problematic when separated by use of graphite-like phases. This combination of attributes makes FMS an excellent complement to graphite-like phases for use in two-dimensional liquid chromatography, where unique selectivity compared to conventional bonded reversed-phase materials, along with good peak shape and separation efficiency are of paramount importance for successful two-dimensional liquid chromatographic separations.

Active Solvent Modulation: A Valve-Based Approach To Improve Separation Compatibility in Two-Dimensional Liquid Chromatography

Two-dimensional liquid chromatography (2D-LC) is increasingly being viewed as a viable tool for solving difficult separation problems, ranging from targeted separations of structurally similar molecules to untargeted separations of highly complex mixtures. In spite of this performance potential, though, many users find method development challenging and most frequently cite the incompatibility between the solvent systems used in the first and second dimensions as a major obstacle. This solvent strength related incompatibility can lead to severe peak distortion and loss of resolution and sensitivity in the second dimension. In this paper, we describe a novel approach to address the incompatibility problem, which we refer to as Active Solvent Modulation (ASM). This valve-based approach enables dilution of 1D effluent with weak solvent prior to transfer to the 2D column but without the need for additional instrument hardware. ASM is related to the concept we refer to as Fixed Solvent Modulation (FSM), with the important difference being that ASM allows toggling of the diluent stream during each 2D separation cycle. In this work, we show that ASM eliminates the major drawbacks of FSM including complex elution solvent profiles, baseline disturbances, and slow 2D re-equilibration and demonstrate improvements in 2D separation quality using both simple small molecule probes and degradants of heat-treated bovine insulin as case studies. We believe that ASM will significantly ease method development for 2D-LC, providing a path to practical methods that involve both highly complementary 1D and 2D separations and sensitive detection.