Dwight Stoll | Gustavus Adolphus College (original) (raw)

Papers by Dwight Stoll

Research paper thumbnail of Trapping‐Enrichment Multi‐dimensional Liquid Chromatography with On‐Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale

Angewandte Chemie, 2022

At the forefront of chemistry and biology research, development timelines are fast-paced and larg... more At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE- Dt -mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension ( 1 D -UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H 2 O and D 2 O washes using an independent pump setup; and 3) a second dimension separation ( 2 D -UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90% using as little as a few micrograms of material.

Research paper thumbnail of In Silico Multifactorial Modeling for Streamlined Development and Optimization of Two-Dimensional Liquid Chromatography

Analytical Chemistry, 2021

Continued adoption of two-dimensional liquid chromatography (2D-LC) in industrial laboratories wi... more Continued adoption of two-dimensional liquid chromatography (2D-LC) in industrial laboratories will depend on the development of approaches to make method development for 2D-LC more systematic, less tedious, and less reliant on user expertise. In this paper, we build on previous efforts in these directions by describing the use of multifactorial modeling software that can help streamline and simplify the method development process for 2D-LC. Specifically, we have focused on building retention models for second dimension (2D) separations involving variables including gradient time, temperature, organic modifier blending, and buffer concentration using LC simulator (ACD/Labs) software. Multifactorial retention modeling outcomes are illustrated as resolution map planes or cubes that enable straightforward location of 2D conditions that maximize resolution while minimizing analysis time. We also illustrate the practicality of this approach by identifying conditions that yield baseline separation of all compounds co-eluting from a first dimension (1D) separation using a single combination of 2D stationary phase and elution conditions. The multifactorial retention models were found to be very accurate for both the 1D and 2D separations, with differences between experimental and simulated retention times of less than 0.5%. Pharmaceutical applications of this approach for multiple heartcutting 2D-LC were demonstrated using IEC-IEC or achiral RPLC-chiral RPLC for 2D separations of multicomponent mixtures. The framework outlined here should help make 2D-LC method development more systematic and streamline development and optimization for a variety of 2D-LC applications in both industry and academia.

Research paper thumbnail of Fast methods for structurally similar compounds using carbon HPLC columns

Lc Gc North America, 2002

Method development can be challenging for structurally similar compounds in reversed-phase liquid... more Method development can be challenging for structurally similar compounds in reversed-phase liquid chromatography (RPLC). A new carbon-clad zirconia phase offers dramatically different chromatographic selectivity for thesetypes of compounds when compared with traditional silica-based bonded phases. This note shows baseline separation of six structurally similar sulfate-steroid conjugates using a zirconia-based ZirChrom®-CARB HPLC column.

Research paper thumbnail of Fast Separation of Nitrosamines

Lc Gc North America, 2002

Interest in the analysis of nitrosamine compounds is increasing as researchers become more aware ... more Interest in the analysis of nitrosamine compounds is increasing as researchers become more aware of their toxicity and presence in the environment, foods, and pharmaceuticals. The extraordinary chemical and thermal stability of zirconia-based stationary phases allows for the rapid separation of these polar, basic compounds with good peak shape and efficiency. This note shows the separation of nine low molecular weight nitrosamines using a DiamondBond-C18 column.

Research paper thumbnail of Implications of dispersion in connecting capillaries for separation systems involving post-column flow splitting

Journal of Chromatography A, 2021

Implications of dispersion in connecting capillaries for separation systems involving post-column... more Implications of dispersion in connecting capillaries for separation systems involving post-column flow splitting.

Research paper thumbnail of Mapping the Separation Landscape in Two-Dimensional Liquid Chromatography: Blueprints for Efficient Analysis and Purification of Pharmaceuticals Enabled by Computer-Assisted Modeling

Analytical Chemistry, 2020

Recent developments in two-dimensional liquid chromatography (2D-LC) now make separation and anal... more Recent developments in two-dimensional liquid chromatography (2D-LC) now make separation and analysis of very complex mixtures achievable. Despite being such a powerful chromatographic tool, current 2D-LC technology requires a series of arduous method development activities poorly suited for a fastpaced industrial environment. Recent introductions of new technologies including active solvent modulation and a support for multicolumn 2D-LC are helping to overcome this stigma. However, many chromatography practitioners believe that the lack of a systematic way to effectively optimize 2D-LC separations is a missing link in securing the viability of 2D-LC as a mainstay for industrial applications. In this work, a computer-assisted modeling approach that dramatically simplifies both offline and online 2D-LC method developments is introduced. Our methodology is based on mapping the separation landscape of pharmaceutically relevant mixtures across both first (1 D) and second (2 D) dimensions using LC Simulator (ACD/Labs) software. Retention models for 1 D and 2 D conditions were built using a minimal number of multifactorial modeling experiments (2 × 2 or 3 × 3 parameters: gradient slope, column temperature, and different column and mobile phase combinations). The approach was first applied to online 2D-LC analysis involving achiral and chiral separations of complex mixtures of enantiomeric species. In these experiments, the retention models proved to be quite accurate for both the 1 D and 2 D separations, with retention time differences between experiments and simulations of less than 3.5%. This software-based concept was also demonstrated for offline 2D-LC purification of drug substances.

Research paper thumbnail of Orthogonal Middle-up Approaches for Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High-Resolution Mass Spectrometry

Analytical Chemistry, 2018

Etanercept is a recombinant Fc-fusion protein widely used to treat rheumatic diseases. This prote... more Etanercept is a recombinant Fc-fusion protein widely used to treat rheumatic diseases. This protein is highly glycosylated and contains numerous O-and N-glycosylation sites. Since glycosylation is recognized as an important critical quality attribute (CQA) that can affect immunogenicity, solubility, and stability of Fc-fusion proteins, it should be thoroughly characterized. In this work, hydrophilic interaction chromatography (HILIC) was combined with high resolution mass spectrometry (HRMS) by using a QTOF/MS instrument to assess glycosylation of etanercept at the middle-up level of analysis (fragments of circa 25-30 kDa). In addition, a combination of different enzymatic digestion procedures (i.e. glycosidase, sialidase and protease) was systematically employed to facilitate spectra deconvolution. With the developed procedure, the main post-translational modifications (PTMs) of etanercept were assessed, and a global overview of the subunit-specific distribution of the glycosylation pattern was obtained at a middle-up level of analysis.

Research paper thumbnail of Robust methods with high efficiency, bonded-carbon HPLC columns

Lc Gc North America, 2002

Loss of stationary phase, retention drift, and short column life are common problems using bonded... more Loss of stationary phase, retention drift, and short column life are common problems using bonded-phase silicas, especially with aggressive mobile phases. A new bonding technology overcomes these problems by attaching C18 groups to a carbon surface with ultra-stable carbon-carbon bonds. This note shows that robust methods with high efficiency are now possible using ultra-stable bonded-carbon columns.

Research paper thumbnail of Direct identification of rituximab main isoforms and subunit analysis by online selective comprehensive two-dimensional liquid chromatography - mass spectrometry

Analytical chemistry, Jan 6, 2015

In this proof-of-concept study, rituximab, a reference therapeutic monoclonal antibody (mAb), was... more In this proof-of-concept study, rituximab, a reference therapeutic monoclonal antibody (mAb), was characterized through the implementation of online, selective comprehensive two-dimensional liquid chromatography (sLCxLC) coupled with mass spectrometry (MS), and using a middle-up approach. In this setup, cation exchange chromatography (CEX) and reversed 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 and 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 de...

Research paper thumbnail of Effect of pressure pulses at the interface valve on the stability of second dimension columns in online comprehensive two-dimensional liquid chromatography

Journal of chromatography. A, Jan 23, 2015

Users of online comprehensive two-dimensional liquid chromatography (LCxLC) frequently acknowledg... more Users of online comprehensive two-dimensional liquid chromatography (LCxLC) frequently acknowledge that the mechanical instability of HPLC columns installed in these systems, particularly in the second dimension, is a significant impediment to its use. Such instability is not surprising given the strenuous operating environment to which these columns are subjected, including the large number (thousands per day) of fast and large pressure pulses resulting from interface valve switches (on the timescale of tens of milliseconds) associated with very fast second dimension separations. There appear to be no published reports of systematic studies of the relationship between second dimension column lifetime and any of these variables. In this study we focused on the relationship between the lifetimes of commercially available columns and the pressure pulses observed at the inlet of the second dimension column that occur during the switching of the valve that interfaces the two dimensions ...

Research paper thumbnail of Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants

Analytical and bioanalytical chemistry, 2015

In this paper, we describe the findings of a study aimed at assessing the detection sensitivity o... more In this paper, we describe the findings of a study aimed at assessing the detection sensitivity of comprehensive two-dimensional high-performance liquid chromatography (LCxLC) separation of a degraded active pharmaceutical ingredient (API) with UV absorption as the detection technique. Specifically, we have examined the impact of the volume and solvent composition (referred to as "interface conditions") of fractions of first-dimension column effluent transferred to the second dimension for further separation on the ability to resolve and detect low-abundance compounds. Historically, LCxLC has been perceived as being inferior to 1D-LC from the point of view of detection sensitivity. In this work, we demonstrate that LCxLC is sufficiently sensitive to be useful in the pharmaceutical context where in general impurities present at 0.05 % (relative to the API concentration) should be quantified. Moreover, we find that this level of sensitivity is only attained under certain con...

Research paper thumbnail of Two dimensional assisted liquid chromatography – a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation, dual detectors, and multivariate curve resolution

Analytica Chimica Acta, 2015

A novel method for quantitation in LC Â LC is proposed based on use of dual detectors. Curve reso... more A novel method for quantitation in LC Â LC is proposed based on use of dual detectors. Curve resolution was carried out on data from both detectors. The combined use of data from both detectors lead to improved quantitative results. In some cases, use of only first dimension data results in improved quantification.

Research paper thumbnail of Principles of Online Comprehensive Multidimensional Liquid Chromatography

Advances in Chromatography, 2012

Research paper thumbnail of Fast, Comprehensive Two-Dimensional HPLC Separation of Tryptic Peptides Based on High-Temperature HPLC

Journal of the American Chemical Society, 2005

Two-dimensional HPLC (2D-LC) has recently received considerable attention. 1 This interest is kin... more Two-dimensional HPLC (2D-LC) has recently received considerable attention. 1 This interest is kindled by both the statistical model of overlap (SMO) theory, which makes it clear that the resolving power of one-dimensional HPLC is grossly insufficient for many purposes, 2 and by the demands of many bio-analytical applications. 1

Research paper thumbnail of Targeted three-dimensional liquid chromatography: A versatile tool for quantitative trace analysis in complex matrices

Journal of Chromatography A, 2010

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' ... more Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.

Research paper thumbnail of Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part II: Applications

Journal of Chromatography A, 2012

An approach to enhancing the resolution of select portions of conventional one-dimensional high p... more An approach to enhancing the resolution of select portions of conventional one-dimensional high performance liquid chromatography (HPLC) separations was developed, which we refer to as selective comprehensive two-dimensional HPLC (sLC×LC). In this first of a series of two papers we describe the principles of this approach, which breaks the long-standing link in on-line multi-dimensional chromatography between the timescales of sampling the first dimension (¹D) separation and the separation of fractions of ¹D effluent in the second dimension. This allows rapid, high-efficiency separations to be used in the first dimension, while still adequately sampling ¹D peaks. Transfer, transient storage, and subsequent second dimension (²D) separations of multiple fractions of a particular ¹D peak produces a two-dimensional chromatogram that reveals the coordinates of the peak in both dimensions of the chromatographic space. Using existing valve technology we find that the approach is repeatable (%RSD of peak area <1.5%), even at very short first dimension sampling times--as low as 1s. We have also systematically studied the critical influence of the volume and composition of fractions transferred from the first to the second dimension of the sLC × LC system with reversed-phase columns in both dimensions, and the second dimension operated isocratically. We find that dilution of the transferred fraction, so that it contains 10-20% less organic solvent than the ²D eluent, generally mitigates the devastating effects of large transfer volumes on ²D performance in this type of system. Several example applications of the sLC × LC approach are described in the second part of this two-part series. We anticipate that future advances in the valve technology used here will significantly widen the scope of possible applications of the sLC × LC approach.

Research paper thumbnail of Dependence of Effective Peak Capacity in Comprehensive Two-Dimensional Separations on the Distribution of Peak Capacity between the Two Dimensions

Analytical Chemistry, 2008

One of the basic tenets of comprehensive two-dimensional chromatography is that the total peak ca... more One of the basic tenets of comprehensive two-dimensional chromatography is that the total peak capacity is simply the product of the first and second dimension peak capacities. As formulated the total peak capacity does not depend on the relative values of the individual dimensions but only on the product of the two. This concept is tested here for the experimentally realistic situation wherein the first dimension separation is undersampled. We first propose that a relationship exists between the number of observed peaks in a two-dimensional separation and the effective peak capacity. We then show here for a range of reasonable total peak capacities (500 to 4000) and various contributions of peak capacity in each dimension (10 to 150) that the number of observed peaks is only slightly dependent on the relative contributions over a reasonable and realistic range in sampling times (equal to the first dimension peak standard deviation, multiplied by 0.2 to 16). Most of this work was carried out under the assumption of totally uncorrelated retention times. For uncorrelated separations the small deviations from the product rule are due to the "edge effect" of statistical overlap theory and a recently introduced factor that corrects for the broadening of first dimension peaks by undersampling them. They predict that relatively more peaks will be observed when the ratio of the first to the second dimension peak capacity is much less than unity. Additional complications are observed when first and second dimension retention times show some correlation but again the effects are small. In both cases deviations from the product rule are measured by the relative standard deviations of the number of observed peaks, which are typically 10 or less. Thus although the basic tenet of two-dimensional chromatography is not exact when the first dimension is undersampled, the deviations from the product rule are sufficiently small as to be unimportant in practical work. Our results show that practitioners have a high degree of flexibility in designing and optimizing experimental comprehensive two-dimensional separations.

Research paper thumbnail of Effect of First-Dimension Undersampling on Effective Peak Capacity in Comprehensive Two-Dimensional Separations

Analytical Chemistry, 2008

The objective of this work is to establish a means of correcting the theoretical maximum peak cap... more The objective of this work is to establish a means of correcting the theoretical maximum peak capacity of comprehensive two-dimensional (2D) separations to account for the deleterious effect of undersampling firstdimension peaks. Simulations of comprehensive 2D separations of hundreds of randomly distributed sample constituents were carried out, and 2D statistical overlap theory was used to calculate an effective first-dimension peak width based on the number of observed peaks in the simulated separations. The distinguishing feature of this work is the determination of the effective firstdimension peak width using the number of observed peaks in the entire 2D separation as the defining metric of performance. We find that the ratio of the average effective first-dimension peak width after sampling to its width prior to sampling (defined as < >) is a simple function of the ratio of the first-dimension sampling time (t s) to the first-dimension peak standard deviation prior to sampling (1 σ): < >) 1+0.21(t s / 1 σ) 2 This is valid for 2D separations of constituents having either randomly distributed or weakly correlated retention times, over the range of 0.2 e t s / 1 σ e 16. The dependence of < > on t s / 1 σ from this expression is in qualitative agreement with previous work based on the effect of undersampling on the effective width of a single first-dimension peak, but predicts up to 35% more broadening of first-dimension peaks than is predicted by previous models. This simple expression and accurate estimation of the effect of undersampling first-dimension peaks should be very useful in making realistic corrections to theoretical 2D peak capacities, and in guiding the optimization of 2D separations.

Research paper thumbnail of Comparison of the Practical Resolving Power of One- and Two-Dimensional High-Performance Liquid Chromatography Analysis of Metabolomic Samples

Analytical Chemistry, 2008

Two-dimensional liquid chromatography (2DLC) has become a mainstay of proteomics research due to ... more Two-dimensional liquid chromatography (2DLC) has become a mainstay of proteomics research due to its higher peak capacity compared to one-dimensional LC (1DLC). Because of the long analysis times typically associated with 2DLC (tens of hours) and its use primarily in proteomics applications, 2DLC in the context of general HPLC has been regarded as a niche technique for use in analysis of mixtures containing hundreds to thousands of components compared to the far more common techniques of isocratic and gradient elution 1DLC. A significant next step in the analytical development of 2DLC is to consider using its higher resolving power to reduce the analysis time of rather &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;simple&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; mixtures, in the range of only tens to hundreds of chemical constituents. The chief objective of this paper is to provide guidance to practitioners who need to decide whether 1DLC or 2DLC gives the superior separation in a given analysis time. Conditional peak capacities are predicted for fully optimized 1DLC and practical 2DLC separations of the low molecular weight constituents of an extract of corn seed at several analysis times using a model based on the chromatographic properties of compounds that are representative of real mixtures of lower molecular weight species. Two important corrections to the ideal 2DLC peak capacity are made to account for both incomplete usage of the separation space and the serious effect of first-dimension undersampling; this allows, we believe for the first time, a fair comparison of the resolving power of 1D- and 2DLC under realistic conditions. The predicted optimum conditions are then used to carry out experimental separations of low molecular weight corn seed extract, and peaks are counted in each 1D- and 2DLC chromatogram. Based on comparisons of both the predicted peak capacities and number of peaks observed in experimental chromatograms, we believe that practical 2DLC will be superior to fully optimized gradient 1DLC for separations lasting more than only about 10 min. This crossover time is much shorter than intuitively expected, and we believe this finding will inevitably have a major impact on the practice of 2DLC in liquid-phase separations in general.

Research paper thumbnail of Analysis of Four-Way Two-Dimensional Liquid Chromatography-Diode Array Data:  Application to Metabolomics

Analytical Chemistry, 2006

Two-dimensional liquid chromatography (2D-LC) is rapidly gaining popularity for the analysis of v... more Two-dimensional liquid chromatography (2D-LC) is rapidly gaining popularity for the analysis of very complex mixtures, including proteomic and metabolomic samples. It provides an effective strategy for separating such samples, because the resolving power of 2D-LC is far superior to that of traditional single-dimension separations. The present work focuses on the development of data analysis methods for the extremely large data sets, on the order of 10 million data points, generated by 2D-LC with diode-array detection (DAD). Specifically, we have applied and adapted chemometric methods to the analysis of maize seedling digests, focusing on compounds related to the biosynthetic pathways of indole-3-acetic acid, the primary growth regulator in plants. The chemometric techniques of window target testing factor analysis (WTTFA), along with parallel factor analysis-alternating least squares (PARAFAC-ALS) were used to analyze 2D-LC-DAD chromatograms of a sample composed of 26 indolic standards, 2 extracts of mutant orange pericarp maize seedlings, 2 extracts of wild-type maize seedlings, and a blank sample. The indolic compounds studied belonged to six spectrally unique groups, and WTTFA was able to specifically identify the presence or absence of any of the 26 indolic standards in the mutant and wild-type samples. A PARAFAC-ALS algorithm and an ALS algorithm with flexible constraints were successfully applied to resolve the spectrally rank deficient data and to demonstrate the quantitative potential of multivariate curve resolution methods. Using this procedure, 95 total peaks were resolved in the data set analyzed. Of those 95 peaks, 45 were found in both the mutant and wild-type maize samples, 16 peaks were unique to the mutant maize samples, 13 peaks were unique to the wild-type maize samples, and 15 peaks were unique to the standard chromatograms. Of the 26 standards included in the data set, several indole acetic acid conjugates were identified and quantified in the maize samples at levels of approximately 0.3-2 µg/g plant material.

Research paper thumbnail of Trapping‐Enrichment Multi‐dimensional Liquid Chromatography with On‐Line Deuterated Solvent Exchange for Streamlined Structure Elucidation at the Microgram Scale

Angewandte Chemie, 2022

At the forefront of chemistry and biology research, development timelines are fast-paced and larg... more At the forefront of chemistry and biology research, development timelines are fast-paced and large quantities of pure targets are rarely available. Herein, we introduce a new framework, which is built upon an automated, online trapping-enrichment multi-dimensional liquid chromatography platform (TE- Dt -mDLC) that enables: 1) highly efficient separation of complex mixtures in a first dimension ( 1 D -UV); 2) automated peak trapping-enrichment and buffer removal achieved through a sequence of H 2 O and D 2 O washes using an independent pump setup; and 3) a second dimension separation ( 2 D -UV-MS) with fully deuterated mobile phases and fraction collection to minimize protic residues for immediate NMR analysis while bypassing tedious drying processes and minimizing analyte degradation. Diverse examples of target isolation and characterization from organic synthesis and natural product chemistry laboratories are illustrated, demonstrating recoveries above 90% using as little as a few micrograms of material.

Research paper thumbnail of In Silico Multifactorial Modeling for Streamlined Development and Optimization of Two-Dimensional Liquid Chromatography

Analytical Chemistry, 2021

Continued adoption of two-dimensional liquid chromatography (2D-LC) in industrial laboratories wi... more Continued adoption of two-dimensional liquid chromatography (2D-LC) in industrial laboratories will depend on the development of approaches to make method development for 2D-LC more systematic, less tedious, and less reliant on user expertise. In this paper, we build on previous efforts in these directions by describing the use of multifactorial modeling software that can help streamline and simplify the method development process for 2D-LC. Specifically, we have focused on building retention models for second dimension (2D) separations involving variables including gradient time, temperature, organic modifier blending, and buffer concentration using LC simulator (ACD/Labs) software. Multifactorial retention modeling outcomes are illustrated as resolution map planes or cubes that enable straightforward location of 2D conditions that maximize resolution while minimizing analysis time. We also illustrate the practicality of this approach by identifying conditions that yield baseline separation of all compounds co-eluting from a first dimension (1D) separation using a single combination of 2D stationary phase and elution conditions. The multifactorial retention models were found to be very accurate for both the 1D and 2D separations, with differences between experimental and simulated retention times of less than 0.5%. Pharmaceutical applications of this approach for multiple heartcutting 2D-LC were demonstrated using IEC-IEC or achiral RPLC-chiral RPLC for 2D separations of multicomponent mixtures. The framework outlined here should help make 2D-LC method development more systematic and streamline development and optimization for a variety of 2D-LC applications in both industry and academia.

Research paper thumbnail of Fast methods for structurally similar compounds using carbon HPLC columns

Lc Gc North America, 2002

Method development can be challenging for structurally similar compounds in reversed-phase liquid... more Method development can be challenging for structurally similar compounds in reversed-phase liquid chromatography (RPLC). A new carbon-clad zirconia phase offers dramatically different chromatographic selectivity for thesetypes of compounds when compared with traditional silica-based bonded phases. This note shows baseline separation of six structurally similar sulfate-steroid conjugates using a zirconia-based ZirChrom®-CARB HPLC column.

Research paper thumbnail of Fast Separation of Nitrosamines

Lc Gc North America, 2002

Interest in the analysis of nitrosamine compounds is increasing as researchers become more aware ... more Interest in the analysis of nitrosamine compounds is increasing as researchers become more aware of their toxicity and presence in the environment, foods, and pharmaceuticals. The extraordinary chemical and thermal stability of zirconia-based stationary phases allows for the rapid separation of these polar, basic compounds with good peak shape and efficiency. This note shows the separation of nine low molecular weight nitrosamines using a DiamondBond-C18 column.

Research paper thumbnail of Implications of dispersion in connecting capillaries for separation systems involving post-column flow splitting

Journal of Chromatography A, 2021

Implications of dispersion in connecting capillaries for separation systems involving post-column... more Implications of dispersion in connecting capillaries for separation systems involving post-column flow splitting.

Research paper thumbnail of Mapping the Separation Landscape in Two-Dimensional Liquid Chromatography: Blueprints for Efficient Analysis and Purification of Pharmaceuticals Enabled by Computer-Assisted Modeling

Analytical Chemistry, 2020

Recent developments in two-dimensional liquid chromatography (2D-LC) now make separation and anal... more Recent developments in two-dimensional liquid chromatography (2D-LC) now make separation and analysis of very complex mixtures achievable. Despite being such a powerful chromatographic tool, current 2D-LC technology requires a series of arduous method development activities poorly suited for a fastpaced industrial environment. Recent introductions of new technologies including active solvent modulation and a support for multicolumn 2D-LC are helping to overcome this stigma. However, many chromatography practitioners believe that the lack of a systematic way to effectively optimize 2D-LC separations is a missing link in securing the viability of 2D-LC as a mainstay for industrial applications. In this work, a computer-assisted modeling approach that dramatically simplifies both offline and online 2D-LC method developments is introduced. Our methodology is based on mapping the separation landscape of pharmaceutically relevant mixtures across both first (1 D) and second (2 D) dimensions using LC Simulator (ACD/Labs) software. Retention models for 1 D and 2 D conditions were built using a minimal number of multifactorial modeling experiments (2 × 2 or 3 × 3 parameters: gradient slope, column temperature, and different column and mobile phase combinations). The approach was first applied to online 2D-LC analysis involving achiral and chiral separations of complex mixtures of enantiomeric species. In these experiments, the retention models proved to be quite accurate for both the 1 D and 2 D separations, with retention time differences between experiments and simulations of less than 3.5%. This software-based concept was also demonstrated for offline 2D-LC purification of drug substances.

Research paper thumbnail of Orthogonal Middle-up Approaches for Characterization of the Glycan Heterogeneity of Etanercept by Hydrophilic Interaction Chromatography Coupled to High-Resolution Mass Spectrometry

Analytical Chemistry, 2018

Etanercept is a recombinant Fc-fusion protein widely used to treat rheumatic diseases. This prote... more Etanercept is a recombinant Fc-fusion protein widely used to treat rheumatic diseases. This protein is highly glycosylated and contains numerous O-and N-glycosylation sites. Since glycosylation is recognized as an important critical quality attribute (CQA) that can affect immunogenicity, solubility, and stability of Fc-fusion proteins, it should be thoroughly characterized. In this work, hydrophilic interaction chromatography (HILIC) was combined with high resolution mass spectrometry (HRMS) by using a QTOF/MS instrument to assess glycosylation of etanercept at the middle-up level of analysis (fragments of circa 25-30 kDa). In addition, a combination of different enzymatic digestion procedures (i.e. glycosidase, sialidase and protease) was systematically employed to facilitate spectra deconvolution. With the developed procedure, the main post-translational modifications (PTMs) of etanercept were assessed, and a global overview of the subunit-specific distribution of the glycosylation pattern was obtained at a middle-up level of analysis.

Research paper thumbnail of Robust methods with high efficiency, bonded-carbon HPLC columns

Lc Gc North America, 2002

Loss of stationary phase, retention drift, and short column life are common problems using bonded... more Loss of stationary phase, retention drift, and short column life are common problems using bonded-phase silicas, especially with aggressive mobile phases. A new bonding technology overcomes these problems by attaching C18 groups to a carbon surface with ultra-stable carbon-carbon bonds. This note shows that robust methods with high efficiency are now possible using ultra-stable bonded-carbon columns.

Research paper thumbnail of Direct identification of rituximab main isoforms and subunit analysis by online selective comprehensive two-dimensional liquid chromatography - mass spectrometry

Analytical chemistry, Jan 6, 2015

In this proof-of-concept study, rituximab, a reference therapeutic monoclonal antibody (mAb), was... more In this proof-of-concept study, rituximab, a reference therapeutic monoclonal antibody (mAb), was characterized through the implementation of online, selective comprehensive two-dimensional liquid chromatography (sLCxLC) coupled with mass spectrometry (MS), and using a middle-up approach. In this setup, cation exchange chromatography (CEX) and reversed 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 and 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 de...

Research paper thumbnail of Effect of pressure pulses at the interface valve on the stability of second dimension columns in online comprehensive two-dimensional liquid chromatography

Journal of chromatography. A, Jan 23, 2015

Users of online comprehensive two-dimensional liquid chromatography (LCxLC) frequently acknowledg... more Users of online comprehensive two-dimensional liquid chromatography (LCxLC) frequently acknowledge that the mechanical instability of HPLC columns installed in these systems, particularly in the second dimension, is a significant impediment to its use. Such instability is not surprising given the strenuous operating environment to which these columns are subjected, including the large number (thousands per day) of fast and large pressure pulses resulting from interface valve switches (on the timescale of tens of milliseconds) associated with very fast second dimension separations. There appear to be no published reports of systematic studies of the relationship between second dimension column lifetime and any of these variables. In this study we focused on the relationship between the lifetimes of commercially available columns and the pressure pulses observed at the inlet of the second dimension column that occur during the switching of the valve that interfaces the two dimensions ...

Research paper thumbnail of Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants

Analytical and bioanalytical chemistry, 2015

In this paper, we describe the findings of a study aimed at assessing the detection sensitivity o... more In this paper, we describe the findings of a study aimed at assessing the detection sensitivity of comprehensive two-dimensional high-performance liquid chromatography (LCxLC) separation of a degraded active pharmaceutical ingredient (API) with UV absorption as the detection technique. Specifically, we have examined the impact of the volume and solvent composition (referred to as "interface conditions") of fractions of first-dimension column effluent transferred to the second dimension for further separation on the ability to resolve and detect low-abundance compounds. Historically, LCxLC has been perceived as being inferior to 1D-LC from the point of view of detection sensitivity. In this work, we demonstrate that LCxLC is sufficiently sensitive to be useful in the pharmaceutical context where in general impurities present at 0.05 % (relative to the API concentration) should be quantified. Moreover, we find that this level of sensitivity is only attained under certain con...

Research paper thumbnail of Two dimensional assisted liquid chromatography – a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation, dual detectors, and multivariate curve resolution

Analytica Chimica Acta, 2015

A novel method for quantitation in LC Â LC is proposed based on use of dual detectors. Curve reso... more A novel method for quantitation in LC Â LC is proposed based on use of dual detectors. Curve resolution was carried out on data from both detectors. The combined use of data from both detectors lead to improved quantitative results. In some cases, use of only first dimension data results in improved quantification.

Research paper thumbnail of Principles of Online Comprehensive Multidimensional Liquid Chromatography

Advances in Chromatography, 2012

Research paper thumbnail of Fast, Comprehensive Two-Dimensional HPLC Separation of Tryptic Peptides Based on High-Temperature HPLC

Journal of the American Chemical Society, 2005

Two-dimensional HPLC (2D-LC) has recently received considerable attention. 1 This interest is kin... more Two-dimensional HPLC (2D-LC) has recently received considerable attention. 1 This interest is kindled by both the statistical model of overlap (SMO) theory, which makes it clear that the resolving power of one-dimensional HPLC is grossly insufficient for many purposes, 2 and by the demands of many bio-analytical applications. 1

Research paper thumbnail of Targeted three-dimensional liquid chromatography: A versatile tool for quantitative trace analysis in complex matrices

Journal of Chromatography A, 2010

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' ... more Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.

Research paper thumbnail of Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part II: Applications

Journal of Chromatography A, 2012

An approach to enhancing the resolution of select portions of conventional one-dimensional high p... more An approach to enhancing the resolution of select portions of conventional one-dimensional high performance liquid chromatography (HPLC) separations was developed, which we refer to as selective comprehensive two-dimensional HPLC (sLC×LC). In this first of a series of two papers we describe the principles of this approach, which breaks the long-standing link in on-line multi-dimensional chromatography between the timescales of sampling the first dimension (¹D) separation and the separation of fractions of ¹D effluent in the second dimension. This allows rapid, high-efficiency separations to be used in the first dimension, while still adequately sampling ¹D peaks. Transfer, transient storage, and subsequent second dimension (²D) separations of multiple fractions of a particular ¹D peak produces a two-dimensional chromatogram that reveals the coordinates of the peak in both dimensions of the chromatographic space. Using existing valve technology we find that the approach is repeatable (%RSD of peak area &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;1.5%), even at very short first dimension sampling times--as low as 1s. We have also systematically studied the critical influence of the volume and composition of fractions transferred from the first to the second dimension of the sLC × LC system with reversed-phase columns in both dimensions, and the second dimension operated isocratically. We find that dilution of the transferred fraction, so that it contains 10-20% less organic solvent than the ²D eluent, generally mitigates the devastating effects of large transfer volumes on ²D performance in this type of system. Several example applications of the sLC × LC approach are described in the second part of this two-part series. We anticipate that future advances in the valve technology used here will significantly widen the scope of possible applications of the sLC × LC approach.

Research paper thumbnail of Dependence of Effective Peak Capacity in Comprehensive Two-Dimensional Separations on the Distribution of Peak Capacity between the Two Dimensions

Analytical Chemistry, 2008

One of the basic tenets of comprehensive two-dimensional chromatography is that the total peak ca... more One of the basic tenets of comprehensive two-dimensional chromatography is that the total peak capacity is simply the product of the first and second dimension peak capacities. As formulated the total peak capacity does not depend on the relative values of the individual dimensions but only on the product of the two. This concept is tested here for the experimentally realistic situation wherein the first dimension separation is undersampled. We first propose that a relationship exists between the number of observed peaks in a two-dimensional separation and the effective peak capacity. We then show here for a range of reasonable total peak capacities (500 to 4000) and various contributions of peak capacity in each dimension (10 to 150) that the number of observed peaks is only slightly dependent on the relative contributions over a reasonable and realistic range in sampling times (equal to the first dimension peak standard deviation, multiplied by 0.2 to 16). Most of this work was carried out under the assumption of totally uncorrelated retention times. For uncorrelated separations the small deviations from the product rule are due to the "edge effect" of statistical overlap theory and a recently introduced factor that corrects for the broadening of first dimension peaks by undersampling them. They predict that relatively more peaks will be observed when the ratio of the first to the second dimension peak capacity is much less than unity. Additional complications are observed when first and second dimension retention times show some correlation but again the effects are small. In both cases deviations from the product rule are measured by the relative standard deviations of the number of observed peaks, which are typically 10 or less. Thus although the basic tenet of two-dimensional chromatography is not exact when the first dimension is undersampled, the deviations from the product rule are sufficiently small as to be unimportant in practical work. Our results show that practitioners have a high degree of flexibility in designing and optimizing experimental comprehensive two-dimensional separations.

Research paper thumbnail of Effect of First-Dimension Undersampling on Effective Peak Capacity in Comprehensive Two-Dimensional Separations

Analytical Chemistry, 2008

The objective of this work is to establish a means of correcting the theoretical maximum peak cap... more The objective of this work is to establish a means of correcting the theoretical maximum peak capacity of comprehensive two-dimensional (2D) separations to account for the deleterious effect of undersampling firstdimension peaks. Simulations of comprehensive 2D separations of hundreds of randomly distributed sample constituents were carried out, and 2D statistical overlap theory was used to calculate an effective first-dimension peak width based on the number of observed peaks in the simulated separations. The distinguishing feature of this work is the determination of the effective firstdimension peak width using the number of observed peaks in the entire 2D separation as the defining metric of performance. We find that the ratio of the average effective first-dimension peak width after sampling to its width prior to sampling (defined as < >) is a simple function of the ratio of the first-dimension sampling time (t s) to the first-dimension peak standard deviation prior to sampling (1 σ): < >) 1+0.21(t s / 1 σ) 2 This is valid for 2D separations of constituents having either randomly distributed or weakly correlated retention times, over the range of 0.2 e t s / 1 σ e 16. The dependence of < > on t s / 1 σ from this expression is in qualitative agreement with previous work based on the effect of undersampling on the effective width of a single first-dimension peak, but predicts up to 35% more broadening of first-dimension peaks than is predicted by previous models. This simple expression and accurate estimation of the effect of undersampling first-dimension peaks should be very useful in making realistic corrections to theoretical 2D peak capacities, and in guiding the optimization of 2D separations.

Research paper thumbnail of Comparison of the Practical Resolving Power of One- and Two-Dimensional High-Performance Liquid Chromatography Analysis of Metabolomic Samples

Analytical Chemistry, 2008

Two-dimensional liquid chromatography (2DLC) has become a mainstay of proteomics research due to ... more Two-dimensional liquid chromatography (2DLC) has become a mainstay of proteomics research due to its higher peak capacity compared to one-dimensional LC (1DLC). Because of the long analysis times typically associated with 2DLC (tens of hours) and its use primarily in proteomics applications, 2DLC in the context of general HPLC has been regarded as a niche technique for use in analysis of mixtures containing hundreds to thousands of components compared to the far more common techniques of isocratic and gradient elution 1DLC. A significant next step in the analytical development of 2DLC is to consider using its higher resolving power to reduce the analysis time of rather &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;simple&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; mixtures, in the range of only tens to hundreds of chemical constituents. The chief objective of this paper is to provide guidance to practitioners who need to decide whether 1DLC or 2DLC gives the superior separation in a given analysis time. Conditional peak capacities are predicted for fully optimized 1DLC and practical 2DLC separations of the low molecular weight constituents of an extract of corn seed at several analysis times using a model based on the chromatographic properties of compounds that are representative of real mixtures of lower molecular weight species. Two important corrections to the ideal 2DLC peak capacity are made to account for both incomplete usage of the separation space and the serious effect of first-dimension undersampling; this allows, we believe for the first time, a fair comparison of the resolving power of 1D- and 2DLC under realistic conditions. The predicted optimum conditions are then used to carry out experimental separations of low molecular weight corn seed extract, and peaks are counted in each 1D- and 2DLC chromatogram. Based on comparisons of both the predicted peak capacities and number of peaks observed in experimental chromatograms, we believe that practical 2DLC will be superior to fully optimized gradient 1DLC for separations lasting more than only about 10 min. This crossover time is much shorter than intuitively expected, and we believe this finding will inevitably have a major impact on the practice of 2DLC in liquid-phase separations in general.

Research paper thumbnail of Analysis of Four-Way Two-Dimensional Liquid Chromatography-Diode Array Data:  Application to Metabolomics

Analytical Chemistry, 2006

Two-dimensional liquid chromatography (2D-LC) is rapidly gaining popularity for the analysis of v... more Two-dimensional liquid chromatography (2D-LC) is rapidly gaining popularity for the analysis of very complex mixtures, including proteomic and metabolomic samples. It provides an effective strategy for separating such samples, because the resolving power of 2D-LC is far superior to that of traditional single-dimension separations. The present work focuses on the development of data analysis methods for the extremely large data sets, on the order of 10 million data points, generated by 2D-LC with diode-array detection (DAD). Specifically, we have applied and adapted chemometric methods to the analysis of maize seedling digests, focusing on compounds related to the biosynthetic pathways of indole-3-acetic acid, the primary growth regulator in plants. The chemometric techniques of window target testing factor analysis (WTTFA), along with parallel factor analysis-alternating least squares (PARAFAC-ALS) were used to analyze 2D-LC-DAD chromatograms of a sample composed of 26 indolic standards, 2 extracts of mutant orange pericarp maize seedlings, 2 extracts of wild-type maize seedlings, and a blank sample. The indolic compounds studied belonged to six spectrally unique groups, and WTTFA was able to specifically identify the presence or absence of any of the 26 indolic standards in the mutant and wild-type samples. A PARAFAC-ALS algorithm and an ALS algorithm with flexible constraints were successfully applied to resolve the spectrally rank deficient data and to demonstrate the quantitative potential of multivariate curve resolution methods. Using this procedure, 95 total peaks were resolved in the data set analyzed. Of those 95 peaks, 45 were found in both the mutant and wild-type maize samples, 16 peaks were unique to the mutant maize samples, 13 peaks were unique to the wild-type maize samples, and 15 peaks were unique to the standard chromatograms. Of the 26 standards included in the data set, several indole acetic acid conjugates were identified and quantified in the maize samples at levels of approximately 0.3-2 µg/g plant material.