Improved hydrophobic subtraction model of reversed-phase liquid chromatography selectivity based on a large dataset with a focus on isomer selectivity.

Reversed-phase (RP) liquid chromatography is an important tool for the characterization of materials and products in the pharmaceutical industry. Method development is still challenging in this application space, particularly when dealing with closely-related compounds. Models of chromatographic selectivity are useful for predicting which columns out of the hundreds that are available are likely to have very similar, or different, selectivity for the application at hand.

Continuous stationary phase gradient preparation on planar chromatographic media using vapor phase deposition of silane.

A new, versatile, and straightforward vapor phase deposition (VPD) approach was used to prepare continuous stationary phase gradients (cSPGs) on silica thin-layer chromatography (TLC) plates using phenyldimethylchlorosilane (PDCS) as a precursor. A mixture of paraffin oil and PDCS was placed at the bottom of an open-ended rectangular chamber, allowing the reactive silanes to evaporate and freely diffuse under a controlled atmosphere. As the volatile silane diffused across the length of the TLC plate, it reacted with the surface silanol groups thus functionalizing the surface in a gradient fashion.

Preparation and characterization of stationary phase gradients on C8 liquid chromatography columns.

Continuous C8 stationary phase gradients are created on commercial Waters Symmetry Shield RP8 columns by strategically cleaving the C8 moieties in a time-dependent fashion. The method relies on the controlled infusion of a trifluoroacetic acid/water/acetonitrile solution through the column to cleave the organic functionality (e.g.

Experimental design and re-parameterization of the Neue-Kuss model for accurate and precise prediction of isocratic retention factors from gradient measurements in reversed phase liquid chromatography.

The present work describes a re-parameterization of the Neue Kuss (NK) model for describing retention in liquid chromatography, and this re-parameterized model is used to fit a large set of isocratic retention measurements with improved convergence properties relative to the original parameterization of the model. Next, an experimental design for retention measurements using mobile phase gradient elution conditions is proposed for the purpose of obtaining accurate and precise NK parameters. Simulated retention data for mobile phase gradient elution conditions with two different levels of noise, as well as an essentially zero noise level were fit with the re-parameterized model.

Characterization of a high throughput approach for large scale retention measurement in liquid chromatography.

Many contemporary challenges in liquid chromatography-such as the need for "smarter" method development tools, and deeper understanding of chromatographic phenomena-could be addressed more efficiently and effectively with larger volumes of experimental retention data than are available. The paucity of publicly accessible, high-quality measurements needed for the development of retention models and simulation tools has largely been due to the high cost in time and resources associated with traditional retention measurement approaches. Recently we described an approach to improve the throughput of such measurements by using very short columns (typically 5 mm), while maintaining measurement accuracy.

Accuracy of retention model parameters obtained from retention data in liquid chromatography.

In liquid chromatography, it is often very useful to have an accurate model of the retention factor, k, over a wide range of isocratic elution conditions. In principle, the parameters of a retention model can be obtained by fitting either isocratic or gradient retention factor data. However, in spite of many of our own attempts to accurately predict isocratic k values using retention models trained with gradient retention data, this has not worked in our hands.

Closed form approximations to predict retention times and peak widths in gradient elution under conditions of sample volume overload and sample solvent mismatch.

Closed form expressions for the prediction of retention times and peak widths for gradient liquid chromatography are particularly useful in understanding, rationalizing and optimizing separations. These expressions are obtained by integrating differential equations, in conjunction with a model of the variation of the retention factor as a function of mobile phase composition. Two of these models, the linear solvent strength (LSS) model and the Neue-Kuss (NK) model are explored in the present work.

Improvements in the predictive accuracy of the hydrophobic subtraction model of reversed-phase selectivity.

The hydrophobic subtraction model (HSM) for characterizing the selectivity of reversed-phase liquid chromatography (LC) columns has been used extensively by the LC community since it was first developed in 2002. Continuing interest in the model is due in part to the large, publicly available set of column descriptors that has been assembled over the past 18 years. In the work described in this report, we sought to refine the HSM with the goal of improving the predictive accuracy of the model without compromising its physico-chemical interpretability.

Simulation of elution profiles in liquid chromatography - IV: Experimental characterization and modeling of solute injection profiles from a modulation valve used in two-dimensional liquid chromatography.

Simulation software for liquid chromatography can accelerate method development capabilities. In two-dimensional chromatography this is particularly attractive because there are more method variables to consider, provided simulations can account for the effects of injecting effluent from the first dimension separation into the second dimension column. In this paper we describe the adaptation of a previously described model (the Forssén model) to enable prediction of the profile of an injection pulse as it exits an Active Solvent Modulation (ASM) valve and enters the second dimension column under a variety of flow rate and sample loop size conditions (a global model).

Fabrication and Characterization of a Reversed-Phase/Strong Cation Exchange Stationary Phase Gradient.

Continuous stationary phase gradients for liquid chromatography (LC) have been recently shown to be a promising method of altering selectivity. In this work, we present the first multicomponent continuous stationary phase gradient for separations involving both reversed-phase (RP) and strong cation exchange (SCX) mechanisms. These columns are fabricated using a two-step methodology based on controlled rate infusion (CRI).

Experimental- and simulation-based investigations of coupling a mobile phase gradient with a continuous stationary phase gradient.

This work seeks to explore and understand the effects of column orientation and degree of modification of continuous stationary phase gradient columns under a mobile phase gradient using both simulations and experiments. Peak parameters such as retention times, peak widths and resolution are obtained for five phenolic compounds on a C-silica gradient stationary phase. Simulations show that peak widths for the solutes are dependent upon the fractional composition of C and orientation of the stationary phase gradient when coupled to a mobile phase gradient.

Rational design of mixtures for chromatographic peak tracking applications via multivariate selectivity.

Chromatographic characterization and parameterization studies targeting many solutes require the judicious choice of operating conditions to minimize analysis time without compromising the accuracy of the results. To minimize analysis time, solutes are often grouped into a small number of mixtures; however, this increases the risk of peak overlap. While multivariate curve resolution methods are often able to resolve analyte signals based on their spectral qualities, these methods require that the chromatographically overlapped compounds have dissimilar spectra.

Destructive stationary phase gradients for reversed-phase/hydrophilic interaction liquid chromatography.

The use of stationary phase gradients for liquid chromatography (LC) is a promising new strategy to allow for specific control over the selectivity of a separation by having a gradual change in the ligand density along the length of the column. Unfortunately, there have been very few, if any, methods to prepare continuous stationary phase gradients on traditional packed LC columns. In this work, destructive methodologies are used to create stationary phase gradients on commercial C columns by infusing trifluoroacetic acid (TFA) onto the column through controlled rate of infusion (CRI).

Simulation of elution profiles in liquid chromatography - III. Stationary phase gradients.

A previously developed liquid chromatographic simulator (see parts I and II) [1-3] is extended to allow for simulations of stationary phase gradients with isocratic and gradient mobile phases. Gradient stationary phases have recently been proposed as means of engineering unique chromatographic selectivities. In the present work, the simulator provides retention times and peak widths that agree with closed form theory for a linear gradient in retention factor and provides accurate retention time predictions for experimentally implemented continuous and discontinuous gradients.

Simulation of elution profiles in liquid chromatography - II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography.

An important research direction in the continued development of two-dimensional liquid chromatography (2D-LC) is to improve the detection sensitivity of the method. This is especially important in applications where injection of large volumes of effluent from the first dimension (D) column into the second dimension (D) column leads to severe D peak broadening and peak shape distortion. For example, this is common when coupling two reversed-phase columns and the organic solvent content of the D mobile phase overwhelms the D column with each injection of D effluent, leading to low resolution in the second dimension.

Analysis of Liquid Chromatography-Mass Spectrometry Data with an Elastic Net Multivariate Curve Resolution Strategy for Sparse Spectral Recovery.

Analysis of liquid chromatography-mass spectrometry (LC-MS) data requires the differentiation between a small number of relevant chemical signals and a larger amount of noise. This is often done based, at least partially, on a threshold which assumes that low intensity m/z signals arise from the noise. This eliminates low-intensity fragments, isotopes, and adducts and may exclude relevant low-intensity compounds all together.

Comparison of multivariate curve resolution strategies in quantitative LCxLC: Application to the quantification of furanocoumarins in apiaceous vegetables.

Comprehensive two-dimensional liquid chromatography (LC × LC) has been gaining popularity for the analysis of complex samples in a wide range of fields including metabolomics, environmental analysis, and food analysis. While LC × LC can provide greater chromatographic resolution than one-dimensional LC (1D-LC), overlapping peaks are often still present in separations of complex samples, a problem that can be alleviated by chemometric curve resolution techniques such as multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS has also been previously shown to assist in the quantitative analysis of LC x LC data by isolating pure analyte signals from background signals which are often present at higher levels in LC x LC compared to 1D-LC.

Multivariate Curve Resolution-Alternating Least Squares Analysis of High-Resolution Liquid Chromatography-Mass Spectrometry Data.

Methods such as liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) are crucial for differentiating compounds with highly similar masses. This is a necessity when analyzing highly complex samples; however, the size of high-resolution LC-HRMS data sets can cause difficulties when applying advanced data analysis techniques. In this work, LC-HRMS analyses of known amphetamine samples and unknown bacterial lipid samples were carried out, and multivariate curve resolution-alternating least squares (MCR-ALS) was applied to the data to obtain mathematical separation of overlapped analyte signals.

Simulation of elution profiles in liquid chromatography-I: Gradient elution conditions, and with mismatched injection and mobile phase solvents.

High-performance liquid chromatography (HPLC) simulators are effective method development tools. The goal of the present work was to design and implement a simple algorithm for simulation of liquid chromatographic separations that allows for characterization of the effect of injection solvent mismatch and injection solvent volume overload. The simulations yield full analyte profiles during solute migration and at elution, which enable a thorough physical understanding of the effects of method variables on chromatographic performance.

Amine Gradient Stationary Phases on In-House Built Monolithic Columns for Liquid Chromatography.

Stationary phase gradients on monolithic silica columns have been successfully and reproducibly prepared and characterized with comparisons made to uniformly modified stationary phases. Stationary phase gradients hold great potential for use in liquid chromatography (LC), both in terms of simplifying analysis as well as providing novel selectivity. In this work, we demonstrate the creation of a continuous stationary phase gradient on in-house synthesized monolithic columns by infusing an aminoalkoxysilane solution through the silica monoliths via controlled rate infusion.

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.

Comprehensive two-dimensional liquid chromatography (LC×LC) is rapidly evolving as the preferred method for the analysis of complex biological samples owing to its much greater resolving power compared to conventional one-dimensional (1D-LC). While its enhanced resolving power makes this method appealing, it has been shown that the precision of quantitation in LC×LC is generally not as good as that obtained with 1D-LC. The poorer quantitative performance of LC×LC is due to several factors including but not limited to the undersampling of the first dimension and the dilution of analytes during transit from the first dimension ((1)D) column to the second dimension ((2)D) column, and the larger relative background signals.

In vivo monitoring of serotonin in the striatum of freely moving rats with one minute temporal resolution by online microdialysis-capillary high-performance liquid chromatography at elevated temperature and pressure.

Online monitoring of serotonin in striatal dialysate from freely moving rats was carried out for more than 16 h at 1 min time resolution using microdialysis coupled online to a capillary HPLC system operating at about 500 bar and 50 °C. Several aspects of the system were optimized toward robust, in vivo online measurements. A two-loop, eight-port rotary injection valve demonstrated better consistency of continuous injections than the more commonly used two-loop, 10-port valve.

Comparison of chemometric methods for the screening of comprehensive two-dimensional liquid chromatographic analysis of wine.

Two chemometric methods are compared for the rapid screening of comprehensive two-dimensional liquid chromatographic (LC×LC) analysis of wine. The similarity index and Fisher ratio methods were both found to be able to distinguish geographical variability and to determine potentially significant peaks for further quantitative and qualitative study. An experimental data set consisting of five different wine samples and multiple simulated data sets were analyzed in the investigation of the screening methods.

Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation--application to the quantitative analysis of furanocoumarins in apiaceous vegetables.

Various implementations of two-dimensional high-performance liquid chromatography are increasingly being developed and applied to the analysis of complex materials, including those encountered in the analysis of foods, beverages, and nutraceuticals. Previously, we introduced the concept of selective comprehensive two-dimensional liquid chromatography (sLC × LC) as a hybrid between the more conventional, but extreme opposite sampling modes of heartcutting (LC-LC) and fully comprehensive (LC × LC) 2D separation. The sLC × LC approach breaks the link between first dimension ((1)D) sampling time and second dimension ((2)D) analysis time that is faced in LC × LC and allows very rapid (as low as 1 s) sampling of highly efficient (1)D separations, while at the same time allowing efficient (2)D separations on the timescale of tens of seconds.