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. The results showed that the re-parameterized fits yielded average (absolute value) prediction errors for the parameters at the highest noise level of 7.2 % for S1,ref, 18 % for S2,ref and 6.2 % for kref (the re-parameterized NK model parameters). These errors were significantly smaller than those for the original parameterization of the NK model, where the errors were 23 % for S1, 25 % for S2 and 160 % for kw (the original NK model parameters). Furthermore, isocratic retention factors predicted using these model parameters were found to have an average magnitude of error of 0.51 % for the re-parameterized model, as opposed to 6800 % for the model with the original parameterization. A further test of this approach was carried out for independent experimental measurements for five solutes on a C18 column. The average magnitude of error of the isocratic retention factors predicted from parameters obtained from fits of gradient data was 1.6 %, provided that the range of organic solvent compositions that the solute sampled in the mobile phase gradient experiments was consistent with the isocratic experiments. These results indicate that the re-parameterization of the NK model allows for significant improvements in the fitting process, and that the proposed experimental design allows for NK parameters to be extracted from mobile phase gradient experiments, with prediction accuracies of isocratic retention factors on the order of 1-2 %.

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. Here, we expand on these closed form expressions to account for effects of sample volume overload and a mismatch between the sample solvent and the initial mobile phase composition for the gradient. We show that there have been errors in expressions reported in the literature, and we have evaluated the accuracy of the predictions from the closed form expressions reported here using a recently developed liquid chromatography simulator. The expressions assume a constant plate height and consider elution across four zones of the gradient profile - elution in the sample solvent, elution in the initial (isocratic) mobile phase caused by the gradient delay volume, elution during a linear gradient, and elution post-gradient at the final (isocratic) mobile phase composition. The expressions generally give reasonably accurate predictions for retention times and peak widths, except for cases where the solute elutes during transitions between the different zones. The average magnitude of the prediction errors for retention time and peak width relative to simulation were 0.093% and 0.40% for the LSS expressions for ten amphetamine solutes at 36 different separation conditions, and 0.22% and 1.8% for the NK expressions for eight alkylbenzene solutes at 36 different separation conditions, respectively.

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. The approach taken here has the following facets. A set of retention measurements for 635 columns and the 16 probe solutes used to characterize new columns using the HSM was assembled. Principal components analysis (PCA) was used as a guide for the development of a refined version of the HSM. Several outlying columns (84) were eliminated from the analysis because they were either inconsistent with the PCA model or were outliers from the original HSM model. With the retention dataset for the 16 probe solutes on the remaining 551 columns, we determined that a six-component model is the most sophisticated form of the model that can be used without overfitting the data. In our refined version of the HSM, the S*σ term has been removed. Two new terms have been added, which more accurately account for the molecular volume of the solute (Vv), and the solute dipolarity (Dd), and the remaining terms have been adjusted to accommodate these changes. The refined model described here provides improved prediction of retention factors, with the model standard error being reduced from 1.0 for the original HSM to 0.35 for the refined model (16 solutes, 551 columns). Furthermore, the number of retention factors with errors greater than 10% are reduced from 231 to 25. A revised metric for column similarity, F, is also proposed as a part of this work.

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). Experimentally measured injection profiles were used to train empirical models capable of generating injection profiles as a function of sample loop volume and flow rate. The resulting parameters were then used to generate an injection profile for a loop volume not used in the training set. The resulting profile agreed well with the experimentally obtained profile for this sample loop. Finally, chromatograms were simulated using previously developed simulation software incorporating the injection profile models developed in this work. Chromatographic peaks were simulated for valerophenone on an Agilent Zorbax Stablebond C18 stationary phase with an acetonitrile/water mobile phase gradient. Results of simulations based on experimental injection profiles, profiles predicted using the Forssén or global models, and rectangular injection profiles were compared. Comparison of the resulting chromatographic peaks revealed good agreement between those produced using experimental profiles or the Forssén or global models, with less than ± 0.3% deviations for retention times and less than ± 10% deviations for the peak widths (expressed as σ).

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). First, destructive CRI creates a gradient of excess silanol groups along a uniform C18 column. Next, these columns are infused with 3-mercaptopropyltrimethoxysilane (MPTMOS), which bonds to the excess silanol groups. The terminal thiols of the MPTMOS ligands are oxidized with H2O2 to create the sulfonate functionality (SO3-) needed for SCX separations. The success of the modification procedure is characterized by thermogravimetric analysis and X-ray photoelectron spectroscopy. The stability of the C18-SO3- gradients were found to have less than 5 % retention loss and the column-to-column reproducibility had a relative standard deviation under 9 %. The peak asymmetry factors for seven biogenic amines were found to be between 1.03 ± 0.04 to 1.30 ± 0.02, which illustrates minimal peak tailing due to poor packing and residual silanol groups. Characterization of the gradient columns using an isocratic mobile phase showed a unique elution order compared to a uniform C18 and SO3- columns. At lower counterion concentrations, more than 48 % of the overall retention on the gradient stationary phase is due to a SCX mechanism. Meanwhile, the RP mechanism was shown to predominate at higher counterion concentrations on the gradient columns (SCX retention contribution less than 40 %). Coupling the stationary phase gradient to a salt gradient in the mobile phase showed that the gradient phase provides a unique, intermediate selectivity to the uniform C18 and SO3- columns. Under an ACN mobile phase gradient, a significant increase (p < 0.003) in the retention times of three biogenic amines (15 - 16 %) was observed when the multicomponent gradient was oriented to have a high SO3- ligand density near the detector. This work serves as a proof-of-concept design for a multicomponent stationary phase gradient to continue fundamental studies into retention and encourage novel applications.

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 C18-silica gradient stationary phase. Simulations show that peak widths for the solutes are dependent upon the fractional composition of C18 and orientation of the stationary phase gradient when coupled to a mobile phase gradient. Also, when compared to a simulated uniform mixed-mode column, peak widths reach a minimum on the gradient column with a coverage higher than 50% C18 where the column is oriented to have the C18 dense region at the end. Experimentally, continuous stationary phase gradients were fabricated to have a total C18 composition of 78% of the original uniform column with an exponential profile using a previously described destructive controlled rate infusion method. Under gradient mobile phase conditions, experimental retention times for the gradient column showed a significant increase compared to the original 100% C18 column. Simulations with a similar C18 composition, however, predicted decreased retention times from the original C18 column. A statistical increase in the retention time of protocatechuic acid and decrease in the peak width of tyrosol, caffeic acid, and coumaric acid were noted when the gradient column was oriented to have the C18 dense region located near the detector. Collectively, combining gradients in both the mobile and stationary phases can yield interesting neighboring ligand effects and peak broadening/focusing effects.

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. In this work, a strategy for grouping compounds into sample mixtures containing solutes with distinct spectral and, optionally, with distinct chromatographic properties, in order to ensure successful solute resolution either chromatographically or with curve resolution methods is proposed. We name this strategy rational design of mixtures (RDM). RDM utilizes multivariate selectivity as a metric for making decisions regarding group membership (i.e., whether to add a particular solute to a particular sample). A group of 97 solutes was used to demonstrate this strategy. Utilizing both estimated chromatographic properties and measured spectra to group these 97 analytes, only 12 groups were required to avoid a situation where two or more solutes in the same group could not be resolved either chromatographically (i.e., they have significantly different retention times) or spectrally (i.e., spectra are different enough to enable resolution by curve resolution methods). When only spectral properties were utilized (i.e., the chromatographic properties are unknown ahead of time) the number of groups required to avoid unresolvable overlaps increased to 20. The grouping strategy developed here will improve the time and instrument efficiency of studies that aim to obtain retention data for solutes as a function of operating conditions, whether for method development or determination of the chromatographic parameters of solutes of interest (e.g., k w ).

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 C18 columns by infusing trifluoroacetic acid (TFA) onto the column through controlled rate of infusion (CRI). The introduction of TFA via CRI while the column is heated at 80 °C promotes acid hydrolysis of the alkylsilane ligand in a gradient fashion. Characterization with scanning electron microscopy and Barrett-Joyner-Halenda pore size distributions of the stationary phase after fabrication of the destructive gradient establishes that the chromatographic support was not damaged during the procedure. The shape of the gradient was examined using thermogravimetric analysis (TGA) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. TGA and ATR-FTIR showed an increase in the percent carbon loss along the length of the column, indicating that there was an increase in the C18 ligand from the front to the end of the column. Two selectivity tests demonstrated a decrease in the hydrophobicity and increase in the silanol activity of the stationary phase gradient from the uniform C18 counterpart. Additionally, the fabrication of the destructive stationary phase gradient resulted in two different surface functionalities allowing hydrophobic and hydrophilic interactions with analyte species depending on the mobile phase composition. Plots of the log of retention factor versus percent acetonitrile illustrated that these stationary phase gradients have two separation mechanisms: reversed-phase (RP) and hydrophilic interaction. Coupling the stationary phase gradient with a mobile phase gradient shows differences in the peak widths and the resolution of phenolic compounds, indicating that the orientation of the stationary phase gradient has the potential to enhance the resolution of a separation. With this methodology, stationary phase gradients can be fabricated on previously used RP columns, allowing for these columns to be repurposed.

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. Calculation of discontinuous gradients implemented using the commercially available POPLC system have shown good agreement with experiment, with the largest deviation of the simulated retention time from experiment of 4.5%. In addition, simulations of a novel continuous amine gradient column show good agreement with experiment, and give insights into synergistic interactions on column. With the exception of solutes that show evidence of synergistic interactions, the simulated retention times are in agreement with the 95% confidence limits of the experimental values.

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 (1D) column into the second dimension (2D) column leads to severe 2D peak broadening and peak shape distortion. For example, this is common when coupling two reversed-phase columns and the organic solvent content of the 1D mobile phase overwhelms the 2D column with each injection of 1D effluent, leading to low resolution in the second dimension. In a previous study we validated a simulation approach based on the Craig distribution model and adapted from the work of Czok and Guiochon [1] that enabled accurate simulation of simple isocratic and gradient separations with very small injection volumes, and isocratic separations with mismatched injection and mobile phase solvents [2]. In the present study we have extended this simulation approach to simulate separations relevant to 2D-LC. Specifically, we have focused on simulating 2D separations where gradient elution conditions are used, there is mismatch between the sample solvent and the starting point in the gradient elution program, injection volumes approach or even exceed the dead volume of the 2D column, and the extent of sample loop filling is varied. To validate this simulation we have compared results from simulations and experiments for 101 different conditions, including variation in injection volume (0.4-80µL), loop filling level (25-100%), and degree of mismatch between sample organic solvent and the starting point in the gradient elution program (-20 to +20% ACN). We find that that the simulation is accurate enough (median errors in retention time and peak width of -1.0 and -4.9%, without corrections for extra-column dispersion) to be useful in guiding optimization of 2D-LC separations. However, this requires that real injection profiles obtained from 2D-LC interface valves are used to simulate the introduction of samples into the 2D column. These profiles are highly asymmetric - simulation using simple rectangular pulses leads to peak widths that are far too narrow under many conditions. We believe the simulation approach developed here will be useful for addressing practical questions in the development of 2D-LC methods.

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. This work describes the use of multivariate curve resolution-alternating least-squares with an additional sparse regression step using elastic net (MCR-ENALS) to distinguish relevant m/z signals without the use of a harsh thresholding step, thus allowing for discovery of low-intensity m/z signals corresponding to the analytes. This strategy is demonstrated first on a unit mass analysis of amphetamines in which relevant m/z signals are found at as low as a 0.1% intensity relative to the molecular m/z peak. The incorporation of MCR-ENALS into our previously reported data reduction strategy for analysis of high-resolution LC-MS is also demonstrated. Analysis based on only 0.3% of the original data set, while retaining low-intensity isotope peaks, was accomplished without the use of thresholding, allowing for the application of MCR-ENALS to the high-resolution LC-MS data.

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. In this work we present the analysis of a dataset from the LC × LC analyses of parsley, parsnip and celery samples for the presence and concentrations of 14 furanocoumarins. Several MCR-ALS implementations are compared for the analysis of LC × LC data. These implementations include analyzing the LC x LC chromatogram alone, analyzing the one-dimensional chromatogram alone, as well as two hybrid approaches that make use of both the first and second dimension chromatograms. Furthermore, we compared manual integration of resolved chromatograms versus a simple summation approach, using the resolved chromatographic peaks in both cases. It is found that manual integration of the resolved LC × LC chromatograms provides the best quantification as measured by the consistency between replicate injections. If the summation approach is desired for automation, the choice of MCR-ALS implementation has a large effect on the precision of the analysis. Based on these results, the concentrations of the 14 furanocoumarins are determined in the three apiaceous vegetable types by analyzing the LC × LC chromatograms with MCR-ALS and manual integration for peak area determination. The concentrations of the analytes are found to vary greatly between samples, even within a single vegetable type.

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. In order to minimize computational strain, a novel strategy was developed which minimizes the number of irrelevant masses analyzed at full resolution. To do this, data were first binned to unit mass resolution, and MCR-ALS was performed. This provided mathematical components for each analyte present plus background components. In the resolved spectral profiles of analyte components, masses above a preset intensity threshold were extracted, discarding all other masses, and expanded to successively higher levels of resolution, applying MCR-ALS at each level. These steps were repeated until 0.001 amu resolution was achieved, as dictated by the resolution of the instrument-in this case, a time-of-flight mass spectrometer. This strategy allowed for the accurate recovery of all known amphetamine compounds and select bacterial lipid extracts while minimizing the size of the data, therefore minimizing computational analysis time and data storage requirements. This relatively simple strategy enables the effective coupling of LC-HRMS with MCR-ALS.

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. The Craig counter-current distribution model (the plate model) is used as the basis for simulation, where a local retention factor is assigned for each spatial and temporal element within the simulation. The algorithm, which is an adaptation of an approach originally described by Czok and Guiochon (Ref. [10]), is sufficiently flexible to allow the use of either linear (e.g., Linear Solvent Strength Theory) or non-linear models of solute retention (e.g., Neue-Kuss (Ref. [36])). In this study, both types of models were used, one for simulating separations of a homologous series of alkylbenzenes, and the other for separations of selected amphetamines. The simulation program was validated first by comparison of simulated retention times and peak widths for five amphetamines to predictions obtained using linear solvent strength (LSS) theory, and to results from experimental separations of these compounds. The simulated retention times for the amphetamines agreed within 0.02% and 2.5% compared to theory and experiment, respectively. Secondly, the program was evaluated for simulating the case where there is a compositional mismatch between the mobile phase at the column inlet and the injection solvent (i.e., the sample matrix). This work involved alkylbenzenes, and retention time and peak width predictions from simulations were within 1.5 and 6.0% of experimental values, respectively, even without correction for extra-column dispersion. The issues of sample/eluent solvent mismatch and solvent volume overload are especially important when considering the challenges of transferring eluent from the first to the second dimension in comprehensive two-dimensional liquid chromatography.

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. The presence of amine and its distribution along the length of gradient and uniformly modified columns were assessed via X-ray photoelectron spectroscopy (XPS). XPS showed a clear gradient in surface coverage along the length of the column for the gradient stationary phases while a near uniform distribution on the uniformly modified stationary phases. To demonstrate the application of these gradient stationary phases, the separations of both nucleobases and weak acids/weak bases on these gradient stationary phases have been compared to uniformly modified and unmodified silica columns. Of particular note, the retention characteristics of 11 gradient columns, 5 uniformly modified columns, and 5 unmodified columns have been tested to establish the reproducibility of the synthetic procedures. Standard deviations of the retention factors were in the range from 0.06 to 0.5, depending on the analyte species. We show that selectivity is achieved with the stationary phase gradients that are significantly different from either uniformly modified amine or unmodified columns. These results indicate the significant promise of this strategy for creating novel stationary phases for LC.

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. A new strategy, 2D assisted liquid chromatography (2DALC), is presented here. 2DALC makes use of a diode array detector placed at the end of each column, producing both multivariate (1)D and two-dimensional (2D) chromatograms. The increased resolution of the analytes provided by the addition of a second dimension of separation enables the determination of analyte absorbance spectra from the (2)D detector signal that are relatively pure and can be used to initiate the treatment of data from the first dimension detector using multivariate curve resolution-alternating least squares (MCR-ALS). In this way, the approach leverages the strengths of both separation methods in a single analysis: the (2)D detector data is used to provide relatively pure analyte spectra to the MCR-ALS algorithm, and the final quantitative results are obtained from the resolved (1)D chromatograms, which has a much higher sampling rate and lower background signal than obtained in conventional single detector LC×LC, to obtain accurate and precise quantitative results. It is shown that 2DALC is superior to both single detector selective or comprehensive LC×LC and 1D-LC for quantitation of compounds that appear as severely overlapped peaks in the (1)D chromatogram - this is especially true in the case of untargeted analyses. We also anticipate that 2DALC will provide superior quantitation in targeted analyses in which unknown interfering compounds overlap with the targeted compound(s). When peaks are significantly overlapped in the first dimension, 2DALC can decrease the error of quantitation (i.e., improve the accuracy by up to 14-fold compared to 1D-LC and up to 3.8-fold compared to LC×LC with a single multivariate detector). The degree of improvement in performance varies depending upon the degree of peak overlap in each dimension and the selectivities of the spectra with respect to one another and the background, as well as the extent of analyte dilution prior to the (2)D column.

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. A six-port loop injector for introducing stimulating solutions (stimulus injector) was placed in-line between the syringe pump and microdialysis probe. We minimized solute dispersion by using capillary tubing (75 µm inside diameter, 70 cm long) for the probe inlet and outlet. In vitro assessment of concentration dispersion during transport with a 30 s time resolution showed that the dispersion standard deviation for serotonin was well within the desired system temporal resolution. Each 30 or 60 s measurement reflects the integral of the true time response over the measurement time. We have accounted for this mathematically in determining the concentration dispersion during transport. The delay time between a concentration change at the probe and its detection is 7 min. The timing of injections from the stimulus injector and the cycle time for the HPLC monitoring of the flow stream were controlled. The electrochemical detector contained a 13 µm spacer to minimize detector dead volume. During in vivo experiments, retention time and separation efficiency were stable and reproducible. There was no statistically significant change over 5.5 h in the electrochemical detector sensitivity factor for serotonin. Dialysate serotonin concentrations change significantly in response to a 120 mM K(+) stimulus. Release of serotonin evoked by a 10 min, 120 mM K(+) stimulation, but not for other K(+) stimuli, exhibited a reproducible, oscillating profile of dialysate serotonin concentration versus time. Infusion of fluoxetine, a serotonin uptake inhibitor, increased dialysate serotonin concentrations and stimulated release magnitude. Transient serotonin increases were observed in response to the stress associated with unexpected handling. This system is simple, efficient, reliable, and suitable for the study of serotonin neurochemistry associated with emotion and behavior.

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. Several statistical analyses are employed in the understanding and verification of the results from the similarity index and Fisher ratio methods. The sum rank difference (SRD) method was used to compare the rankings of the two different methods as applied to the different data sets and to determine the amount of variability associated with the ranking of the peak differences. The major advantage the similarity index method offers is that it is an unsupervised method; no a priori knowledge of the samples (i.e., class identification) is required, while the Fisher ratio method is supervised. Both methods are rapid and require little user intervention other than the determination of a threshold for inclusion/exclusion of compounds from further analysis.

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. In this paper, we improve upon our previous sLC × LC work by demonstrating the ability to perform the processes of (1)D sampling and (2)D separation in parallel. This significantly improves the flexibility of the technique and allows targeted analysis of analytes that elute close together in time in the (1)D separation. To demonstrate the value of this added capability, we have developed a sLC × LC method using multi-wavelength ultraviolet absorbance detection for the quantitative analysis of six target furanocoumarin compounds in extracts of celery, parsley, and parsnips. We show that (2)D separations of (1)D effluent containing the target compounds of interest reveal the presence of unanticipated interferent peaks that would otherwise compromise the quantitative accuracy of the method. We also demonstrate the application of the chemometric method iterative key set factor analysis with alternating least-squares to sLC × LC to mathematically resolve target compounds that are only slightly separated chromatographically but not sufficiently resolved for accurate quantitation.