Gradient Elution Reversed-phase Liquid Chromatography Research Articles

Principles and potential of solvent gradient size-exclusion chromatography for polymer analysis

The properties of a polymeric material are influenced by its underlying molecular distributions, including the molecular-weight (MWD), chemical-composition (CCD), and/or block-length (BLD) distributions. Gradient-elution liquid chromatography (LC) is commonly used to determine the CCD. Due to the limited solubility of polymers, samples are often dissolved in strong solvents. Upon injection of the sample, such solvents may lead to broadened or poorly shaped peaks and, in unfavourable cases, to “breakthrough” phenomena, where a part of the sample travels through the column unretained. To remedy this, a technique called size-exclusion-chromatography gradients or gradient size-exclusion chromatography (gSEC) was developed in 2011. In this work, we aim to further explore the potential of gSEC for the analysis of the CCD, also in comparison with conventional gradient-elution reversed-phase LC, which in this work corresponded to gradient-elution reversed-phase liquid chromatography (RPLC). The influence of the mobile-phase composition, the pore size of the stationary-phase particles, and the column temperature were investigated. The separation of five styrene/ethyl acrylate copolymers was studied with one-dimensional RPLC and gSEC. RPLC was shown to lead to a more-accurate CCD in shorter analysis time. The separation of five styrene/methyl methacrylate copolymers was also explored using comprehensive two-dimensional (2D) LC involving gSEC, i.e. SEC × gSEC and SEC × RPLC. In 2D-LC, the use of gSEC was especially advantageous as no breakthrough could occur.

Separation and identification of polymeric dispersants in detergents by two-dimensional liquid chromatography

Polymeric dispersants are an important ingredient in many consumer products. Their separations and identifications in final product formulation can be very challenging due to the presence of multiple polymeric dispersants at different levels and the presence of other polymeric and small-molecule components. In this study, using nearly comprehensive two-dimensional liquid chromatography (2D-LC), various water-soluble polymer and co-polymer dispersants were separated with aqueous size exclusion chromatography (SEC) in the first dimension (1D) and gradient elution reversed-phase liquid chromatography (RPLC) in the second dimension (2D). Detection of the polymeric dispersants was accomplished by evaporative light scattering detector (ELSD). A large ID (8.0 mm) SEC column used in common one-dimensional SEC practices was directly adopted in the 2D setup for rapid method development. A close representation of fully comprehensive 2D separation was achieved even with 60% of 1D eluent diverted to waste, demonstrating the flexibility and versatility of having SEC in 1D for two dimensional separation of polymers. Important method parameters, such as 2D column dimensions and flow rate, gradient conditions, and buffer pH were studied. Practical aspects of routine industrial applications such as solvent consumption and analysis time were also considered. This method was exploited for quick identification of polymeric dispersants in commercial detergent samples. Nine detergent samples were screened and polymeric dispersants and additional polymer features were detected in the samples.

Reversed-phase liquid chromatography-mass spectrometry of some organic tellurium compounds

The chromatographic behavior of some organic tellurium compounds (OTCs) was studied under the conditions of high-performance liquid chromatography with mass spectrometric detection. The retention characteristics (retention factor, relative retention, and differential molar Gibbs energy difference) of the compounds were obtained under the conditions of gradient elution reversed-phase liquid chromatography. The chemical ionization mass spectra of OTCs at atmospheric pressure were considered. It was shown that there was a relation between the structure of the compounds studied and their chromatographic retention.

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

We studied the run-to-run repeatability of the retention times of both non-ionizable and basic compounds chromatographed using buffered eluents. The effect of flow rate, organic modifier and other additives, buffer type/concentration, stationary phase type, batch-to-batch preparation of the initial eluent, gradient time, sample type and intra-day changes on retention repeatability were examined. We also assessed the effect of column storage solvent conditions on the inter-day repeatability. Although retention repeatability is strongly influenced by many parameters (flow rate, solvent compressibility compensation, precision of temperature control, and buffer/stationary phase type), our primary finding is that with a reasonable size column (15 cm × 4.6 mm (i.d.)) two column volumes of re-equilibration with initial eluent suffices to provide acceptable repeatability (no worse than 0.004 min) for both non-ionizable and basic analytes under a wide variety of conditions. Under ideal conditions (e.g. the right buffer, flow rate, etc.) it is possible to obtain truly extraordinary repeatability often as good as 0.0004 min. These absolute fluctuations in retention translate to worst case changes in resolution of 0.2 units and average changes of only 0.02 units.

Fast gradient elution reversed-phase liquid chromatography with diode-array detection as a high-throughput screening method for drugs of abuse: II. Data analysis

In Part I of this work, we developed a method for the detection of drugs of abuse in biological samples based on fast gradient elution liquid-chromatography coupled with diode array spectroscopic detection (LC-DAD). In this part of the work, we apply the chemometric method of target factor analysis (TFA) to the chromatograms. This algorithm identifies the target compounds present in chromatograms based on a spectral library, resolves nearly co-eluting components, and differentiates between drugs with similar spectra. The ability to resolve highly overlapped peaks using the spectral data afforded by the DAD is what distinguishes the present method from conventional library searching methods. Our library has a mean list length (MLL) of 1.255 and a discriminating power of 0.997 when both retention index and spectral factors are considered. The algorithm compares a library of 47 different compounds of toxicological relevance to unknown samples and identifies which compounds are present based on spectral and retention index matching. The application of a corrected retention index for identification rather than raw retention times compensates for long-term and column-to-column retention time shifts and allows for the use of a single library of spectral and retention data. Training data sets were used to establish the search and identification parameters of the method. A validation data set of 70 chromatograms was used to calculate the sensitivity (correct identification of positives) and specificity (correct identification of negatives) of the method, which were found to be 92% and 94%, respectively.

Retention Prediction of Peptides Based on Uninformative Variable Elimination by Partial Least Squares

A quantitative structure-retention relationship analysis was performed on the chromatographic retention data of 90 peptides, measured by gradient elution reversed-phase liquid chromatography, and a large set of molecular descriptors computed for each peptide. Such approach may be useful in proteomics research in order to improve the correct identification of peptides. A principal component analysis on the set of 1726 molecular descriptors reveals a high information overlap in the descriptor space. Since variable selection is advisable, the retention of the peptides is modeled with uninformative variable elimination partial least squares, besides classic partial least squares regression. The Kennard and Stone algorithm was used to select a calibration set (63 peptides) from the available samples. This set was used to build the quantitative structure-retention relationship models. The remaining 27 peptides were used as independent external test set to evaluate the predictive power of the constructed models. The UVE-PLS model consists of 5 components only (compared to 7 components in the best PLS model), and has the best predictive properties, i.e., the average error on the retention time is less than 30 s. When compared also to stepwise regression and an empirical model, the obtained UVE-PLS model leads to better and much better predictions, respectively.

Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography

A new approach to high speed, comprehensive online dual gradient elution 2DLC (LC × LC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the “analytic dynamic range” problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.

Possibility of Calculating System Maps Using Gradient Elution Reversed-Phase Liquid Chromatography

Gradient elution is explored as a rapid method for calculating system constants of the solvation parameter model for reversed-phase liquid chromatography. Linear solvent-strength theory is used to relate gradient retention times to system properties and to predict isocratic retention factors for the calculation of system maps. The combination of linear solvent strength theory and the solvation parameter model provides only a poor model for gradient retention times. A significant source of uncertainty in these models is the variation of the solute-dependent ratio (column hold-up time / gradient steepness parameter), and to a lesser extent, non-linearity in the plots of retention factors (log k) against mobile phase composition. Reasonable agreement between gradient predicted and experimental isocaratic retention factors was obtained when plots of the isocratic retention factors (log k) against mobile phase composition are linear. But even in this case, small systematic differences in the calculated system constants with increasing volume fraction of the strong solvent were observed. When plots of the isocratic retention factors against mobile phase composition are non-linear, the retention factors estimated by linear solvent strength theory yield system constants that contain significant error, producing fictitious system maps. Consequently, none of the methods studied is recommended for the fast and reliable calculation of system maps or solute descriptors by gradient elution reversed-phase liquid chromatography.

Determination of phthalates in water samples using polystyrene solid-phase extraction and liquid chromatography quantification

A method for the determination of butylbenzylphthalate (BBP) and di-(2-ethylhexyl)phthalate (DEHP) in water samples has been developed and validated. The phthalates were extracted from water samples using solid-phase extraction on polystyrene columns. Quantification was achieved by gradient elution reversed-phase liquid chromatography with UV-detection at 280 nm. The method has been validated for BBP and DEHP in the concentration range of 1–200 and 0.1–10 μg l −1, respectively. The internal standards were dibutylphthalate (DBP) and di- n-octylphthalate (DnOP). The within-day repeatability of BBP and DEHP were 0.5–1.5 and 2.0–6.5% relative standard deviation (RSD), respectively. The accuracy determined as recovery was 99–104% for both compounds. However, DEHP can only be determined with high accuracy in the range 0.1–3 μg l −1. The solubility of DEHP appears to be the limiting factor. The between-day repeatability was 1.1 and 4.2% RSD at the 1 μg l −1 level for BBP and DEHP, respectively. The concentration limits of detection were 0.05 (BBP) and 0.1 (DEHP) μg l −1.

Simultaneous determination of 5′-deoxy-5-fluorouridine, 5-fluorouracil and 5,6-dihydro-5-fluorouracil in serum by liquid chromatography with diode array UV detection

5,6-Dihydro-5-fluorouracil (5-FUH 2), the main catabolite of the anticancer drug 5-fluorouracil (5-FU), can be quantitated in human serum simultaneously with the pro-drug 5′-deoxy-5-fluorouridine (5′-dFUR), the parent drug 5-FU and the anabolites 5-fluorouridine and 2′-deoxy-5-fluorouridine by gradient elution-reversed phase liquid chromatography coupled to diode array UV detection. The analytes were extracted (after protein precipitation with ammonium sulphate) with ethyl acetate-isopropanol (90:10, v/v). The average recovery for 5-FUH 2 was 78.0% at 0.5 μg/ml level. 5-FUH 2 response was linear over three concentration decades and its detection limit was 200 ng/ml of serum. The analytical procedure has been used for simultaneous monitoring of 5′-dFUR, 5-FU and 5-FUH 2 levels in the serum of a patient under 5′-dFUR chemotherapy.

Reduction of reequilibration time following gradient elution reversed-phase liquid chromatography

A simple and convenient method for the reduction of column reequilibration time following gradient elution reversed-phase liquid chromatography is described. This method utilizes the addition of a constant volume of 3% 1-propanol to the mobile phase throughout the solvent gradient to provide consistent solvation of the reversed-phase stationary phase. Reductions in reequilibration time of up to 78% have been observed. The effect of alkyl chain bonding density on reequilibration volume is also examined. A maximum in the mobile phase volume necessary to reequilibrate the column is found at a bonding density of about 2.9 mumol/m2. The relationship of reequilibration volume to bonding density supports the partitioning model of retention for reversed-phase liquid chromatography.

Continuous infrared spectroscopic analysis of isocratic and gradient elution reversed-phase liquid chromatography separations

Utilisation de la methode (HPLC-IR en continu) avec des solutions aqueuses, en utilisant un nouveau nebuliseur capable de deposer et d'eliminer rapidement la phase mobile