Gradient Reversed-phase Liquid Chromatography Research Articles

On the fractionation of lignin oligomers by stepwise gradient reversed-phase liquid chromatography

With the increased interest in lignin valorization, the analytical challenge to separate a complex mixture of a vast number of phenolics has made chromatography an indispensable step in lignin analysis. High-resolution separations, such as gas chromatography, reversed-phase liquid chromatography and supercritical fluid chromatography have typically been targeting low-molecular-weight compounds, while larger lignin oligomers have received less attention. These compounds have proven to be difficult to separate due to the inherent complexity of the high-molecular-weight fraction of lignins, in fact, even high-resolving linear reversed-phase gradients elute them as one wide zone. To tackle this, in this study we show that a crude fractionation of lignin oligomers can be achieved by applying stepwise reversed-phase gradients. A commonly employed reversed-phase system with water:acetonitrile mobile phase is evaluated for this task. Special attention was devoted to uncovering the molecular level explanation of the retention phenomenon. Our results indicate that separation is mainly governed by reversed-phase retention phenomena without any major exclusion or viscosity-related effects, shown by great fits to linear retention models (R2avg = 0.9599 for five different oligomers) and apparent differences in retentivity between different stationary phases. The influence of the gradient shape was demonstrated by the comparison of stepwise gradients with different number and frequency of steps, leading to the conclusion that gradients with a low number of steps yield fewer, but better resolved fractions, while finer multi-step gradients can be used to distinguish more fractions.

Anomalous Retention Prediction Using Modelling Software in Gradient Reversed-Phase Liquid Chromatography: Why it Can Occur and How to Prevent It

Anomalous Retention Prediction Using Modelling Software in Gradient Reversed-Phase Liquid Chromatography: Why it Can Occur and How to Prevent It

Identification and quantification of polymeric impurity in block copolymer by one-dimensional and two-dimensional liquid chromatography coupled to high-resolution mass spectrometry and evaporative light scattering detector

Identifying and quantifying polymeric impurities in a polymeric material is critical for understanding material quality and performance, but it remains a challenge requiring developing new characterization methods. In this work, a comprehensive two-dimensional liquid chromatography method with simultaneous evaporative light scattering and high-resolution mass spectrometry detection was developed to separate and identify a polymeric impurity in alkyl alcohol-initiated polyethylene oxide/polybutylene oxide diblock copolymer. Size exclusion chromatography was implemented in the first dimension, and gradient reversed-phase liquid chromatography using a large-pore C4 column was applied in the second dimension using an active solvent modulation valve as the interface to minimize the polymer breakthrough. The two-dimensional separation significantly reduced the complexity of the mass spectra data compared to the one-dimensional separation, and the combination of retention time and mass spectral interpretation led to the successful identification of the water-initiated triblock copolymer impurity. This identification was confirmed by comparison with the synthesized triblock copolymer reference material. A one-dimensional LC method with evaporative light scattering detection was employed to quantify the triblock impurity. The impurity level in three samples made with the different processes was determined to be in the range of 9–18 wt% using the triblock reference material as the standard.

A comprehensive investigation of the peak capacity for the reversed-phase gradient liquid-chromatographic analysis of intact proteins using a polymer-monolithic capillary column

The present study reports on the analysis of different factors affecting the magnitude of the peak capacity for intact protein separations conducted in gradient reversed-phase liquid chromatography. Experiments were conducted using a 200 µm i.d. capillary styrene-co-divinylbenzene monolithic column that was developed in-house and was characterized by a mode globule cluster size of 1.2 µm and a mode macropore size of 1.0 µm (based on scanning electron microscopy). The monolith yielded a minimum plate-height value of 13.3 µm for uracil. The use of trifluoroacetic acid instead of formic acid as ion-pairing agent generally led to better peak symmetry, narrower peak widths which effect is protein-dependent, and improved loadability characteristics. The peak capacity has been systematically assessed at different flow rates and gradient duration. The highest peak capacity of 247 was obtained at a flow rate of 1 µL min−1 and a gradient time of 120 min, which corresponds to an optimal tG/t0 ratio of ∼60. While the optimum van Deemter velocity for intact proteins was approximated to be 0.065 µL min−1, the highest peak capacity was achieved at approximately 20-fold higher flow rate, depending on the gradient duration applied and the molecular weight of the proteins. The optimum velocity increased with decreasing gradient time and is a compromise between the magnitude of the mass-transfer contribution (decreasing the peak capacity with velocity) affected by molecular diffusion, and the increase in peak capacity induced by the more favorable gradient-volume ratio.

Two-dimensional liquid chromatography with active solvent modulation for studying monomer incorporation in copolymer dispersants.

A pseudo-comprehensive two-dimensional liquid chromatography approach with size exclusion chromatography in the first dimension and gradient reversed-phase liquid chromatography in the second dimension was successfully developed for the characterization of vinyl acetate/acrylic acid copolymers and vinyl acetate/itaconic acid/acrylic acid terpolymers. Active solvent modulation was exploited to prevent the polymer breakthrough in the second dimension separation caused by the strong solvent used in the first dimension. The conditions of the active solvent modulation valve were optimized to achieve sufficient on-line dilution and to completely prevent polymer breakthrough without adding excessive time to the modulation cycle. Using this approach, copolymers made with different monomer ratios and processes were studied. Heterogeneous composition distribution due to insufficient monomer incorporation was detected in some of the copolymer samples. We demonstrated that with active solvent modulation, the two-dimensional liquid chromatography approach is no longer limited to water-soluble polymers and can be used for a broader range of polymers and copolymers.

Analysis of hydrophobically modified ethylene oxide urethane rheology modifiers by comprehensive two dimensional liquid chromatography

Hydrophobically modified ethylene oxide urethane (HEUR) rheology modifiers are polymers used widely in waterborne coatings and other consumer products. The incorporation of hydrophobic end-groups significantly affects the thickening performance of HEURs. Therefore, understanding the degree of incorporation of hydrophobic end-groups on the polymer chain can help gain important insights on the structure/property relationship for HEUR rheology modifiers.A comprehensive two-dimensional liquid chromatography (2D-LC) method was developed for separating polymeric HEUR rheology modifiers based on the number of hydrophobic end-groups incorporated on the polymer chain. Our 2D-LC method uses a size exclusion chromatography (SEC) separation in the first dimension (1D) using an ultra-high performance Advance Polymer Chromatography (APC) column and a gradient reversed-phase liquid chromatography (RPLC) separation in the second dimension (2D). A low flow rate was used in the first dimension SEC separation to minimize the strong organic eluent transferred to the second dimension LC column. A small inner diameter (ID) LC column was used for the second dimension to reduce the total solvent consumption for routine analysis of process samples. The 1D flow rate, 2D transfer volume and gradient profile were all optimized to avoid polymer breakthrough and to achieve optimal separation of the polymer chains based on end-groups. Three types of polymer chains with 0, 1, and 2 terminal hydrophobes were successfully separated using this method. The total run time was further reduced by implementing flow programing in the first dimension separation. The 2D-LC method was applied to study how the polymerization process recipes affected hydrophobe incorporation and how the hydrophobe incorporation correlated with thickening efficiency. The method was also used to separate HEUR rheology modifier with branched polymer architecture.

Determination of reversed-phase high performance liquid chromatography based octanol-water partition coefficients for neutral and ionizable compounds: Methodology evaluation

Reversed-phase liquid chromatography (RPLC) based octanol-water partition coefficient (logP) or distribution coefficient (logD) determination methods were revisited and assessed comprehensively. Classic isocratic and some gradient RPLC methods were conducted and evaluated for neutral, weak acid and basic compounds. Different lipophilicity indexes in logP or logD determination were discussed in detail, including the retention factor logkw corresponding to neat water as mobile phase extrapolated via linear solvent strength (LSS) model from isocratic runs and calculated with software from gradient runs, the chromatographic hydrophobicity index (CHI), apparent gradient capacity factor (kg’) and gradient retention time (tg). Among the lipophilicity indexes discussed, logkw from whether isocratic or gradient elution methods best correlated with logP or logD. Therefore logkw is recommended as the preferred lipophilicity index for logP or logD determination. logkw easily calculated from methanol gradient runs might be the main candidate to replace logkw calculated from classic isocratic run as the ideal lipophilicity index. These revisited RPLC methods were not applicable for strongly ionized compounds that are hardly ion-suppressed. A previously reported imperfect ion-pair RPLC method was attempted and further explored for studying distribution coefficients (logD) of sulfonic acids that totally ionized in the mobile phase. Notably, experimental logD values of sulfonic acids were given for the first time. The IP-RPLC method provided a distinct way to explore logD values of ionized compounds.

Determination of the serine palmitoyl transferase inhibitor myriocin by electrospray and Q-trap mass spectrometry.

Myriocin is a potent inhibitor of serine-palmitoyl-transferase, the first and rate-determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC-MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14-OH-myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid-phase extraction, separated by gradient reversed-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14-OH-myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r2 = 0.9996). The intra- and between-day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra-tracheal delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n = 4) to 11.7 (median; 7.6-22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.

Photodegradation of aqueous argatroban investigated by LC/MSn: Photoproducts, transformation processes and potential implications

Argatroban (ARGA), used as intravenous anticoagulant drug, has been reported to photodegrade under light exposure, requiring specific precautions at handling, storage and administration. Thus, for the first time, aqueous ARGA photodegradation under aerobic conditions has been described in terms of photoproducts, phototransformation processes and potential implications. ARGA significant photoproducts were successfully separated and characterized by gradient reversed-phase liquid chromatography coupled with high-resolution multistage mass spectrometry (LC/HR–MSn). Hitherto still not available in literature, ARGA in-depth fragmentation study was conducted so as to thoroughly sort out the main mechanisms specific to the molecule and therefore, to propose a fragmentation pattern relevant to the identification of ARGA related substances. Thereafter, in view of the structural characteristics of the photoproducts formed, ARGA photodegradation pathways could be worked out, showing that whether by direct photolysis or through photosensitization, the methyltetrahydroquinoline nitrogen and that of guanidine group would be mainly involved in photolysis initiation reactions, through one-electron oxidation along with proton loss. Desulfonation, cyclisation affording compounds of diazinane type, and/or rearrangements with transfer of the methyltetrahydroquinoline group toward the guanidine function were observed accordingly. Having a good insight into ARGA photodegradation pathways allows for consistent measures in view of mitigating or avoiding the drug decay and the related potential effects.

Efficiency of a solvatic sorption model for the prediction of retention times in linear gradient reversed-phase liquid chromatography working with different stationary phases

Efficiency of a solvatic sorption model for the prediction of retention times in linear gradient reversed-phase liquid chromatography working with different stationary phases

Characterization and fractionation of PS-b-PMMA diblock copolymer synthesized via click chemistry

Detailed molecular characterization and fractionation were carried out for a polystyrene-block-poly(methylmethacrylate) (PS-b-PMMA) diblock copolymer prepared by linking PS-alkyne and PMMA-azide through a click reaction. PS and PMMA block precursors were prepared by atom transfer radical polymerization and anionic polymerization, and end-functionalized to have alkyne and azide group, respectively. As-synthesized block copolymer was characterized by solvent gradient reversed-phase liquid chromatography (RPLC) with multiple-detection for both separation and identification of byproducts. As-synthesized PS-b-PMMA contains various byproducts including unreacted precursor homo-polymers as well as byproducts formed during the synthesis of precursor polymers. To fractionate pure PS-b-PMMA out, two-step HPLC fractionation was employed. In the first step, PMMA homo-polymer was removed from the as-synthesized polymer using a C18 bonded silica column. In the second step, PS homo-polymer was removed to obtain pure PS-b-PMMA using a bare silica column. In both steps, multiple-injection method was used to facilitate the large scale fractionation procedure. A two-dimensional liquid chromatography method, solvent gradient RPLC for the first dimension and SEC for the second dimension was also conducted to map the molecular characteristics of the block copolymer and its byproducts.

Gradient liquid chromatographic retention time prediction for suspect screening applications: A critical assessment of a generalised artificial neural network-based approach across 10 multi-residue reversed-phase analytical methods

For the first time, the performance of a generalised artificial neural network (ANN) approach for the prediction of 2492 chromatographic retention times (tR) is presented for a total of 1117 chemically diverse compounds present in a range of complex matrices and across 10 gradient reversed-phase liquid chromatography-(high resolution) mass spectrometry methods. Probabilistic, generalised regression, radial basis function as well as 2- and 3-layer multilayer perceptron-type neural networks were investigated to determine the most robust and accurate model for this purpose. Multi-layer perceptrons most frequently yielded the best correlations in 8 out of 10 methods. Averaged correlations of predicted versus measured tR across all methods were R2=0.918, 0.924 and 0.898 for the training, verification and test sets respectively. Predictions of blind test compounds (n=8–84 cases) resulted in an average absolute accuracy of 1.02±0.54min for all methods. Within this variation, absolute accuracy was observed to marginally improve for shorter runtimes, but was found to be relatively consistent with respect to analyte retention ranges (~5%). Finally, optimised and replicated network dependency on molecular descriptor data is presented and critically discussed across all methods. Overall, ANNs were considered especially suitable for suspects screening applications and could potentially be utilised in bracketed-type analyses in combination with high resolution mass spectrometry.

Suspect screening of large numbers of emerging contaminants in environmental waters using artificial neural networks for chromatographic retention time prediction and high resolution mass spectrometry data analysis

The recent development of broad-scope high resolution mass spectrometry (HRMS) screening methods has resulted in a much improved capability for new compound identification in environmental samples. However, positive identifications at the ng/L concentration level rely on analytical reference standards for chromatographic retention time (tR) and mass spectral comparisons. Chromatographic tR prediction can play a role in increasing confidence in suspect screening efforts for new compounds in the environment, especially when standards are not available, but reliable methods are lacking. The current work focuses on the development of artificial neural networks (ANNs) for tR prediction in gradient reversed-phase liquid chromatography and applied along with HRMS data to suspect screening of wastewater and environmental surface water samples. Based on a compound tR dataset of >500 compounds, an optimized 4-layer back-propagation multi-layer perceptron model enabled predictions for 85% of all compounds to within 2min of their measured tR for training (n=344) and verification (n=100) datasets. To evaluate the ANN ability for generalization to new data, the model was further tested using 100 randomly selected compounds and revealed 95% prediction accuracy within the 2-minute elution interval. Given the increasing concern on the presence of drug metabolites and other transformation products (TPs) in the aquatic environment, the model was applied along with HRMS data for preliminary identification of pharmaceutically-related compounds in real samples. Examples of compounds where reference standards were subsequently acquired and later confirmed are also presented. To our knowledge, this work presents for the first time, the successful application of an accurate retention time predictor and HRMS data-mining using the largest number of compounds to preliminarily identify new or emerging contaminants in wastewater and surface waters.

Wide injection zone compression in gradient reversed-phase liquid chromatography

Chromatographic zone broadening is a common issue in microfluidic chromatography, where the sample volume introduced on column often exceeds the column void volume. To better understand the propagation of wide chromatographic zones on a separation device, a series of MS Excel spreadsheets were developed to simulate the process. To computationally simplify these simulations, we investigated the effects of injection related zone broadening and its gradient related zone compression by tracking only the movements of zone boundaries on column. The effects of sample volume, sample solvent, gradient slope, and column length on zone broadening were evaluated and compared to experiments performed on 0.32mm I.D. microfluidic columns. The repetitive injection method (RIM) was implemented to generate experimental chromatograms where large sample volume scenarios can be emulated by injecting two discrete small injection plugs spaced in time. A good match between predicted and experimental RIM chromatograms was observed. We discuss the performance of selected retention models on the accuracy of predictions and use the developed spreadsheets for illustration of gradient zone focusing for both small molecules and peptides.

Application of screening experimental designs to assess chromatographic isotope effect upon isotope-coded derivatization for quantitative liquid chromatography-mass spectrometry.

Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography–mass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, 13C6-, 15N2-, or 15N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and Plackett–Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of 15N or 13C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus 15N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with 15N- or 13C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects.

Prediction of Retention in Gradient Reversed-Phase Liquid Chromatography for Phenyl isothiocyanate-Derivatives of Amino Acids

Aim: To study the applicability of the solvatic retention model for the prediction of retention times in gradient elution of phenylisothiocyanate derivatives of natural amino acids. Methodology: The solvatic retention model of reversed-phase liquid chromatography has been used to predict retention of phenylisothiocyanate derivatives of 25 natural amino acids under conditions of gradient elution. Retention factors have been calculated from molecular parameters of the structures of analytes and the characteristics of a column and an eluent using the ChromSword software package. Results: The modeled initial estimation of retention time was found to be within 4% of the experimentally determined retention time. Fine-tuning of the model parameters using data from experimental runs further improved the accuracy of the model. Conclusion: A step-by-step method which includes the first-guess prediction of initial conditions from structural formula and fine tuning parameters of the retention model using data from successive runs can save time in method development and optimization of the Research Article American Chemical Science Journal, 4(1): 14-23, 2014 15 separation of target compounds.

Stability-indicating LC method for the determination of cephalothin in lyophilized powder for injection

A stability-indicating gradient reversed phase liquid chromatography (RP-LC) method has been developed for the quantitative determination of cephalothin (CET), an antimicrobial compound, in the presence of its impurities and degradation products generated from forced degradation studies.

Approaches to model the retention and peak profile in linear gradient reversed-phase liquid chromatography

The optimisation of the experimental conditions in gradient reversed-phase liquid chromatography requires reliable algorithms for the description of the retention and peak profile. As in isocratic elution, the linear relationship between the logarithm of the retention factor and the solvent contents is only acceptable in relatively small concentration ranges of modifier. However, more complex models may not allow an analytical integration of the general equation for gradient elution. Alternative approaches for modelling the retention in linear gradient elution are here proposed. Those based on the quadratic logarithmic model and a model proposed for normal liquid chromatography yielded accurate predictions of the retention time for a wide range of initial concentrations of organic modifier and gradient slopes, with errors usually below 1–2%. Based on the half-width changes of chromatographic peaks along one or more gradients, an approach is also reported to predict the peak profile with low errors (usually below 2−3%). The proposed approaches were applied to two sets of probe compounds (diuretics and flavonoids), eluted with acetonitrile–water gradients. The changes in retention and peak shape in isocratic and gradient elution are illustrated through diagrams that define triangular regions including all possible values of retention factors or peak half-widths (or widths) inside the selected working ranges.

Assessment of recombinant human parathyroid hormone: correlation of LC methods with bioassays

Reversed-phase liquid chromatography (RP-LC) and size exclusion liquid chromatography (SE-LC) methods were validated for the assessment of recombinant human parathyroid hormone (rhPTH 1-34). The gradient RP-LC method was carried out on a Zorbax 300 SB C(18) column (150 mm × 4.6 mm i.d.), maintained at 40 °C. The mobile phase A consisted of 0.1 M sodium sulphate buffer, pH 2.3, and the mobile phase B was acetonitrile. The SE-LC method was carried out on a BioSep-SEC-S 2000 column (300 mm × 7.8 mm i.d.), maintained at 25 °C. The mobile phase consisted of 0.1 M phosphoric acid buffer, pH 2.5, run isocratically at a flow rate of 0.7 mL min(-1). Chromatographic separation was obtained with retention times of 12.2 min, and 13.2 min, and was linear over the concentration range of 1-250 μg mL(-1) (r(2) = 0.9997) and 2-300 μg mL(-1) (r(2) = 0.9993), respectively, for RP-LC and SE-LC, with photodiode array (PDA) detection at 214 nm. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.49% and 100.22%, with bias lower than 1.12% and 0.81% respectively. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p < 0.05). Chromatographic methods were applied for the content/potency assessment of rhPTH and related proteins in biopharmaceutical injectable dosage forms, and the results were correlated with those of in vitro and in vivo bioassays. It is concluded that the employment of the methods in conjunction allows a great improvement in monitoring stability, contributing to evaluate alternatives which improve the quality control and thereby assure the therapeutic efficacy of the biotechnology-derived medicine.

Evaluation of mobile phase gradient supercritical fluid chromatography for impurity profiling of pharmaceutical compounds

The use of gradient supercritical fluid chromatography (SFC) for the impurity profiling of pharmaceutical products is not widely practiced. Historically, the limited advancement in SFC instrumentation and the lag in column development have resulted in marginal sensitivity, selectivity and reproducibility when compared with high performance liquid chromatography (HPLC). Using a recently developed commercial module, which allows an ordinary HPLC to be converted to a SFC system, a significant improvement in sensitivity (up to ~12-fold) has been obtained over previous studies. This has allowed for the first time a "real-world" head-to-head comparison of SFC to HPLC for impurity profiling of pharmaceutical products in a regulated environment. Retention time reproducibility and low level impurity detection were found to be comparable to reversed phase liquid chromatography (RPLC), that is, single digit %relative standard deviations (RSDs) were obtained for impurities present at less than 0.1 area%. Furthermore, these results were obtained with drug loading levels (≤2 mg/mL) that are not only comparable to those employed with HPLC, but are dictated by the limited solubility of many drug candidates. The elution of impurities was generally found to be orthogonal to that obtained with RPLC, but it was still challenging to find SFC conditions that would separate all of the components in the mixtures studied. In terms of enhancing selectivity, small amounts of mobile phase additives (0.1-1%) and temperature optimization were found to have a greater impact in SFC method development versus RPLC. However, unlike gradient RPLC, the relative changes in baseline noise and slope were found to be a complex function of the experimental conditions, with the largest differences in noise levels being generally observed for the widest and steepest gradients. It is likely that this gradient related noise is more apparent now because other sources of noise in SFC have been reduced significantly.