Gradient Elution In Liquid Chromatography Research Articles

A protocol for fabrication of polymer monolithic capillary columns and tuning the morphology targeting high-resolution bioanalysis in gradient-elution liquid chromatography.

Polymer monolithic stationary phases are designed as a continuous interconnected globular material perfused by macropores. Like packed column, where separation efficiency is related to particle diameter, the efficiency of monoliths can be enhanced by tuning the size of both the microglobules and macropores. This protocol described the synthesis of poly(styrene-co-divinylbenzene) monolithic stationary phases in capillary column formats. Moreover, guidelines are provided to tune the macropore structure targeting high-throughput and high-resolution monolith chromatography. The versatility of these columns is exemplified by their ability to separate tryptic digests, intact proteins, and oligonucleotides under a variety of chromatographic conditions. The repeatability of the presented column fabrication process is demonstrated by the successful creation of 12 columns in three different column batches, as evidenced by the consistency of retention times (coefficients of variance [c.v.]=0.9%), peak widths (c.v.=4.7%), and column pressures (c.v.=3.1%) across the batches.

Capacitively coupled contactless conductivity detection to account for system-induced gradient deformation in liquid chromatography

The time required for method development in gradient-elution liquid chromatography (LC) may be reduced by using an empirical modelling approach to describe and predict analyte retention and peak width. However, prediction accuracy is impaired by system-induced gradient deformation, which can be especially prominent for steep gradients. As the deformation is unique to each LC instrument, it needs to be corrected for if retention modelling for optimization and method transfer is to become generally applicable. Such a correction requires knowledge of the actual gradient profile. The latter has been measured using capacitively coupled “contactless” conductivity detection (C4D), featuring a low detection volume (approximately 0.05 μL) and compatibility with very high pressures (80 MPa or more). Several different solvent gradients, from water to acetonitrile, water to methanol, and acetonitrile to tetrahydrofuran, could be measured directly without the addition of a tracer component to the mobile phase, exemplifying the universal nature of the approach. Gradient profiles were found to be unique for each solvent combination, flowrate, and gradient duration. The profiles could be described by convoluting the programmed gradient with a weighted sum of two distribution functions. Knowledge of the exact profiles was used to improve the inter-system transferability of retention models for toluene, anthracene, phenol, emodin, sudan-I and several polystyrene standards.

Peak width in liquid chromatography with exponential retention and linear program preceded by isocratic hold

Expression for peak width in gradient elution liquid chromatography with exponential dependence of solute retention on linearly programmed solvent composition preceded by initial isocratic hold has been derived. A special case of previously defined balanced hold has been considered and compared with published results.

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.

Development and validation of apalutamide-related substances method in solid dosage forms using HPLC.

A related-substances method was developed for the anticancer drug formulation apalutamide 60 mg tablets and validated using a liquid chromatography gradient elution method. All of the impurities and degradants were separated using the Luna Omega 5μm Polar C18 , (250 × 4.6) mm HPLC column with a 1.0 ml min-1 flow rate. The detection was done at 225 nm by injecting the 10μl of injection volume, controlling the sample temperature at 10°C and maintaining the column compartment temperature at 30°C. The total run time was 85 min. A 0.01m disodium phosphate dihydrate pH4.20 ± 0.05 buffer mixed with acetonitrile in the ratio of 73:27 (v/v) was used as mobile phase A. Mobile phase B consisted of water and acetonitrile in the ratio 30:70 (v/v). The proposed method was validated as per the current regulatory guidelines. The method precisions (RSD) at 100% specification level were 1.41, 1.74, 1.84, and 1.66% for the four impurities. The accuracy results were obtained between 96.0 and 106.3% for the limit of quantitation to the 150% level. The standard and sample solutions stability were established for 44 h at 10°C. The correlation coefficient (r) value was >0.999 for all four impurities, indicating good linearity between the concentration and peak response: 0.9999, 0.9999, 0.9999 and 1.0000. These results show the method's linearity. The three filter compatibility was proved and it was concluded that 0.45 μm Nylon, PTFE and PVDF filters are suitable. The robustness of the method was established by varying the conditions. The method specificity was proved and the forced degradation data reveal the method's stability-indicating nature.

Recycling gradient-elution liquid chromatography for the analysis of chemical-composition distributions of polymers

Synthetic polymers typically show dispersity in molecular weight and potentially in chemical composition. For the analysis of the chemical-composition distribution (CCD) gradient liquid chromatography may be used. The CCD obtained using this method is often convoluted with an underlying molecular-weight distribution (MWD). In this paper we demonstrate that the influence of the MWD can be reduced using very steep gradients and that such gradients are best realized utilizing recycling gradient liquid chromatography (LC↻LC). This method allows for a more-accurate determination of the CCD and the assessment of (approximate) critical conditions (if these exist), even when high-molecular-weight standards of narrow dispersity are not readily available. The performance and usefulness of the approach is demonstrated for several polystyrene standards, and for the separation of statistical copolymers consisting of styrene/methyl methacrylate and methyl methacrylate/butyl methacrylate. For the latter case, approximate critical compositions of the copolymers were calculated from the critical compositions of two homopolymers and one copolymer of known chemical composition, allowing for a determination of the CCD of unknown samples. Using this approach it is shown that the copolymers elute significantly closer to the predicted critical compositions after recycling of the gradient. This is most clear for the lowest-molecular-weight copolymer (Mw = 4.2 kDa), for which the difference between measured and predicted elution composition decreases from 7.9% without recycling to 1.4% after recycling.

Computer aided optimization of multilinear gradient elution in liquid chromatography

Analytical expressions for retention time and peak compression factor are deduced by assuming quadratic solvent strength model and multilinear gradient elution. Based on these expressions, a program for the optimization of multilinear gradient profile is written with Visual Basic for Applications in Excel using genetic algorithm. The program is applied to search for a gradient profile for the separation of twelve compounds that are degraded from lignin. It is shown that the predicted and experimental chromatograms are well consistent. A better separation of the compounds is achieved under an S-shaped multilinear gradient profile than that obtained under linear gradient profile.

Analysis of gradient elution chromatography using the transport model

A transport model is considered to describe gradient elution in liquid chromatography in packed beds with the linear isotherm dependent on the mobile phase modulator. By applying a coordinate transformation, the model is solved analytically using the Laplace transform approach. The moment generating property of the Laplace domain solution is used to derive analytical expressions for the first three moments of the response to rectangular injections. These moments are instructive for analyzing the retention time, band broadening and asymmetry of elution profiles. Compared to isocratic elution, the derivation of analytical solutions and moments for gradient elution is more complicated, because the retention behavior of the solutes depends on the varying mobile phase modulator. Several case studies are evaluated theoretically. To gain confidence on the derived analytical results, a high-resolution finite volume scheme is also applied to solve the same model equations numerically. The analytical solutions and moments provided are utilized to predict the effects of starting and ending times of gradient, magnitude of modulator concentration variation, gradient slopes, and mass transfer coefficient on retention and peak shape. The analytical moment expressions derived can be used to determine retention and mass transfer parameters from experimental peaks and to predict elution behaviors if these parameters are known.

Peak compression in linear gradient elution liquid chromatography

An expression for peak compression factor based on temporal peak widths is proposed which can be measured directly. The relationship between it and peak compression factor based on spatial peak widths is given. The experimental data presented in the Neue's work are reevaluated. The peak compression factor is recalculated by taking into account the curvature in the plot of logarithmic retention factor (lnk) vs. mobile phase composition (φ) and the variation in plate height (H) with φ. The lnk vs. φ plot is accounted for by quadratic solvent strength model (QSSM). The lnH vs. φ plot is accounted for by QSSM and a quadratic equation that accounts for the plot of logarithmic peak width obtained under isocratic elution (lnWI) vs. φ. By taking these two factors into account, the discrepancy between experimental and predicted values of peak width is reduced. This result confirms the Neue's conclusion that the anomalous peak broadening reported previously may be an artifact of the procedure to determine the theoretical value of peak width. Moreover, by assuming linear gradient elution, constant column efficiency, and general solvent strength, the relationship between peak compression factors obtained under ideal and real situation respectively is discussed.

A linear mass concentration detector for solvent gradient polymer separations

Characterization of copolymers requires the measurement of two distributions-molecular weight (MW) and chemical composition (CC). Molecular weight distributions (MWD) are traditionally determined using size exclusion chromatography (SEC) run under isocratic solvent conditions. Chemical composition distributions (CCD) are often determined using liquid adsorption chromatography (LC) with solvent gradients. The use of solvent gradients, however, often limits options of compatible detectors. A gradient compatible, universal linear mass concentration detector is a longstanding unmet need. Many industrially-relevant polymers lack chromophores or other discriminating moieties requiring detectors with a universal response. Differential refractive index (dRI) is incompatible with gradient elution due to its small dynamic range. Charged aerosol detectors (CAD) and evaporative light scattering detectors (ELSD) are probably the most promising options for gradient elution detection, but both suffer from a nonlinear mass concentration response. Silicon photonic microring resonators are optical sensors that are responsive to changes in the local refractive index (RI). The substantial dynamic range of this technology makes it attractive for refractive index-based detection during solvent gradient elution. Previously, the microring resonator platform was used as a SEC detector to characterize the MWD of broadly dispersed polystyrene (PS) standards. In this study, we demonstrate the gradient compatibility of the microring resonator platform for polymer detection by quantifying the CCD of polymer blend components. Control experiments were run with UV and ELSD detection, highlighting the uniqueness of the platform as a linear mass concentration detector with a universal detector response.

Peak dispersion in gradient elution: An insight based on the plate model

Gradient elution in liquid chromatography reduces the analysis time, improves the efficiency and increases the peak capacity. The study of this chromatographic mode has been based mainly on kinetic dispersion models. The plate model has been applied to a lesser extent, despite being the basis for the concepts of plate height and chromatographic efficiency. In this work, a general equation describing peak dispersion in HPLC gradient elution is derived from the plate model. This equation is studied and validated for three types of gradients: (i) a reference gradient without ramp in which the retention factor varies with time identically throughout the column, (ii) a gradient of stationary phase in which the nature of the stationary phase varies continuously inside the column, resulting in a ramp of constant retention factor over time, and finally, (iii) a mobile phase gradient, which produces a retention factor ramp that varies over time. In the latter case, the results are similar to those derived from the mass-transport equation in linear solvent gradients when the linear solvent strength model is applied. The final equations are expressed based on the initial and final instantaneous retention factors, thus they can be applied independently of the deviation of the elution model, being fully compatible with isocratic elution. Results predicted with the proposed equations are identical to those obtained by numerical resolution of the elution differential equation system. The additional compression due to the presence of a ramp of modifier is also verified. However, useful compressions will appear only when the retention factor changes with time. Finally, the study indicates that the extra-column variance undergoes a compression process when the retention factor varies over time, whether or not there is a ramp inside the column.

Apparent efficiency of serially coupled columns in gradient elution liquid chromatography: Extension to the combination of any column formats

It was shown earlier that in serially coupled column systems, not only retention (selectivity) but peak width as well can change and the apparent peak width depends on the order of the columns. A generic equation has been developed to describe the apparent efficiency of serially coupled columns in gradient elution liquid chromatography mode. In a previous work, the theoretical basis for a two-column system was developed and by extending and generalizing this model, it is now possible to predict the apparent peak width for the combination of any column segments in any formats (columns possessing various efficiencies and lengths). (Identical retention and selectivity have been assumed to develop this model.) At the end, column order can be a useful parameter to set the desired efficiency of coupled systems.

Optimized purification of a fusion protein by reversed-phase high performance liquid chromatography informed by the linear solvent strength model

Fusion protein systems are commonly used for expression of small proteins and peptides. An important criterion for a fusion protein system to be useful is the ability to separate the protein of interest from the tag. Additionally, because no protease cleaves fusion proteins with 100% efficiency, the ability to separate the desired peptide from any remaining uncleaved protein is also necessary. This is likely to be the more difficult task as at least a portion of the sequence of the fusion protein is identical to that of the protein of interest. When a high level of purity is required, gradient elution reversed-phase HPLC is frequently used as a final purification step. Shallow gradients are often advantageous for maximizing both the purity and yield of the final product; however, the relationship between relative retention times at shallow gradients and those at steeper gradients typically used for analytical HPLC are not always straightforward. In this work, we report reversed-phase HPLC results for the fusion protein system consisting of the N-terminal domain of ribosomal protein L9 (NTL9) and the 36-residue villin headpiece subdomain (HP36) linked by a recognition sequence for the protease factor Xa. This system represents an excellent example of the difficulties in purification that may arise from this unexpected elution behavior at shallow gradients. Additionally, we report on the sensitivity of this elution behavior to the concentration of the additive trifluoroacetic acid in the mobile phase and present optimized conditions for separating HP36 from the full fusion protein by reversed-phase HPLC using a shallow gradient. Finally, we suggest that these findings are relevant to the purification of other fusion protein systems, for which similar problems may arise, and support this suggestion using insights from the linear solvent strength model of gradient elution liquid chromatography.

Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography-Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-μm Particles.

In recent years protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficult to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kspi range, accommodates the use of capillary columns in excess of one meter packed with sub-2 μm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at the higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.

Qualitative screening of absorbed indoloquinazoline alkaloids and their metabolites in rat plasma after the oral administration of Wu-Zhu-Yu decoction by high-resolution mass spectrometry with multiple data mining algorithms.

A rapid and sensitive liquid chromatography with high-resolution mass spectrometry method with multiple data processing algorithms was developed and applied for the metabolite profiling of evodiamine and its analogous alkaloids in rat plasma after the administration of Wu-Zhu-Yu decoction. All samples were purified using hydrophilic-lipophilic balanced solid-phase extraction cartridges and analyzed by a Sciex TripleTOF 5600(+) mass spectrometer with a 35 min liquid chromatography gradient elution. High-resolution full-scan mass spectrometry and information-dependent acquisition tandem mass spectrometry data were analyzed using multiple data processing approaches. The results indicated that the detected eight prototype alkaloids could be metabolized to 58 metabolites through both phase I and phase II reactions. Oxidation was demonstrated to be the principle metabolic pathway of the parent compounds. The study contributes to the understanding of the absorption and metabolism of the alkaloids in Wu-Zhu-Yu decoction and provides a detailed analysis of scientific data.

The HPLC Fingerprint Analysis of SelectedCirsiumSpecies with Aid of Chemometrics

Twelve Cirsium sp. methanolic extracts were analyzed using high-performance liquid chromatography gradient elution method with run time of 45 min. Four Kinetex (150 × 4.6 mm) chromatographic columns (C18 5 µm, C18 2.6 µm, pentafluorophenyl 5 µm, phenyl-hexyl 5 µm) and mobile phase consisting of methanol/water/formic acid 1% were used. Eight standards (naringin, vanilic acid, chlorogenic acid, caffeic acid, rutin, luteolin, apigenin, p-coumaric acid) were analyzed in the same conditions to confirm their presence in all of Cirsium methanolic extracts. The obtained chromatograms were compared and the similarity between them was evaluated using the similarity indices (Pearson's correlation coefficient, determination coefficient and congruence coefficient), distance indices (Euclidean, Manhattan and Chebyshev distance) and multi-scale structural similarity (MS-SSIM). Obtained results were confirmed using the principal component analysis (PCA). The attempt of identification of two unknown Cirsium species was performed using the similarity, distance indices and PCA analysis.

Quantitative structure–retention relationships applied to development of liquid chromatography gradient-elution method for the separation of sartans

QSRR are mathematically derived relationships between the chromatographic parameters determined for a representative series of analytes in given separation systems and the molecular descriptors accounting for the structural differences among the investigated analytes. Artificial neural network is a technique of data analysis, which sets out to emulate the human brain's way of working. The aim of the present work was to optimize separation of six angiotensin receptor antagonists, so-called sartans: losartan, valsartan, irbesartan, telmisartan, candesartan cilexetil and eprosartan in a gradient-elution HPLC method. For this purpose, ANN as a mathematical tool was used for establishing a QSRR model based on molecular descriptors of sartans and varied instrumental conditions. The optimized model can be further used for prediction of an external congener of sartans and analysis of the influence of the analyte structure, represented through molecular descriptors, on retention behaviour. Molecular descriptors included in modelling were electrostatic, geometrical and quantum-chemical descriptors: connolly solvent excluded volume non-1,4 van der Waals energy, octanol/water distribution coefficient, polarizability, number of proton-donor sites and number of proton-acceptor sites. Varied instrumental conditions were gradient time, buffer pH and buffer molarity. High prediction ability of the optimized network enabled complete separation of the analytes within the run time of 15.5min under following conditions: gradient time of 12.5min, buffer pH of 3.95 and buffer molarity of 25mM. Applied methodology showed the potential to predict retention behaviour of an external analyte with the properties within the training space. Connolly solvent excluded volume, polarizability and number of proton-acceptor sites appeared to be most influential paramateres on retention behaviour of the sartans.

Retention prediction and separation optimization under multilinear gradient elution in liquid chromatography with Microsoft Excel macros

A package of Excel VBA macros have been developed for modeling multilinear gradient retention data obtained in single or double gradient elution mode by changing organic modifier(s) content and/or eluent pH. For this purpose, ten chromatographic models were used and four methods were adopted for their application. The methods were based on (a) the analytical expression of the retention time, provided that this expression is available, (b) the retention times estimated using the Nikitas-Pappa approach, (c) the stepwise approximation, and (d) a simple numerical approximation involving the trapezoid rule for integration of the fundamental equation for gradient elution. For all these methods, Excel VBA macros have been written and implemented using two different platforms; the fitting and the optimization platform. The fitting platform calculates not only the adjustable parameters of the chromatographic models, but also the significance of these parameters and furthermore predicts the analyte elution times. The optimization platform determines the gradient conditions that lead to the optimum separation of a mixture of analytes by using the Solver evolutionary mode, provided that proper constraints are set in order to obtain the optimum gradient profile in the minimum gradient time. The performance of the two platforms was tested using experimental and artificial data. It was found that using the proposed spreadsheets, fitting, prediction, and optimization can be performed easily and effectively under all conditions. Overall, the best performance is exhibited by the analytical and Nikitas-Pappa's methods, although the former cannot be used under all circumstances.

Gradient elution liquid chromatography mass spectrometry determination of acetylcorynoline in rat plasma and its application to a pharmacokinetic study

1. Acetylcorynoline is the major alkaloid component derived from Corydalis bungeana herbs. A sensitive and selective liquid chromatography mass spectrometry method for determination of acetylcorynoline in rat plasma was developed over the range of 5–1000 ng/mL to characterize the pharmacokinetic properties.2. Chromatographic separation was achieved on a C18 (2.1 mm× 150 mm, 5 μm) column with acetonitrile 0.1% formic acid in water as mobile phase with gradient elution. The flow rate was set at 0.4 mL/min. After addition of carbamazepine as internal standard (IS), protein precipitation by acetonitrile–methanol (9:1, v/v) was used as sample preparation. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring mode was used for quantification using target ions m/z 410 for acetylcorynoline and m/z 237 for the IS.3. Mean recoveries of acetylcorynoline in rat plasma were in the range of 72.3–87.6%. Matrix effects for acetylcorynoline were measured to be between 88.7% and 93.5%. Coefficient of variation of intra-day and inter-day precision were both <13%. The accuracy of the method ranged from 95.8% to 112.1%. The analyte was stable under auto-sampler, room temperature, freeze-thaw and long-term (20 days), the bias in concentration was within ±15% of their nominal values.4. The LC-MS method for the determination of acetylcorynoline in rat plasma utilizing 100 µL of plasma with an LLOQ of 5.0 ng/mL developed and validated, it was sensitive, selective and simple. This method was successfully applied in pharmacokinetic study of acetylcorynoline after intravenous administration of single dosage 3 mg/kg in rats.

Refractive index-based detection of gradient elution liquid chromatography using chip-integrated microring resonator arrays.

Refractive index-based sensors offer attractive characteristics as nondestructive and universal detectors for liquid chromatographic separations, but a small dynamic range and sensitivity to minor thermal perturbations limit the utility of commercial RI detectors for many potential applications, especially those requiring the use of gradient elutions. As such, RI detectors find use almost exclusively in sample abundant, isocratic separations when interfaced with high-performance liquid chromatography. Silicon photonic microring resonators are refractive index-sensitive optical devices that feature good sensitivity and tremendous dynamic range. The large dynamic range of microring resonators allows the sensors to function across a wide spectrum of refractive indices, such as that encountered when moving from an aqueous to organic mobile phase during a gradient elution, a key analytical advantage not supported in commercial RI detectors. Microrings are easily configured into sensor arrays, and chip-integrated control microrings enable real-time corrections of thermal drift. Thermal controls allow for analyses at any temperature and, in the absence of rigorous temperature control, obviates extended detector equilibration wait times. Herein, proof of concept isocratic and gradient elution separations were performed using well-characterized model analytes (e.g., caffeine, ibuprofen) in both neat buffer and more complex sample matrices. These experiments demonstrate the ability of microring arrays to perform isocratic and gradient elutions under ambient conditions, avoiding two major limitations of commercial RI-based detectors and maintaining comparable bulk RI sensitivity. Further benefit may be realized in the future through selective surface functionalization to impart degrees of postcolumn (bio)molecular specificity at the detection phase of a separation. The chip-based and microscale nature of microring resonators also make it an attractive potential detection technology that could be integrated within lab-on-a-chip and microfluidic separation devices.