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• A model-based approach was shown for gradient elution liquid-liquid chromatography. • The elution profiles of the modifier and solutes were described with a stage model. • Only a few experiments were necessary for model parameter determination. • Pre-selected gradient shapes were optimized by maximizing productivity and yield. • The approach is applicable for systems with constant stationary phase composition. Liquid-liquid chromatography (LLC) is a separation technique that utilizes a biphasic solvent system as the mobile and stationary phases. The components are separated solely due to their different distributions between the two liquid phases. Gradient change in the mobile phase composition during the chromatographic process is a powerful method for improving the resolution of separation or shortening the process time. Gradient elution readily applies to LLC with biphasic solvent systems in which the stationary phase composition remains nearly constant when the mobile phase composition changes. This work proposes a model-based approach to optimize gradients in LLC and circumvent tedious trial-and-error experiments. The solutes' distribution constant depends on the mobile phase composition. Thus, the distribution constants were described as a function of the content of one of the solvents (= modifier) in the mobile phase. The dispersive and mass-transfer effects in the tubing and the column are modeled with a stage model. Only a few experiments are required to determine the model parameters. After the validation of the model and its parameters, the model can be used for LLC gradient optimization. The proposed approach was demonstrated for a gradient LLC separation of a mixture of four cannabinoids. Two different gradient shapes, one-step and linear gradient, were considered. For a pre-selected minimal purity requirement, the gradient was optimized for maximum process efficiency, defined as the product of productivity and yield. An experiment conducted with the optimized gradient conditions was in good agreement with the simulation, showing the potential of the proposed method. [ABSTRACT FROM AUTHOR]

• 11 free fatty acids were simultaneously determined by LC-MS/MS. • Microvolume serum pretreated by two-step derivatisation strategy. • High throughput analysis of samples can be achieved. • Method was developed and validated with good results. A stable isotope dilution–liquid chromatography–tandem mass spectrometry method based on a derivatisation strategy involving an N,N'-carbonylimidazole solution (CDI) with 4-(dimethylamino)-benzenemethanamine was developed for the determination of 11 free fatty acids (FFAs) in human blood samples. Serum samples were subjected to liquid‒liquid extraction and centrifuged, and the supernatant was collected for a two-step derivatisation reaction with a CDI and 4-(dimethylamino)-aniline acetonitrile solution. The derivatised solution was separated on a ACQUITY UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) column with a mobile phase consisting of water–acetonitrile in gradient elution and then detected by tandem mass spectrometry using electrospray ionisation (ESI) and multiple reaction monitoring (MRM) in positive ion mode and quantified using the isotope internal standard method. The effects of the derivatisation reaction time, temperature and concentration of derivatisation reagents on the response values of the analytes were investigated. The optimal conditions were as follows: 1.0 mg mL−1 CDI acetonitrile solution at 25 °C for 25 min, followed by a reaction with a 1.0 mg mL−1 4-(dimethylamino)-benzenemethanamine acetonitrile solution at 70 °C for 30 min. Under the optimal conditions, the limits of detection (LODs) of the 11 FFAs were in the range of 3.0–14.0 ng mL−1; the limits of quantification (LOQs) were in the range of 8.0–45.0 ng mL−1; and the mean recoveries ranged from 83.4 to 112.8%, with intraday and interday precisions ranging from 0.7 to 9.1% and 3.7–9.5%, respectively. The experimental method is simple in terms of the pretreatment operation, accurate and reliable, and can be applied to the sensitive determination of FFAs in human blood samples. [ABSTRACT FROM AUTHOR]

• Open circular columns (OCC) and open planar columns (OPC) have the highest quality factor (q max). • q max of OCC and OPC is three times larger than that of a core-shell packed column (CSPC). • Peak capacity (n) of LC analysis in a given time at a given pressure is proportional to q max. • OCC and OPC can generate 3 times larger n than CSPC can at the same pressure in the same time. • The challenges of practical implementation of OCC and OPC considered. The largest peak capacity (n) that LC analysis can generate in isocratic or gradient elution analysis of a given sample in a given time at a given pressure is proportional to the quality factor (q max) of its column structure. In this study, the multi-channel structures with open pseudo-planar channel s (OPPC) and open circular channels (OCC) where compared with PC2 – a typical core-shell column packed with 2 μm particles. These columns have q max of 1.27, 1.17 and 0.41, respectively. The former two q max are the highest among all known column structures – about 3 times higher than q max of PC2. This means that the OPPC and OCC can generate about 3 times higher n compared to what a PC2 can in the same analysis time (t anal) at the same pressure, or they require about 81 times shorter t anal (81 is the 4th power of 3) to generate the same n as a PC2 can at the same pressure. However, while PC2 is a commercially available column, there are substantial challenges in manufacturing the OPPC and OCC that can compete with PC2 in practical applications. In order to be competitive with PC2, the OPPC and OCC should have sub-1μm characteristic dimensions (e.g., the inter-pillar distance, g , in OPPC-based pillar array columns, internal diameters of OCC). Thus, in order to compete with PC2 in one scenario, an OPPC requires g ≤ 0.14 μm. Additionally, to be competitive with PC2, OPPC and OCC should be able to sustain the same high pressure. Highlighting the challenges of their design and manufacturing might help to develop the manufacturable columns substantially superior to the packed ones. [ABSTRACT FROM AUTHOR]

• Multistep pH-peak-focusing LC with a hydrophilic polymer gel column was developed. • Synergism of acac and phen exhibits efficient retention for rare earth metal ions. • Each metal ion is enriched at a pH border formed between two zones of different pH. • Semi-preparative separation of La, Ce, Nd, Eu, Y, Tb, and Yb was achieved. Multistep pH-peak-focusing liquid chromatography with a column packed with a hydrophilic polymer gel (a cross-linked hydroxylated methacrylic polymer gel) was developed for separation of rare earth metal ions. Metal ions in a sample solution introduced to the column are chromatographically extracted into the stationary gel phase at the top of the column equilibrated with a basic solution used as the first mobile phase containing acetylacetone and 1,10-phenanthroline by synergistic extraction effect. After the sample solution is introduced, the mobile phases are delivered into the column by stepwise gradient elution in order of decreasing pH. Each metal ion is concentrated at a pH border formed between the zones of different pH in the column and moves toward the outlet of the column with the pH border. Mutual separation of La(III), Ce(III), Nd(III), Eu(III), Y(III), Tb(III), and Yb(III) was achieved by the present method for an 1-mL sample injection with the column of which the inner volume is 11.8 mL. The multistep pH-peak-focusing liquid chromatography with a hydrophilic polymer gel column developed in this study has great potential as a useful method for the separation of rare earth metal ions on a preparatory scale. [ABSTRACT FROM AUTHOR]

• Chromatographic properties of phosphorylated peptides have been investigated using extensive proteomics-derived datasets. • Retention shifts induced by phosphorylation under reversed-phase separation conditions increase in the order HFBA < TFA < Formic acid < Acetic acid. • Retention shifts induced by phosphorylation under reversed-phase separation conditions increase in the order Tyr < Thr < Ser. • Composition and sequence-dependent factors determining retention shifts have been established. The development of a peptide retention prediction model for reversed-phase chromatography applications in proteomics is reported for peptides carrying phosphorylated Ser, Thr and Tyr-residues. The major retention features have been assessed using a collection of over 10,000 phosphorylated/non-phosphorylated peptide pairs identified in a series 1D and 2D LC-MS/MS acquisitions using formic acid as ion pairing modifier. Single modification event on average results in increased peptide retention for phosphorylation of Ser (+ 1.46), Thr (+1.33), Tyr (+0.93% acetonitrile, ACN) on gradient elution scale for Luna C18(2) stationary phase. We established several composition and sequence specific features, which drive deviations from these average values. Thus, single phosphorylation of serine results in retention shifts ranging from -2.4 to 5.5% ACN depending on position of the residue, nature of nearest neighbour residues, peptide length, hydrophobicity and pI value, and its propensity to form amphipathic helical structures. We established that the altered ion-pairing environment upon phosphorylation is detrimental for this variability. Hydrophobicity of ion-pairing modifier directly informs the magnitude of expected shifts: (most hydrophilic) 0.5 % acetic acid (larger positive shift upon phosphorylation) > 0.1 % formic acid (positive) > 0.1 % trifluoroacetic (negative) > 0.1 % heptafluorobutyric acid (larger negative shift). The effect of phosphorylation has been also evaluated for several separation conditions used in the first dimension of 2D LC applications: high pH reversed-phase (RP), hydrophilic interaction liquid chromatography (HILIC), strong cation- and strong anion exchange separations. [ABSTRACT FROM AUTHOR]

• Multimodal chromatography for standard and multispecific antibody formats. • Mechanistic chromatography model calibrated for 59 full-length IgG derivatives. • Thermodynamic parameters predicted from antibody-specific descriptors. • Multiscale models validated at pH 5.0 to 7.0 for a priori process development. Multimodal chromatography has emerged as a powerful method for the purification of therapeutic antibodies. However, process development of this separation technique remains challenging because of an intricate and molecule-specific interaction towards multimodal ligands, leading to time-consuming and costly experimental optimization. This study presents a multiscale modeling approach to predict the multimodal chromatographic behavior of therapeutic antibodies based on their sequence information. Linear gradient elution (LGE) experiments were performed on an anionic multimodal resin for 59 full-length antibodies, including five different antibody formats at pH 5.0, 6.0, and 7.0 that were used for parameter determination of a linear adsorption model at low loading density conditions. Quantitative structure-property relationship (QSPR) modeling was utilized to correlate the adsorption parameters with up to 1374 global and local physicochemical descriptors calculated from antibody homology models. The final QSPR models employed less than eight descriptors per model and demonstrated high training accuracy (R² > 0.93) and reasonable test set prediction accuracy (Q² > 0.83) for the adsorption parameters. Model evaluation revealed the significance of electrostatic interaction and hydrophobicity in determining the chromatographic behavior of antibodies, as well as the importance of the HFR3 region in antibody binding to the multimodal resin. Chromatographic simulations using the predicted adsorption parameters showed good agreement with the experimental data for the vast majority of antibodies not employed during the model training. The results of this study demonstrate the potential of sequence-based prediction for determining chromatographic behavior in therapeutic antibody purification. This approach leads to more efficient and cost-effective process development, providing a valuable tool for the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

• Comprehensive 2D RPCCC × RPLC chromatographic analysis of Rheum palmatum L. • 2D bioassay profilings were combined with 2D chromatogram for 2D bioscreening. • Separation and characterization of 11 constituents from Rheum palmatum L. • Two components were identified as natural α -glucosidase inhibitors for the first time. In the present work, comprehensive two-dimensional reversed-phase countercurrent chromatography × reversed-phase liquid chromatography combined (2D RPCCC × RPLC) with 2D microfraction bioactive evaluation was employed to screen and isolate α -glucosidase inhibitors from Rheum palmatum L. Countercurrent chromatography was employed to improve 2D analysis and preparative separation. A selected biphasic solvent system composed of petroleum ether/ethyl acetate/methanol/water with gradient elution mode was used for the first dimension RPCCC separation (1D RPCCC). Solid-phase extraction was applied to eliminate interfering polar compounds before the second dimension analysis (2D RPLC). 76 components were shown in 2D contour plot in UV 280 nm. 11 Candidates were separated by a scaled-up CCC and identified by 1H NMR and 13C NMR, including anthraquinones, flavonoids, stilbenes, phenols, and glucoside derivatives. In addition, it was found that two components, resveratrol-4′- O -(6″-galloyl)glucoside (36) and lyciumaside (43) were identified as natural α -glucosidase inhibitors in Rheum palmatum L. for the first time. [ABSTRACT FROM AUTHOR]

• Self-disproportionation of enantiomers (SDE) on a silica column was examined. • Isotherm coefficients were correlated with the modifier concentration. • Gradient elution allowed enhancement of SDE and separation selectivity. • Dynamic model was formulated and used to predict the process. The influence of mobile phase composition on the efficiency of enantiomer separation by achiral chromatography (ACh) was investigated. The separation was induced by the phenomenon of self-disproportionation of enantiomers (SDE) triggered by their homo and hetero-chiral interactions in an achiral environment. Typically, SDE occurs in apolar mobile phases of weak elution strength, which causes the separation time to extend and the process productivity to deteriorate. To mitigate that effect, we altered the content of a strong solvent (modifier) in the mobile phase by use of a solvent gradient in which the target enantiomer was separated in the presence of the weak solvent, whereas the unresolved mixture of enantiomers was eluted by increasing the modifier content in the mobile phase. This enabled accelerating the solute elution while preserving the separation selectivity. The approach was examined for the separation of nonracemic mixtures of two structurally different compounds that exhibited the SDE effect in ACh, i.e., metalaxyl (MX) and methyl p -tolyl sulfoxide (MTSO). The target compound of the separation was the more abundant enantiomer in the enantiomeric mixture. The process realization was preceded by the determination of the effect of the modifier content on the separation yield for enantiomeric mixtures of MX and MTSO of different enantiomeric excess (ee). In the case of MX, yield of the pure target enantiomer varied from 2 %, for the maximum concentration of the modifier, to 45 % for the minimum modifier concentration and the largest ee used in the experiments. In the case of MTSO, the yield varied from minimum 40 % to maximum 66 %. To predict the process, we employed a dynamic model, in which underlying thermodynamic dependencies were implemented. [ABSTRACT FROM AUTHOR]

During the manufacturing of therapeutic antibodies, effective Protein A chromatography as initial column step is crucial to simplify the remaining purification effort for subsequent polishing steps. This is particularly relevant for molecules with high impurity content so that desired product purity can be attained. The present study demonstrates beneficial effects on impurity removal when applying kosmotropic salts, e.g., sodium sulfate or sodium chloride, in the elution phase. Initially, a screen using negative linear pH gradient elution evaluated the impact of the kosmotropic salts in comparison to no additive and chaotropic urea using three mAbs and three common resins. Retaining acceptable yield, the kosmotropic salts improved resolution of monomer and impurities and reduced the contents of process-related host cell proteins and DNA as well as of product-related low and high molecular weight forms, despite some resin- and mAb-dependent variations. Moreover, a decrease in hydrolytic activity measured by a new assay for polysorbase activity was observed. In contrast, urea was hardly effective. The findings served to establish optimized step elution conditions with 0.25 M of sodium sulfate for a challenging mAb with complex format (bispecific 2 + 1 CrossMab) displaying high relative hydrophobicity and impurity levels. With yield and purity both in the range of 90 %, the contents of all impurity components were reduced, e.g., low molecular weight forms by two-fold and polysorbase activity by four-fold. The study indicates the potential of kosmotropic salts to establish efficient and comprehensive impurity separation by Protein A for facilitated downstream processing and economic manufacturing of complex antibodies. [ABSTRACT FROM AUTHOR]

• Re-parameterized Neue-Kuss model is reported. • Re-parameterization improves accuracy and precision of results. • Experimental design for gradient elution is reported. • Reported design allows for accurate prediction of isocratic retention factors. 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 S 1,ref , 18 % for S 2,ref and 6.2 % for k ref (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 S 1 , 25 % for S 2 and 160 % for k w (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 %. [ABSTRACT FROM AUTHOR]

• Linear pH gradients applied to multimodal chromatography for 64 diverse antibodies. • Investigated standard and multispecific antibodies formats. • Developed Gaussian process regression model utilizing antibody-specific descriptors. • Acquired structure-function relationship of multimodal antibody-ligand interactions. Multimodal chromatography has emerged as a promising technique for antibody purification, owing to its capacity to selectively capture and separate target molecules. However, the optimization of chromatography parameters remains a challenge due to the intricate nature of protein-ligand interactions. To tackle this issue, efficient predictive tools are essential for the development and optimization of multimodal chromatography processes. In this study, we introduce a methodology that predicts the elution behavior of antibodies in multimodal chromatography based on their amino acid sequences. We analyzed a total of 64 full-length antibodies, including IgG1, IgG4, and IgG-like multispecific formats, which were eluted using linear pH gradients from pH 9.0 to 4.0 on the anionic mixed-mode resin Capto adhere. Homology models were constructed, and 1312 antibody-specific physicochemical descriptors were calculated for each molecule. Our analysis identified six key structural features of the multimodal antibody interaction, which were correlated with the elution behavior, emphasizing the antibody variable region. The results show that our methodology can predict pH gradient elution for a diverse range of antibodies and antibody formats, with a test set R² of 0.898. The developed model can inform process development by predicting initial conditions for multimodal elution, thereby reducing trial and error during process optimization. Furthermore, the model holds the potential to enable an in silico manufacturability assessment by screening target antibodies that adhere to standardized purification conditions. In conclusion, this study highlights the feasibility of using structure-based prediction to enhance antibody purification in the biopharmaceutical industry. This approach can lead to more efficient and cost-effective process development while increasing process understanding. [ABSTRACT FROM AUTHOR]

• A novel LC‒MS/MS method detects bronopol in aquatic tissues with high sensitivity. • Optimized extraction and cleanup steps yield reliable quantification of bronopol. • Calibration curves show excellent linearity for fish, shrimp, crab, and shellfish matrices (r > 0.99). • The developed method successfully addressed the challenge of extracting bronopol from aquatic products. • Successful validation supports method's potential for safeguarding aquatic product quality and public health. Fish farming plays a vital role in providing food, nutrition, and employment globally. However, this industry faces security challenges, necessitating the use of fungicides and preservatives, such as bronopol, to increase product yields. Bronopol (2‑bromo-2-nitropropan-1,3-diol; CAS:52–51–7) is widely used in various fields, including food production, cosmetics, and, more recently, aquaculture. Currently, there is a limited number of techniques available for detecting bronopol in aquaculture products. This is primarily due to bronopol's instability, susceptibility to degradation, and tendency to form precipitates that pose challenges in extraction from aquaculture products. For this issue, this study presents a comprehensive method for detecting bronopol content in aquaculture tissues using liquid chromatography-tandem mass spectrometry (LC‒MS/MS). The methodology was optimized, involving extraction with Cu-Zn precipitant, cleanup using a small HLB column, separation on a T3 column, and gradient elution with water and acetonitrile mobile phases. The quantitative approach was employed without the use of an internal standard, following the external standard method. The spiked recoveries at 3 fortification levels (0.1, 0.2, and 1 mg/kg) ranged from 87.1 % to 108.1 % with relative standard deviations RSD ≤ 9.0 %. By applying this method to fresh fish, shrimp, crab, and shellfish samples from a local supermarket, no residues of bronopol were detected, ensuring the reliability of the results. The simplicity, rapidity, and high sensitivity of the method make it a suitable alternative to conventional techniques for bronopol detection. Moreover, the successful validation of the method's recovery and precision supports its potential application in monitoring and preventing the misuse of bronopol in aquaculture, thereby safeguarding aquaculture product quality and protecting public health. [ABSTRACT FROM AUTHOR]

• A simultaneous analysis method for seven N -nitrosamines by LC-MS/MS was developed. • On-line in-tube solid-phase microextraction conditions of N -nitrosamines were optimized. • The proposed method is automated, simple, rapid, selective, and sensitive. • This method was utilized to analyze N -nitrosamine impurities in metformin tablets. Contamination of active pharmaceutical ingredients (APIs) and pharmaceutical preparations with carcinogenic N -nitrosamines has led to recalls of these products and supply shortages to patients. The present study describes the development of a highly sensitive method for simultaneous analysis of seven N -nitrosamines using on-line in-tube solid-phase microextraction (IT-SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine their actual contamination in metformin tablets. Using a Carboxen 1006 PLOT capillary as the extraction device for IT-SPME, these compounds were efficiently extracted and concentrated 6‒24-fold by subjecting 40 µL of sample to 25 repeated draw/eject cycles at a rate of 0.2 mL/min. The seven N -nitrosamines were separated within 11 min by gradient elution with 0.1 % formic acid solution and acetonitrile as the mobile phase using a CAPCELL PAK C18 MGII column and detected by multiple reaction monitoring in positive ion mode. The calibration curve showed linearity in the range 0.2‒50 ng/mL and detection limits (S/ N = 3) in the range 3‒112 pg/mL. The intra-day and inter-day precisions were less than 5.5 % and 7.0 % (n = 6), respectively, with accuracies ranging from 93‒117 %. Following ultrasonic extraction with water, centrifugation and filtration of the supernatant liquid through a membrane filter, the N -nitrosamine impurities in metformin tablets could be analyzed by IT-SPME/LC‒MS/MS. Their limits of quantification (S/ N = 10) were 0.1‒5.1 pg/mg API and recoveries ranged from 87‒102 %. Analysis of eight metformin tablets from eight manufacturers showed that 5.8‒7.5 pg/mg N -nitrosodimethylamine were present in three tablets, with no other N -nitrosamines detected in any of the eight tablets. This method may be useful in testing for N -nitrosamine impurities in pharmaceutical preparations. [ABSTRACT FROM AUTHOR]

• A supercritical fluid-based method for extraction and analysis of indole alkaloids. • A more specific two-step SFE method towards alkaloids of complex natural plants origin. • C8SCX as adsorbent in SFE provide additional electrostatic attraction towards alkaloids. • An SFC-MS/MS method was utilized for analysis of indole alkaloids within 8 min. The development of an approach based on simultaneous supercritical fluid extraction-sample cleanup, followed by supercritical fluid chromatography/tandem mass spectrometry (SFE-SFC-MS/MS) was as a tool for the extraction, separation and characterization of indole alkaloids of Uncaria rhynchophylla. A two-step SFE method was designed. A mixture of the U. rhynchophylla sample and an adsorbent named C18SCX with the ratio of 1:1 (w/w) was placed into an extraction cell. The extraction temperature was 40 °C and the pressure was 25 Mpa. In the first step, 10 % EtOH as the co-solvent was used to extract for 60 min, which was considered as a cleanup process to remove non-alkaloid components. In the second step, 0.1 % DEA was added to 10 % EtOH and it extracted for 60 min to obtain the desired extract. By introducing an additional adsorbent, the specificity of SFE towards alkaloids was greatly improved. An SFC-MS/MS method was then utilized for analysis of the SFE extract. Using 2-EP as stationary phase with the gradient elution of 0–10 min, 5–25 % EtOH (+0.05 % DEA) in CO 2 , column temperature 40 °C, and back pressure 13.8 Mpa, 10 peaks were separated within 8 min. Further MS/MS analysis confirmed that nine of the 10 peaks in the SFE extract were indole alkaloids. This study developed a supercritical fluid-based method specifically towards extraction and analysis of alkaloids, which is helpful to the study of alkaline compounds in complex samples. [ABSTRACT FROM AUTHOR]

• A new methanol linear gradient CCC was introduced based on HepEMWat solvent systems. • Tedious preparations of solvent systems and waste of solvent was avoided. • This method can increase the range of K values separated in a single separation. • 12 compounds were highly enriched from Sinopodophyllum hexandrum in one-step separation. Counter-current chromatography (CCC) is a unique, liquid-liquid partition chromatography process. Both the mobile and stationary phases are liquids, so no solid support matrix is used. CCC has gained wide acceptance as a preparative technique in a variety of fields. Because the mobile and stationary phases are both liquids, gradient elution is difficult to perform with CCC. Phase equilibrium must be maintained, so any change in the composition of one phase may induce a compositional change in the other. In this work, a new linear gradient elution method was developed for CCC. Biphasic solvent systems containing heptane, ethyl acetate, methanol, and water (HepEMWat) in various ratios were prepared and used to optimize both isocratic and linear gradient CCC separation with methanol. We first separated a test mixture of four standard compounds with partition coefficients ranging from 0.8 to 7.8. The separation resembled a reversed-phase process, and elution was performed while progressively decreasing the polarity of the mobile phase. Target molecules with small partition coefficients eluted first in the lower phase of the optimized HepEMWat solvent system. Elution of constituents with large partition coefficients was quite slow under isocratic conditions. Separation time was significantly reduced when elution was performed with a linear gradient using methanol and the optimal HepEMWat system. Elution with a 3:7:4:6 (v/v/v/v) HepEMWat system took approximately 200 min. This included an 80-min isocratic step, followed by gradient elution with methanol from 0% to 30%. The optimized methanol linear gradient CCC method was then used to separate a complex mixture of natural products isolated from Sinopodophyllum hexandrum (Royle) Ying roots. Twelve compounds with a wide range of polarities were well-resolved in a single separation. We have developed a convenient and cost-effective strategy for the separation of complex mixtures. No tedious mobile phase preparation step is required. The volume of unused mobile phase is minimal, so little solvent is wasted. The method is an important advance for the separation of mixtures that contain many compounds with a large range of polarities and partition coefficients, which are common features of natural products. [ABSTRACT FROM AUTHOR]

• a dimensionless measure of the particle size gradient steepness was introduced. • about 15–20% gain in efficiency can be expected with particle size gradient. • too large particle size gradient can result in lower efficiencies than monodisperse packing. • large molecules (peptides, proteins) can in principle see the highest gain in efficiency. In this paper, the benefits of using columns packed with particles of decreasing size (particle size gradient) in liquid chromatography was investigated from a theoretical point of view. It is indeed well known that such columns may be useful in gradient elution, since the decrease of particle size along the chromatographic column can provide extra peak focusing effect. In the present contribution, several parameters (i.e., mobile phase gradient steepness, retention times and operating pressures) were considered and the kinetic performance of various types of columns packed with particle size gradient were evaluated. In the best case, about 15–20% gain in efficiency can be expected at a given retention time when utilizing a particle size gradient, compared to constant particle size. Conversely, when fixing efficiency, the analysis time can be decreased by about 15% with an optimal particle size gradient. However, it is also important to keep in mind that a too large a particle size gradient can result in lower efficiencies than a column packed with monodisperse packing. We have introduced the g d value, which is a dimensionless measure of the particle size gradient steepness that measures the relative variation of particle diameter throughout the column with respect to the average. We finally observed that g d = 0.3 - 0.4 provides the highest gain under practically useful conditions. [ABSTRACT FROM AUTHOR]

• Simulations of both gradient and uniform mixed-mode LC columns are described. • Peak focusing is noted on stationary phase gradients under mobile phase gradients. • Orientation of stationary phase gradients effects peak width and retention time. • Experimental and simulation differences may suggest neighboring ligand effects. This work seeks to explore and understand the effects of column orientation and degree of modification of continuous stationary phase gradient columns under a mobile phase gradient using both simulations and experiments. Peak parameters such as retention times, peak widths and resolution are obtained for five phenolic compounds on a C 18 -silica gradient stationary phase. Simulations show that peak widths for the solutes are dependent upon the fractional composition of C 18 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% C 18 where the column is oriented to have the C 18 dense region at the end. Experimentally, continuous stationary phase gradients were fabricated to have a total C 18 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% C 18 column. Simulations with a similar C 18 composition, however, predicted decreased retention times from the original C 18 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 C 18 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. [ABSTRACT FROM AUTHOR]

• Simultaneous gradient elution separation of siRNA duplexes and lipids via IP-RPLC. • Investigated 5 UHPLC columns and 4 ion-pairing agents. • Best retention conditions achieved with phenyl column and DBAA. • Optimal temperature depends on melting characteristics of siRNA duplex. • Forced-stress testing and analysis of lipid nanoparticle formulation of siRNA. RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products. [ABSTRACT FROM AUTHOR]

• Bispecific antibodies exhibit three-peak elution on high-resolution CEX resins. • Reversible on-column interconversion of three molecular BiSAb forms occurs. • A phenomenological model predicts the gradient elution behavior. The cation exchange chromatographic behavior of three homologous bivalent bispecific antibodies (BiSAb) is characterized for two different resins, Source 15S and ProPac WCX-10, having different base matrix, particle size, pore structure, and ligand chemistry. For both resins, elution with a salt gradient results in multiple peaks for each of the three BiSAb molecules at short residence times. The peaks gradually merge into two peaks and then into one peak eluting at intermediate salt concentrations when the residence time is gradually increased. Re-injecting fractions of each the individual peaks obtained at short residence time results in nearly the same multiple peak elution pattern. This behavior, which is contrary to the behavior normally encountered in ion exchange chromatography, appears to be related to the reversible, surface -catalyzed interconversion between different conformational states of each BiSAb that interact with different strength with the chromatographic surface. This behavior is qualitatively independent of pH in the range 5–8.5, protein load in the range 0.06–5.0 mg/ml, and gradient slope, and is not associated with the formation of aggregates. Gradually increasing temperature, however, reduces the multiple peak behavior eventually resulting into a single peak at 55 °C A phenomenological model is developed that predicts the experimental behavior over a broad range of conditions using fitted rate and equilibrium constants. [ABSTRACT FROM AUTHOR]

• HPLC-CAD developed for direct chain length characterization of polySia. • HPLC-CAD was shown to provide the actual chain length of polySia. • CAD as an alternative for time consuming DMB-derivatization. • Application of HPLC-CAD for polySia quantification in a broad concentration range. Polysialic acid (polySia) is widely investigated in various biopharmaceutical applications (e.g. treatment of inflammatory neurodegenerative diseases), whereby a certain polySia chain length with an average degree of polymerization 20 (polySia avDP20) shows most promising effects. In this study, a rapid analytical method using a HPLC and charged aerosol detector (CAD) for the direct chain length characterization of biopharmaceutically relevant polySia was developed. It was evaluated as a fast alternative to the commonly used 1,2-diamino-4,5-methylenedioxybenzene (DMB) HPLC application. In contrast to HPLC-FLD, the CAD-application provides the actual chain length of polySia within ∼3 h. The reliability of the HPLC-CAD was evaluated with a commercial reference sample of known chain length and biotechnologically produced LC polySia (long chain polySia with a DP ∼130). Moreover, HPLC-CAD was successfully applied in the direct detection of oligo- and polySia until DP ∼65 and can be used to monitor the thermal hydrolysis and subsequent chromatographic isolation of polySia avDP20 (average degree of polymerization 20) without DMB sample derivatization. In addition, CAD was successfully applied for polySia quantification using a modified elution gradient. It was tested as a fast alternative to commonly used thiobarbituric acid (TBA) assay. A differentiation between LC polySia and smaller, hydrolysed polySia chains was intended and possible. For LC polySia and polySia avDP20, a quadratic relation between polySia mass-concentration and CAD signal was observed. In case of LC polySia, a quadratic dependency with a determination coefficient of R2 = 0.99 in a broad concentration range between 0.025 and 15 mg mL−1 was determined. Quantification of polySia avDP20 was found to have quadratic dependency with a determination coefficient of R2 = 0.99 in a concentration range between 0.02 and 0.25 mg mL−1. The HPLC-CAD was tested for quantification with polySia references of known concentration and showed high accordance with a concentration deviation ≤6.7%. The CAD quantification method was also applied in the polySia avDP20 production process and was compared to the TBA assay. Results of a correlation plot showed a high determination coefficient of R2 = 0.98. Overall, HPLC-CAD analysis was successfully tested as a suitable characterization and quantification application in the biopharmaceutical production of polySia. [ABSTRACT FROM AUTHOR]

What is known in the literature as the fundamental equation for gradient elution (FEGE) was previously proven only for conventional gradient LC – uniform (the same at any distance from the inlet) static (fixed in time) solvent velocity (u m) in a column of uniform and static internal structure, cross-section and thermodynamic properties. A published alternative to the FEGE – the general migration equation – is valid for any column-based linear chromatography (GC, LC, SFC etc.). It allows one to theoretically or numerically predict a solute migration time to any location along the column. Starting from that general equation, several migration equations in gradient LC under different operational conditions including non-uniform non-static u m , Neue-Kuss retention model and others have been developed in this report. It has been shown that the conditions of validity of the FEGE can be expanded to include non-uniform u m. On the other hand, the FEGE is not valid for other unconventional operations of LC including gradient LC with dynamic (changing in time) u m. This implies that FEGE cannot be applied to, e.g., gradient LC operating at constant pressure where, due to the change in solvent composition, the solvent viscosity changes causing the change in u m with time. Applications of newly developed equations to other unconventional operations of gradient LC were also considered. Several new time parameters of the mobile phase flow were identified, interpreted, and evaluated. [ABSTRACT FROM AUTHOR]

• Develop and validate a UPLC-MS/MS method for the identification of vericiguat • Astragaloside IV significantly reduces C max of vericiguat • Drug-drug interaction might happen between vericiguat and astragaloside IV • Astragaloside IV might decrease vericiguat exposure via P-glycoprotein Vericiguat (VER) is a novel soluble guanylate cyclase stimulator treating symptomatic chronic heart failure (HF), and it is a substrate of both transporters P-glycoprotein and breast cancer resistance protein (BCRP). Astragaloside IV (ASIV) is the main active ingredient in Radix Astragali (Huangqi), a traditional Chinese medicine widely used for HF treatment in China. ASIV's effect on the protein expression of P-glycoprotein and BCRP has been observed, its impact on VER metabolism remain uncertain. In the present study, male Sprague-Dawley rats were administered with 20 mg/kg ASIV and 1 mg/kg VER to study their pharmacokinetics. Blood samples were subject to liquid-liquid extraction, and riociguat was employed as the internal standard (IS). The analytical method involved a C18 column (XSelect® HSS T3 column, 2.1 × 100 mm, 2.5 μm) with a mobile phase of 0.1% formic acid and acetonitrile for gradient elution. The flow rate of the mobile phase was set at 0.2 mL/min, and 5 µL of the sample was used for analysis. The positive ion multi-response monitoring mode was utilized with a transition of m/z 427.4→109.1 for VER and m/z 423.3→109.1 for the IS. The method exhibited good linearity within the concentration range of 0.1 to 300 ng/mL (r = 0.9987), and all the validation processes were conducted in accordance with the requirements of biological analysis. The pharmacokinetic results revealed that ASIV did not significantly alter the main parameters of VER, except for C max , which decreased by 33.2% (P < 0.05). Overall, our study successfully established a selective, sensitive and repeatable ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis for detecting VER in rat plasma. [ABSTRACT FROM AUTHOR]

• General equations of Part 1 applied to linear gradients in uniform columns. • Equations for retention time, band compression, peak focusing, peak separation and others developed and analyzed. • LSS gradient elution LC is treated as a special case of general linear gradients independent of programming rates. The unified approach to studies of different separation techniques (GC, LC, etc.) adopted in Part 1 continued herein. As before, the column temperature in GC, the solvent composition in LC, etc., are represented by the concept of solute mobilization (y). General equations for dynamic (moving) linear y- gradients (gradients in y) in non-uniform columns developed in Part 1 are reduced herein to special cases of uniform columns. Only the uniform columns, the ideal sample introduction, the positive y -rates, the negative y -gradients, and the eluting solutes are considered. Equations for solute band compression, peak width, peak focusing, peak separation and others derived. All equations are expressed via dimensionless parameters eliminating unessential factors like the mobile phase type, column type and dimensions, etc. The conventional gradient elution LC is treated as a special case of the separations with y -gradients. Effects of the y -gradients on solute retention time, on band compression, on peak focusing and on peak separation are analyzed. The equations developed herein, while describing simple and familiar concepts (retention time, peak width, etc.), are, unfortunately, cumbersome in many cases. The good news is that the equations are exact and the conditions of existence of the equations are explicitly formulated so that there is no question where the equations can and where thy cannot be used. [ABSTRACT FROM AUTHOR]

• The impact of injection volume when using ultra-short columns was studied in a systematic manner. • Three different chromatographic modes were evaluated: IEX, RPLC and HILIC. • Relatively large volumes can be injected in RPLC and IEX modes without loss in performance. • The injected volume was found to be particularly critical in HILIC mode. The impact of injected sample volume on apparent efficiency has been studied for very short columns in a systematic way. For large molecules such as therapeutic proteins, it was found that relatively large volumes can be injected onto ultra-short RPLC and IEX columns (i.e. L < 50 mm) without significantly affecting the quality of the separation. This favourable behavior is due to the on-off elution mechanism of large molecules and to the fact that therapeutic protein samples are formulated in aqueous-based media, which is the weakest solvent in RPLC and IEX. Therefore, their peak is strongly focused at the column inlet even when large volume is injected, and pre-column peak dispersion is compensated. However, ultra-short HILIC columns do not seem to be favorable, as they require for very low injection volume to avoid detrimental peak splitting and breakthrough effects. Such peak distortion is related to the inherent solvent mismatch between sample diluent (aqueous) and mobile phase strength (highly organic in HILIC). When studying mass load, the ranking of the elution modes was the same, and the largest relative mass could be injected onto IEX columns (as large as 10% sample to sorbent mass), without affecting the separation quality. [ABSTRACT FROM AUTHOR]

• A GMA-DVB polymer stationary phase with multiple polar groups. • The phase shows negligible bleed under high fraction of aqueous solution. • Synergistic effect of lysine and amide groups of such polar phase. A novel polymer-based polar stationary phase for hydrophilic interaction chromatography (HILIC) is described. It was obtained by grafting lysine and acrylamide onto poly (glycidyl methacrylate-divinylbenzene) (GMA-DVB) microspheres via ring-opening reaction of epoxy groups and free radical polymerization with pendant double bonds of the microspheres. Multiple types of polar groups including zwitterionic (carboxylate and amine), amide and diol onto the microspheres make them highly hydrophilic. It showed typical HILIC character and good separation performance towards model polar analytes. Negligible bleed level under gradient elution mode (up to 50% fraction of water) was observed. It also exhibited specific separation selectivity to ionic analytes and simultaneous separation of anions and cations could be achieved in ideal electrostatic selectivity elution order, e.g. I−< NO 3 −

• An interface for automated closed-loop method development of LC × LC was designed. • Retention times were predicted in shifting gradients in the second dimension. • Within a few iterations, a separation of a complex mAb sample was improved. • The algorithm is modular by design and can be applied to other samples. Method development in comprehensive two-dimensional liquid chromatography (LC × LC) is a complicated endeavor. The dependency between the two dimensions and the possibility of incorporating complex gradient profiles, such as multi-segmented gradients or shifting gradients, renders method development by "trial-and-error" time-consuming and highly dependent on user experience. In this work, an open-source algorithm for the automated and interpretive method development of complex gradients in LC × LC-mass spectrometry (MS) was developed. A workflow was designed to operate within a closed-loop that allowed direct interaction between the LC × LC-MS system and a data-processing computer which ran in an unsupervised and automated fashion. Obtaining accurate retention models in LC × LC is difficult due to the challenges associated with the exact determination of retention times, curve fitting because of the use of gradient elution, and gradient deformation. Thus, retention models were compared in terms of repeatability of determination. Additionally, the design of shifting gradients in the second dimension and the prediction of peak widths were investigated. The algorithm was tested on separations of a tryptic digest of a monoclonal antibody using an objective function that included the sum of resolutions and analysis time as quality descriptors. The algorithm was able to improve the separation relative to a generic starting method using these complex gradient profiles after only four method-development iterations (i.e., sets of chromatographic conditions). Further iterations improved retention time and peak width predictions and thus the accuracy in the separations predicted by the algorithm. [ABSTRACT FROM AUTHOR]

• Ultrasound has been found useful for speeding up the membrane-assisted solvent extraction process. • Simultaneous isolation of several classes of drug-facilitated sexual assaults from urine in 10 min. • Highly sensitive assessment of drug-facilitated sexual assaults with good applicability in the forensic laboratory. A simple and highly efficient ultrasound assisted membrane-assisted solvent extraction (MASE) pre-treatment method for urine has been developed and validated for the simultaneous determination of twenty-two drugs involved in drug-facilitated sexual assaults (DFSAs) by liquid chromatography-tandem mass spectrometry. MASE was performed with 4.0 mL of urine (pH adjusted at 12), 400 μL of hexane as an organic solvent inside the polypropylene membrane, and ultrasonication (45 kHz, 120 W) for 10 min. A pre-concentration factor of 40 was achieved after evaporation (N 2 stream) and re-dissolution in 100 µL of methanol. Analytes were separated using a Zorbax Eclipse Plus C18 column under gradient elution with aqueous 10 mM NH 4 HCO 3 (pH 8.0) and methanol as mobile phases. Matrix-matched calibrations allowed the assessment of DFSA drugs of quite different octanol–water partition coefficients (K o/w), from 1.32 101 for pregabalin to 2.45 105 for clomipramine (Log P values from 1.12 (pregabalin) to 5.39 (clomipramine)). The limit of detection (LOD) was between 0.0075 to 0.37 µg L−1, with analytical recoveries ranging from 73 to 103%, and relative standard deviations (RSDs) within the 2–20% range. The applicability of the method was demonstrated after analysing urine samples under forensic investigation. [ABSTRACT FROM AUTHOR]

• Ultra-short columns offer some clear advantages for the analysis of large biomolecules. • 10–20 mm columns are ideal for protein analysis. • Chromatographic instrumentation still needs to be improved to realise the full potential of short columns. • Ultra-short columns can be used in any chromatographic mode under gradient elution conditions. Today, reverse phase liquid chromatography (RPLC) analysis of proteins is almost exclusively performed on conventional columns (100–150 mm) in gradient elution mode. However, it was shown many years ago that large molecules present an on/off retention mechanism, and that only a very short inlet segment of the chromatographic column retains effectively the large molecules. Much shorter columns - like only a few centimetres or even a few millimetres - can therefore be used to efficiently analyse such macromolecules. The aim of this review is to summarise the historical and more recent works related to the use of very short columns for the analysis of model and therapeutic proteins. To this end, we have outlined the theoretical concepts behind the use of short columns, as well as the instrumental limitations and potential applications. Finally, we have shown that these very short columns were also possibly interesting for other chromatographic modes, such as ion exchange chromatography (IEX), hydrophilic interaction chromatography (HILIC) or hydrophobic interaction chromatography (HIC), as analyses in these chromatographic modes are performed in gradient elution mode. [ABSTRACT FROM AUTHOR]

• The performance of multiple dynamic traveling wave profiles was evaluated. • Optimal resolution was obtained using a static TW, but the ramp performed similarly. • Regarding SNR, optimum waveform profiles were species dependent. • Increases in ion abundance were observed when using a ramped TW for select species. • Tailored waveforms to optimize TW-SLIM separations require continued refinement. The amenability of traveling wave ion mobility spectrometry (TWIMS) to extended separation pathlengths has prompted a recent surge of interest concerning the technique. While promising, the optimization of ion transmission, particularly when analyzing increasingly disparate species, remains an obstacle in TWIMS. To address this issue, we evaluated a suite of dynamic TW profiles using an original TW structures for lossless ion manipulations (TW-SLIM) platform developed at Washington State University. Inspired by the range of gradient elution profiles used in traditional chromatography, three distinct square TW profiles were evaluated: a static approach which represents a traditional waveform, a dual approach which consists of two distinct TW profiles within a given separation event; and a ramp approach which varies TW speed and amplitude at a fixed rate during separation. The three waveform profiles were evaluated in terms of their impact on separation (quantified as resolution) and sensitivity (quantified using signal-to-noise ratio (SNR), and ion abundance). Concerning separation, the highest resolution (R) was observed when operating with the static waveform (R = 7.92); however, the ramp waveform performed comparably (R = 7.70) under similar conditions. Regarding SNR, optimum waveform profiles were species dependent. Bradykinin2+ displayed the largest gains in SNR (36.6% increase) when ramping TW speed, while the gains were greatest (33.5% increase) for tetraoctylammonium when modulating TW amplitude with the static waveform. Lastly, significant (>10%) increases in the abundance of tetraoctylammonium ions were observed exclusively when utilizing a ramped waveform. The present set of experiments outline the results and challenges related to optimizing separations using alternative TW profiles and provides insight concerning TW-SLIM method development which may be tailored to enhance select analytical metrics. [ABSTRACT FROM AUTHOR]

• Isomeric separation of underivatized N -glycans on nano ZIC-HILIC-LC-MS/MS. • Optimizing the chromatographic conditions improves separation of N -glycan isomers. • Lower retention of sialylated and oligomannose N -glycans at acidic mobile phase pH. • Lower resolution of isomers on ZIC-HILIC as mobile phase pH becomes more basic. • Higher temperature improves isomeric separation of native N -glycans on ZIC-HILIC. Changes in the expression of glycan isomers have been implicated in the development and progression of several diseases. However, the analysis of structurally diverse isomeric N- glycans by LC−MS/MS is still a major analytical challenge, particularly due to their large number of possible isomeric conformations. Common approaches derivatized the N -glycans to increase their hydrophobicity and to gain better detection in the MS system. Unfortunately, glycan derivatization is time-consuming and, in many cases, adds complexity because of the multiple reaction and cleaning steps, incomplete chemical labeling, possible degradation, and unwanted side reactions. Thus, analysis of native glycans, especially for samples with low abundance by LC−MS/MS, is desirable. Normal phase chromatography, which employs HILIC stationary phase, has been commonly employed for the identification and separation of labeled glycans. In this study, we focused on achieving efficient isomeric separation of native N -glycans using a nano ZIC-HILIC column commonly employed to separate labeled glycans and glycopeptides. Underivatized sialylated and oligomannose N -glycans derived from bovine fetuin and Ribonuclease B were initially utilized to optimize chromatographic conditions, including column temperature, pH of mobile phases, and gradient elution time. The optimized condition was then applied for the isomeric separation of native N -glycans derived from alpha-1 acid glycoprotein, as well as from biological samples. Finally, we confirmed the stability and reproducibility of the ZIC-HILIC column by performing run-to-run comparisons of the full width at half height (FWHM) and retention time on different N -glycans. The variability in FWHM was less than 0.5 min, while that of retention time was less than 1.0 min with %RSD less than 1.0%. [ABSTRACT FROM AUTHOR]

• A novel configuration of SFC having three pump units for mobile phase was created. • It enabled additive concentration control independent of modifier ratio in gradient. • It reduced adverse effects caused by excess additive in gradient elution. Mobile phase additives are used to improve retention behavior in chromatography. In supercritical fluid chromatography (SFC), for which supercritical fluid carbon dioxide (SF-CO 2) is used as the main mobile phase, additives can only be added into the modifier. For that reason, when gradient analysis is performed by changing the modifier ratio to SF-CO 2 , the additive concentration in the mobile phase increases in parallel with the modifier ratio. In a preliminary study performed using the conventional SFC system, ammonium acetate was necessary to improve the peak shape of a polar steroid, dehydroepiandrosterone sulfate (DHEA-S), while the peak intensity of a non-polar steroid, progesterone, decreased by 78% compared to that in the absence of the additive in mobile phase when gradient elution was performed. Since ammonium acetate had both favorable and unfavorable effects on sensitive and simultaneous analysis of these two steroid compounds, a compromise between these effects had to be sought. A three-pump configuration of SFC was developed by adding a pump unit to SFC instrument, which enabled control of the additive concentration independently of the modifier ratio, for the purpose of investigating the additive effect in detail using both steroids as model compounds. The putative cause of the decrease in peak intensity of progesterone was excessively elevated additive concentration in gradient analysis. When the additive concentration in the mobile phase was controlled to ensure that it did not increase during gradient analysis, the peak intensities of progesterone, cortisol, corticosterone, and testosterone were 55%, 40%, 25%, and 17% higher than when the additive concentration was not controlled, respectively. On the other hand, the peak intensity of DHEA-S was almost identical between the conditions, with an increase of 2% with three-pump instrument. The three-pump configuration showed the potential to solve problems relating to the use of modifier additives by keeping their concentration constant in gradient SFC analysis. [ABSTRACT FROM AUTHOR]

• A retention increase of macromolecules on IEX column with pressure rise was observed. • Up to 80% increase in retention was measured for isocratic elutions. • Gradient elutions of oligonucleotides, proteins and plasmid DNA showed similar trends. • The observed pressure effect depends on the molecule's size. • The separation resolution changed with different column pressure. Shorter analysis times and greater resolving power are contributing factors for transfer of separation methods from an HPLC to a UHPLC system when performing analysis in biopharmaceutical or clinical research. The effect of pressure on separations in reversed phase chromatography is well described, however such investigations on ion exchange columns were previously not conducted. In this study we describe the effect of pressure on retention properties of proteins, oligonucleotides and plasmid DNA in ion exchange chromatography. Different column inlet pressures were obtained by coupling restriction capillaries with column outlet and performing separations at a constant temperature and mobile phase flow rate. Macromolecules were separated in isocratic mode as well as with various linear gradients of salt concentration at a constant pH value. The measured retention time increase was up to 80% for isocratic and 20% for gradient separations for a 500 bar increase in pressure. The effect of pressure was validated on a separate instrument after few months from initial experiments. The influence of pressure on retention properties seems to be dependent on the size, shape and flexibility of the macromolecule and causes different retention shifts when separating a sample with diverse analytes. Such changes in retention time can sometimes exceed the criteria set by European Pharmacopoeia (Ph. Eur.) for the allowable method adjustment and are thus considered to be a result of a different separation method. Therefore, the pressure effect that follows method transfer from HPLC to UHPLC conditions should not be neglected even for gradient separations in ion exchange chromatography, as the resulting retention change may cause revalidation of the separation method. [ABSTRACT FROM AUTHOR]

• Pulses largely reduce retention of hydrophobic solutes at low modifier contents. • Intra-column delays should be considered to get good predictions close to the pulses. • Designs with pulses provide model parameters that agree with isocratic experiments. • Out-of-domain predictive capability of pulse designs is excellent. • Analysis times and solvent consumption are also competitive. The advantages and disadvantages of the use of isocratic experimental designs including transient increments of organic solvent (i.e., pulses) in the mobile phase(s) of lowest elution strength are explored with modelling purposes. For retained solutes, this type of mixed design offers similar or better predictive capability than gradient designs, shorter measurement time than pure isocratic designs, and retention model parameters that agree with those derived from pure isocratic experiments, with similar uncertainties. The predicted retention times are comparable to those offered by models adjusted from pure isocratic designs, and the solvent waste is appreciably lower. Under a practical standpoint, mixed designs including pulse(s) can be easily constructed by replacing the slowest isocratic runs with runs containing a pulse of short duration at an intermediate time. This allows the elution of the fastest solutes with appreciable retention in the initial sector of the elution program, previous to the pulse, and the elution of the slow solutes after the pulse, also in acceptable times. The fitting of the retention data obtained with pulses is simpler compared to gradient elution, and involves solving the integral equation of gradient elution, simplified by the presence of isocratic sectors. Experiments involving pulses reveal the existence of discrepancies in the predictions for solutes eluting in the nearby of the pulse, offered by the fundamental equation of gradient elution when this is solved using numerical integration. The correction of such discrepancies implies the inclusion of intra-column delays, in the arrival of changes in the concentration of organic modifier in the gradient to the instantaneous position of the solute, along the whole migration. [ABSTRACT FROM AUTHOR]

• Rapid footprint of the carbonyl compounds in hand scent was achieved. • Two-valves- IT-SPME-nanoLC-DAD was proposed to get the chromatographic profiles. • Rapid sample treatment with extraction and DNPH derivatization was optimized. • Nanoparticle based capillary column was used for the analyte preconcentration. In the present work, the footprint of carbonyl compounds in hand scent was achieved by a miniaturized method consisting of sampling with cotton gauze, extraction and derivatization using 2,4-dinitrophenylhydrazine (DNPH) and preconcentration, separation and detection by in-tube solid-phase microextraction (IT-SPME) coupled to nano-liquid chromatography/Uv–vis diode array detection. The coupling IT-SPME-nanoLC-DAD was solved by using a two-valve system: the first valve for loading the sample and the second one to perform IT-SPME. To this aim, a nanoparticle-based capillary column was employed. Firstly, the transfer time from the load loop to the NP-based capillary column in the IT-SPME system was optimized. Additionally, the conditioning and clean-up steps were also studied. For the chromatographic separation of DNPH derivatives, gradient elution mode (acetonitrile/water) and a C 18 nanocolumn were employed. The detection limits achieved were between 0.5 and 1.5 μg/L and % rsd was lower than 5% for quantification limits. The proposed methodology gave rise to different chromatographic profiles of carbonyl compounds in the hand scent of several volunteers. These profiles were obtained by estimating the relative peak area of selected carbonyls in hand scent. Nonanal, decanal and dodecanal and other low polarity carbonyl compounds (unknown hydrazones) were detected in the odor profiles. [ABSTRACT FROM AUTHOR]

• An electrodialytic KOH generator suited for small-bore ion chromatography. • The generator has much lower void volume and match well with sIC. • Wide KOH concentration range and high purity of KOH eluent obtained. An electrodialytic potassium hydroxide eluent generator (EDG) well-suited to small-bore (2 mm i.d.) ion chromatography (sIC) system is described. The basic configuration of EDG cartridge is much similar to that of conventional counterpart except lower void volume, which was measured to be ˜9 μL relative to ˜ 31 μL of conventional one. The corresponding dispersion volume was computed to be ˜58 μL and ˜106 μL, respectively. Lower void volume will improve the sharpness of a step gradient (or steeper rise curve), which is helpful to gradient elution. The device can bear the pressure up to at least 20.5 MPa and the effective KOH concentration that can be generated is ranging from 0.15 mM to 100 mM at the typical flow rate of sIC (0.4 mL/min). High purity of the produced KOH eluent and excellent running reproducibility of the device were observed, as indicated by the typical suppressed background (0.36 μS/cm) and <0.11% (or <0.23%) of the relative standard deviation of the retention times for common inorganic anions achieved under isocratic (or gradient) mode. [ABSTRACT FROM AUTHOR]

• Theory of band broadening along conical columns is developed in gradient elution. • Conically shaped 2.1 mm/4.2 mm i.d.× 15 cm long columns were prepared. • Data confirm the performance loss (<15%) under isocratic conditions. • Flow direction is from the wide to the narrow end for maximum peak capacity. • Conical columns reduce peak tailing caused by column wall/end non-idealities. The chromatographic performance (speed, efficiency, and gradient peak capacity for the same analysis time) of conical columns are investigated from fundamental and experimental viewpoints. A stainless steel, conically shaped column (2.1 mm i.d/4.2 mm i.d. × 15 cm long, 0.4° opening angle) was prepared in-house and packed with 5 μm XBridge-C 18 fully porous particles. Its performance was compared to that of a conventional 3.0 mm × 15 cm cylindrical column packed with the same batch of particles. Both Giddings' theory of non-uniform columns and experiments agree and show that, irrespective of flow direction, the conical column is 15% less efficient than the conventional column. Remarkably, Blumberg's theory of band broadening in gradient elution mode predicts that conical columns may outperform conventional cylindrical columns if the ratio of their outlet i.d. to their inlet i.d. is 0.95 and 0.80 for small molecule and peptide mixtures, respectively. The maximum relative gain is marginal as it does not exceed a few percents. The theory reveals that the flow direction should be from the wide to the narrow end of the conical column in order to deliver the highest peak capacity. In agreement with the theory, the observed losses in absolute peak capacity for the same analysis time are 14.5% (narrow to wide end) and only 11.0% (wide to the narrow end) for small molecules (n -alkanophenones). They are 14.2% (narrow to wide end) and only 8.5% (wide to the narrow end) for peptide samples (bombesin). Additionally, conical columns reduce peak tailing with respect to standard columns. They are suitable column technology for ultra-fast gradient separations as they also minimize sample dispersion through the narrow i.d. outlet frit. [ABSTRACT FROM AUTHOR]

• Relations between gradient retention time and block copolymer structure are derived. • Block copolymers elute close to stronger adsorbing homopolymer. • Results were verified by experiment. • Elution behavior of block copolymer is similar to appropriate homopolymer. The retention of binary diblock copolymers in solvent gradient interaction chromatography (SGIC) was investigated theoretically and compared with experimental results. The isocratic and gradient elution behavior of binary block copolymers can be described by the equations of an equivalent homopolymer having a suitable critical eluent composition and size to pore size ratio. For diblock copolymers the adsorption-desorption transition occurs at a limiting interaction energy, which is characterized by a limiting interaction parameter c A,lim , like the critical interaction parameter of homopolymers. Both, the limiting interaction parameter and the critical interaction parameter are independent of pore size. However, while for homopolymers the critical energy is also independent of molecular size, the limiting interaction parameter of block copolymers depends on the size and composition of the macromolecule. Therefore, even for block copolymers of a fixed composition a molar mass independent elution cannot be realized at isocratic conditions. Since binary block copolymers in solvent gradients elute very close to the stronger adsorbing homopolymer, separation of the block copolymer from the stronger adsorbing homopolymer becomes difficult. A slightly better separation is expected to occur in larger pores. The close elution of block copolymers and homopolymers in gradient elution was confirmed for poly(methy methacrylate)-block-poly(styrene)s in chloroform-THF gradients on a bare silica column. [ABSTRACT FROM AUTHOR]

Highlights • Modulation of retention in microemulsion liquid chromatography is described. • The contents of reagents for best retention, selectivity and resolution are given. • The viability and adequacy of isocratic and gradient elution are discussed. • Reported optimization strategies to facilitate method development are outlined. • Methodology used to prepare stable microemulsion systems is described. Abstract Microemulsions (MEs) are stable, isotropically clear solutions consisting of an oil and water stabilized by a surfactant and a co-surfactant. Oil-in-water microemuslion liquid chromatography (MELC) is a relatively new chromatographic mode, which uses an O/W ME as mobile phase. Retention, selectivity and efficiency can be modified by changing the concentration of the ME components and the ratio between the aqueous and oil phases. This work makes a critical survey on the information found in the literature about the mobile phase compositions that lead to the creation of successful O/W ME mobile phases, as well as the effect of pH for ionizable compounds and temperature. The viability of performing the analyses using isocratic and gradient elution is also considered. The complexity of the composition of a successful ME, and the fact that the different factors interact each other, may require many manipulations during method development to achieve an acceptable separation for complex mixtures. This is the reason of the proposal from several authors of a standard ME as starting point when developing a method for a new separation with no previous reports. Based on these initial conditions, the interest of several authors in applying computer-assisted approaches to optimize the composition of ME mobile phases, and reduce significantly the time and reagent consumption for method development, is described. Some practical tips are given to prepare stable ME mobile phases that yield reproducible results. [ABSTRACT FROM AUTHOR]

Highlights • Sample dispersion through a frit in narrow-bore columns was measured. • Four independent experimental approaches were used to increase data accuracy. • Peak volume variance due to state-of-the-art column frit were from 0.15 to 0.50 μL2. • Frit dispersion is larger than post-column dispersion of least dispersive UHPLC systems. • Observed high throughput LC gradient peak capacity is only 50% of the theoretical value. Abstract The impact of porous frits (2.1 mm i.d. × 1 mm thick, 0.2 μm porosity, 20% void, 0.69 μL) in short 0.5–5 cm long × 2.1 mm i.d. columns packed with sub-2 μm superficially porous particles on gradient performance was investigated using a low dispersive i-class ACQUITY UPLC system (25 cm × 75 μm outlet tube + 250 nL optical cell). In order to maximize data accuracy, the sample dispersion through a single frit was measured from four independent methods: (1) plate height subtraction, (2) peak capacity versus efficiency plot, (3) flow reversal, and (4) direct measurement. Frit dispersion increases non-linearly with increasing flow rate. The corresponding volume variances of small molecules were measured at 0.16 ± 0.04, 0.24 ± 0.05, and 0.31 ± 0.06 μL2 at flow rates of 0.1, 0.2, and 0.3 mL/min, respectively. These observed variances are lower than and consistent with the maximum volume variance of 0. 692 ∼ 0.5 μL2 expected for a mixer-like behavior. The peak capacity of short columns were then calculated for mixtures of peptides using a general model of gradient elution by considering (or not) the actual analyte dispersion taking place in the outlet frit, in the post-column connecting tube, and in the detection cell. The results show that the sole presence of the outlet frit is responsible for about 50% loss in peak capacity (relative to the expected gradient performance in absence of frit and post-column tube) for a 1 cm long column operated under standard gradient steepness. The actual structure and volume of column frits needs adjustment if one wants to take full advantage of the true performance of short high-throughput columns packed with sub-2 μm particles. [ABSTRACT FROM AUTHOR]

Highlights • Theory was developed to predict peak width for any coupled column system. • Columns with different individual plate heights and lengths have been assumed. • It was found that the last column has the largest weight in the outlet width. Abstract 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. [ABSTRACT FROM AUTHOR]

Highlights • Theory was developed to predict the peak widths for coupled column systems. • In gradient elution mode, the order of the columns impacts the apparent peak width. • “Peak sharpening” may occur when the most efficient column is the last in the assembly. • Theory and predictions have been experimentally verified. • Limits of apparent efficiency is discussed assuming infinite plate count for one column. Abstract The goal of this work was to understand the variation of apparent efficiency when serially coupling columns with identical stationary phase chemistries, but with differences in their kinetic performance. For this purpose, a mathematical treatment was developed both for isocratic and gradient modes to assess the change in plate numbers and peak widths when coupling arbitrary several columns. To validate the theory, experiments were also carried out using various mixtures of compounds, on columns packed with different particle sizes, to mimic highly efficient (new, not used) and poorly efficient columns (used one with many injections). Excellent agreement was found between measured and calculated peak widths. The average error in prediction was about 5% (which may be explained by the additional volume of the coupling tubes). In isocratic mode, the plate numbers are not additive when the coupled columns possess different efficiencies, and a limiting plate count value can be calculated depending on the efficiency and length of the individual columns. Theoretical efficiency limit can also be determined assuming one column in the row with infinite efficiency. In gradient elution mode, the columns’ order has a role (non-symmetrical system). When the last column has high enough efficiency, the gradient band compression effect may outperform the competing band broadening caused by dispersive and diffusive processes (peak sharpening). Therefore, in gradient mode, the columns should generally be sequentially placed according to their increasing efficiency. [ABSTRACT FROM AUTHOR]

• Rapid iterative optimization of multimodal ligands with improved mAb selectivity. • Ligand structure mapping: Predicted descriptors of a virtual library guides synthesis. • Chromatographic mapping: HTPD screening of protein binding vs. pH and conductivity. • Quantified multimodal interactions: Isocratic retention factor analysis. • A workflow connecting ligand structure, HTPD binding, and chromatogram modelling. Multimodal chromatography resins are becoming a key tool in the purification of biomolecules. The main objective of this research was the establishment of an iterative framework for the rapid development of new multimodal resins to provide novel selectivity for the future purification challenges. A large chemically diverse virtual library of 100 multimodal Capto™ MMC ligand analogues was created, and a broad array of chemical descriptors were calculated for each ligand in silico. Principal component analysis (PCA) was used to map the chemical diversity and guide selection of ligands for synthesis and coupling to the Capto ImpRes agarose base matrix. Twelve new ligands were prepared in two groups: 'group one' consist of L00-L07 and 'group two' consist of L08-L12. These ligands are diverse in the influence of varied secondary interactions such as hydrophobic interactions, H-bonding, etc. Additional resin prototypes were also prepared to look at the chromatographic impact of ligand density variation. High-throughput plate-based studies were performed for parallel resin screening for batch-binding of six model proteins at different chromatographic binding pH and sodium chloride concentration conditions. Principal component analysis of the binding data provided a chromatographic diversity map leading to the identification of ligands with improved binding. Further, the new ligands have improved separation resolution between a monoclonal antibody (mAb1) and product related impurities, a Fab fragment and high molecular weight (HMW) aggregates, using linear salt gradient elutions. To quantify the importance of secondary interactions, analysis of the retention factor of mAb1 on the ligands at various isocratic conditions lead to estimations of (a) the total number of water molecules and counter salt ions released during adsorption, and (b) hydrophobic contact area (HCA). The iterative mapping approach of chemical and chromatography diversity maps described in the paper proves to be a promising method for identifying new chromatography ligands for biopharmaceutical purification challenges. [ABSTRACT FROM AUTHOR]

• Equation for peak width in gradient LC with solvent composition program having initial isocratic hold has been derived. • Special case of the hold of balanced duration considered. • Newly derived equation for the balanced hold is equivalent to previously published one. 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. [ABSTRACT FROM AUTHOR]

Computer-aided method-development programs require accurate models to describe retention and to make predictions based on a limited number of scouting gradients. The performance of five different retention models for hydrophilic-interaction chromatography (HILIC) is assessed for a wide range of analytes. Gradient-elution equations are presented for each model, using Simpson’s Rule to approximate the integral in case no exact solution exists. For most compound classes the adsorption model, i.e. a linear relation between the logarithm of the retention factor and the logarithm of the composition, is found to provide the most robust performance. Prediction accuracies depended on analyte class, with peptide retention being predicted least accurately, and on the stationary phase, with better results for a diol column than for an amide column. The two-parameter adsorption model is also attractive, because it can be used with good results using only two scanning gradients. This model is recommended as the first-choice model for describing and predicting HILIC retention data, because of its accuracy and linearity. Other models (linear solvent-strength model, mixed-mode model) should only be considered after validating their applicability in specific cases. [ABSTRACT FROM AUTHOR]

A propylene–ethylene random copolymer was fractionated by preparative temperature rising elution fractionation (TREF). The structural heterogeneity of the bulk sample and its TREF fractions was studied by high temperature liquid chromatography with a solvent gradient elution from 1-decanol to 1,2,4-trichlorobenzene. HPLC alone cannot resolve those propylene-ethylene copolymers with high ethylene content in the bulk sample, due to their low weight fractions in the bulk sample and a small response factor of these components in the ELSD detector, as well as their broad chemical composition distribution. These components can only be detected after being separated and enriched by TREF followed by HPLC analysis. Chemical composition separations were achieved for TREF fractions with average ethylene contents between 2.1 and 22.0 mol%, showing that copolymers with higher ethylene contents were adsorbed stronger in the Hypercarb column and eluted later. All TREF fractions, except the 40 °C fraction, were relatively homogeneous in both molar mass and chemical composition. The 40 °C fraction was rather broad in both molar mass and chemical composition distributions. 2D HPLC showed that the molar masses of the components containing more ethylene units were getting lower for the 40 °C fraction. HPLC revealed and confirmed that co-crystallization influences the separation in TREF of the studied propylene-ethylene copolymer. [ABSTRACT FROM AUTHOR]

Gradient retention times are difficult to project from the underlying retention factor ( k ) vs. solvent composition ( φ ) relationships. A major reason for this difficulty is that gradients produced by HPLC pumps are imperfect – gradient delay, gradient dispersion, and solvent mis-proportioning are all difficult to account for in calculations. However, we recently showed that a gradient “back-calculation” methodology can measure these imperfections and take them into account. In RPLC, when the back-calculation methodology was used, error in projected gradient retention times is as low as could be expected based on repeatability in the k vs. φ relationships. HILIC, however, presents a new challenge: the selectivity of HILIC columns drift strongly over time. Retention is repeatable in short time, but selectivity frequently drifts over the course of weeks. In this study, we set out to understand if the issue of selectivity drift can be avoid by doing our experiments quickly, and if there any other factors that make it difficult to predict gradient retention times from isocratic k vs. φ relationships when gradient imperfections are taken into account with the back-calculation methodology. While in past reports, the accuracy of retention projections was >5%, the back-calculation methodology brought our error down to ∼1%. This result was 6–43 times more accurate than projections made using ideal gradients and 3–5 times more accurate than the same retention projections made using offset gradients (i.e., gradients that only took gradient delay into account). Still, the error remained higher in our HILIC projections than in RPLC. Based on the shape of the back-calculated gradients, we suspect the higher error is a result of prominent gradient distortion caused by strong, preferential water uptake from the mobile phase into the stationary phase during the gradient – a factor our model did not properly take into account. It appears that, at least with the stationary phase we used, column distortion is an important factor to take into account in retention projection in HILIC that is not usually important in RPLC. [ABSTRACT FROM AUTHOR]

Coupling the modified Soczewiñski model and one gradient run, a gradient method was developed to build a linear solvation energy relationship (LSER) for normal phase chromatography. The gradient method was tested on dinitroanilinopropyl (DNAP) and silica columns with hexane/dichloromethane (DCM) mobile phases. LSER models built based on the gradient separation agree with those derived from a series of isocratic separations. Both models have similar LSER coefficients and comparable goodness of fit, but the LSER model based on gradient separation required fewer trial and error experiments. [ABSTRACT FROM AUTHOR]

• Parameters for seven isotherm formalisms fit to mAb batch data for Capto MMC and Capto Adhere. • Unique column elution behavior accurately predicted for both multimodal resins using isotherm parameters from batch data. • Removing low protein concentration data from batch isotherms improved Capto MMC predictions. • Extended steric mass action model outperforms complex multimodal isotherm formalisms. • Salt-protein activity coefficient fully accounts for hydrophobic interactions. In this work, we have examined an array of isotherm formalisms and characterized them based on their relative complexities and predictive abilities with multimodal chromatography. The set of isotherm models studied were all based on the stoichiometric displacement framework, with considerations for electrostatic interactions, hydrophobic interactions, and thermodynamic activities. Isotherm parameters for each model were first determined through twenty repeated fits to a set of mAb – Capto MMC batch isotherm data spanning a range of loading, ionic strength, and pH as well as a set of mAb – Capto Adhere batch data at constant pH. The batch isotherm data were used in two ways—spanning the full range of loading or consisting of only the high concentration data points. Predictive ability was defined through the model's capacity to capture prominent changes in salt gradient elution behavior with respect to pH for Capto MMC or unique elution patterns and yield losses with respect to gradient slope for Capto Adhere. In both cases, model performance was quantified using a scoring metric based on agreement in peak characteristics for column predictions and accuracy of fit for the batch data. These scores were evaluated for all twenty isotherm fits and their corresponding column predictions, thereby producing a statistical distribution of model performances. Model complexity (number of isotherm parameters) was then considered through use of the Akaike information criterion (AIC) calculated from the score distributions. While model performance for Capto MMC benefitted substantially from removal of low protein concentration data, this was not the case for Capto Adhere; this difference was likely due to the qualitatively different shapes of the isotherms between the two resins. Surprisingly, the top-performing (high accuracy with minimal number of parameters) isotherm model was the same for both resins. The extended steric mass action (SMA) isotherm (containing both protein-salt and protein-protein activity terms) accurately captured both the pH-dependent elution behavior for Capto MMC as well as loss in protein recovery with increasing gradient slope for Capto Adhere. In addition, this isotherm model achieved the highest median score in both resin systems, despite it lacking any explicit hydrophobic stoichiometric terms. The more complex isotherm models, which explicitly accounted for both electrostatic and hydrophobic interaction stoichiometries, were ill-suited for Capto MMC and had lower AIC model likelihoods for Capto Adhere due to their increased complexity. Interestingly, the ability of the extended SMA isotherm to predict the Capto Adhere results was largely due to the protein-salt activity coefficient, as determined via isotherm parameter sensitivity analyses. Further, parametric studies on this parameter demonstrated that it had a major impact on both binding affinity and elution behavior, therein fully capturing the impact of hydrophobic interactions. In summary, we were able to determine the isotherm formalisms most capable of consistently predicting a wide range of column behavior for both a multimodal cation-exchange and multimodal anion-exchange resin with high accuracy, while containing a minimized set of model parameters. [ABSTRACT FROM AUTHOR]

• Two-dimensional separations enable peak purity analysis of pharmaceutical peptides. • Strategy to derive second-dimension gradient conditions. • Preferred gradient conditions discovered by retention modelling. The importance of therapeutic peptides continues to increase in the marketplace for treating a range of diseases including diabetes and obesity. Quality control analyses for these pharmaceutical ingredients usually depends on reversed-phase liquid chromatography, and it is critically important to ensure that no impurities coelute with the target peptide at levels that would compromise the safety or effectiveness of the drug products. This can be challenging due to the broad range of properties of impurities that can be present on one hand (e.g., amino acid substitutions, chain cleavages, etc.), and the similarity of other impurities on the other hand (e.g., d -/ l -isomers). Two-dimensional liquid chromatography (2D-LC) is a powerful analytical tool that is well suited to address this particular problem; the first dimension can be used to detect impurities over a broad range in properties, while the second dimension can be used to focus specifically on those species that might coelute with the target peptide in the first dimension. While hundreds of papers have been published on the use of 2D-LC for proteomics applications, there are very few papers that have focused on its use for characterisation of therapeutic peptides. This paper is the second in a two-part series. In Part I of the series, we studied several different column / mobile phase combinations that could be useful in 2D-LC separations of therapeutic peptides, with a focus on selectivity, peak shape, and complementarity to other combinations, particularly for isomeric peptides under mass spectrometry-friendly conditions (i.e., volatile buffers). In this second part in the series, we describe a strategy to derive second-dimension (2D) gradient conditions that both, ensure elution from the 2D column, and increase the likelihood of resolving peptides with very similar properties. We find that a two-step process yields conditions that place the target peptide in the middle of the 2D chromatogram. This process begins with two scouting gradient elution conditions in the second dimension of a 2D-LC system, followed by building and refining a retention model for the target peptide using a third separation. The process is shown to be generically useful by developing methods for four model peptides, and application to a sample of degraded model peptide to demonstrate its utility for resolving impurities in a real sample. [ABSTRACT FROM AUTHOR]

• Internal recycling of product-containing fractions impacts the performances of MCSGP. • The role of the gradient slope in this phase is elucidated for PEGylated proteins. • We investigated: isocratic elution, constant slope, steeper gradient in the recycling. • For PEGylated lysozyme, a steeper gradient compresses the recycling phase. • For an industrial protein, the dual gradient increases the yield from 79 to 99%. Protein PEGylation, i.e. functionalization with poly(ethylene glycol) chains, has been demonstrated an efficient way to improve the therapeutic index of these biopharmaceuticals. We demonstrated that Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) is an efficient process for the separation of PEGylated proteins (Kim et al., Ind. and Eng. Chem. Res. 2021, 60, 29, 10764–10776), thanks to the internal recycling of product-containing side fractions. This recycling phase plays a critical role in the economy of MCSGP as it avoids wasting valuable product, but at the same time impacts its productivity extending the overall process duration. In this study, our aim is to elucidate the role of the gradient slope within this recycling stage on the yield and productivity of MCSGP for two case-studies: PEGylated lysozyme and an industrially relevant PEGylated protein. While all the examples of MCSGP in the literature refer to a single gradient slope in the elution phase, for the first time we systematically investigate three different gradient configurations: i) a single gradient slope throughout the entire elution, ii) recycling with an increased gradient slope, to shed light on the competition between volume of the recycled fraction and required inline dilution and iii) an isocratic elution during the recycling phase. The dual gradient elution proved to be a valuable solution for boosting the recovery of high-value products, with the potential for alleviating the pressure on the upstream processing. [ABSTRACT FROM AUTHOR]