Make-up Flow Research Articles

LCxSFC Valve Technologies: Guidelines toward a Successful Online Modulation.

Online two-dimensional liquid chromatography can be used under various separation modes but is sometimes limited in terms of orthogonality, especially for the analysis of neutral compounds. The use of SFC in the second dimension offers a wide choice of mobile and stationary phases, suggesting retention properties complementary to those of the first dimension. Initial works on online LCxSFC coupling published in the literature highlighted the first difficulties of solvent compatibility or potential problems with bubbles created by CO2 in loops. The present work highlights the impact of the interface between the LC and SFC dimensions on the performance of the 2D separation. Six different configurations have been evaluated, differing by the addition of a makeup flow in the first dimension, a division, or a separation of flow paths in the second dimension. Their performances with empty loops eliminated the need for complex trapping columns, regardless of the LC mobile phase nature. Injection in partial fill mode was possible without any problems of CO2 bubble formation, solvent miscibility, pressure, or modulation repeatability in each of the studied configurations. Selected configurations even allowed for the use of a mobile phase split upstream of the valve, so that the online LCxSFC configuration could be as flexible as LCxLC instrumentation in terms of the first dimension flow rate or second dimension injected volume. The obtained results allowed for building guidelines presenting the best interfaces to set up depending on the nature of the mobile phases in both dimensions. Two applications on monomers of lignin and industrial polymers confirmed preliminary results on configuration testing and illustrated that online LCxSFC could now be easily implemented. Precision tests on retention time were conclusive and are promising for the future use of this technique in industrial routines.

اختبار أداء محطة تحلية مياه البحر بالزويتينة

The present study is applied on the seawater multi-stage flash (MSF) desalination plant that is currently under operation in E-Zuetina operations plant located in Libya. The plant contains 21 evaporator stages at capacity of 10025 (ton/day). The presented operating data has been collected during a visit of the plant, a mathematical model for multistage flash (MSF) desalination plants was developed. The model was based on basic principles of physics and chemistry that describe the stages occurring in the desalination process. The input plant parameters that are known to affect the operation of the MSF desalination plant and its performance were taken into account in the construction of the model. These parameters included make-up flow, brine recycle flow, seawater flow, seawater temperature, seawater concentration, steam temperature and the plant load. For each stage, the developed model was used for predicting the temperatures and pressure of the brine, distillate, cooling brine, and the flow rates of brine outlet and distillate production. The developed model was evaluated with the MSF plant vendor simulation results and its actual operating data. The evaluation indicated that model predictions matched well with the vendor simulation results and the plant operating data. The developed model is sufficiently accurate and model predictions can be relied upon. Therefore, it may be recommended for determining optimum set point of a running MSF desalination plant at different loads to maximize the water production or minimize energy consumption. It can also be used to calculate controller set points for different loads of the plant. Keywords:E-Zuetina MSF Desalination Plant, case study, stage model, brine heater.

Coupled kinetic and hydrodynamic model for a carbonator reactor of calcium looping process: Sulfur dioxide effect

The application of the calcium looping (CaL) process for CO2 capture has gained recognition as a highly efficient technology in mitigating greenhouse gases emission. In this study, by considering the characteristics of the adsorbent and the hydrodynamics of the fluidized bed in the fast fluidization regime, a fractal-like random pore kinetic model and a 1D hydrodynamic model were combined together. In order to validate the kinetic model, experimental carbonation data obtained from a lab-scale fluidized bed reactor under realistic operational condition and in the absence/presence of SO2 gas were used. The results demonstrated a satisfactory agreement between the model and experimental data, as indicated by the maximum absolute error of 1.23% and 1.78% in the absence and presence of SO2, respectively. Based on sensitivity analysis, the efficiency of the carbonator reactor was found to be predominantly affected by the solid inventory in the bed and the gas inlet velocity. Also, the results of this analysis indicate that in the presence of SO2, a higher make-up flow rate leads to a more pronounced enhancement in the efficiency of the carbonator reactor, as compared to the absence of SO2 in the reaction environment. Results of modelling indicated that the presence of SO2 necessitated a 36.5-fold increase in the minimum-required make-up flow to attain comparable efficiency obtained in the condition of the absence of SO2. Furthermore, the results of modelling as a function of number of cycles demonstrated a substantial decline in the efficiency of the carbonator reactor, which in the absence of SO2, the efficiency decreased from 68.13% to 18.81%, while in the presence of SO2, it decreased from 45.53% to 4.75% over 100 sequential cycles.

Degradation of polysorbate investigated by a high-performance liquid chromatography multi-detector system with charged aerosol and mass detection

Polysorbate 80 is widely used as a formulation component in biopharmaceutical drug products. Recent studies have shown that polysorbate 80 is readily degraded either through direct or indirect means. The degradation of polysorbate 80 causes a concern for the long-term stability of biopharmaceutical drug product, as the breakdown products of polysorbate 80 have been shown to cause adverse effects, such as formation of sub-visible and visible particles and mAb aggregation. Understanding the path and extent of degradation is of a paramount importance for the formulator during formulation development. A multi-detector HPLC system using charged aerosol and mass detection was developed and optimized for the characterization of polysorbate 80 standards. The system included a post-column make-up flow, i.e. an inverse gradient, that enabled constant eluent composition at the detectors. The inverse gradient eliminated the main source of variability for the charged aerosol detector response, thereby enabling the calculation of the mass balance between polysorbate components with different degrees of esterification. Extracted ion chromatograms of the mass detector combined with their respective retention times were used to qualitatively characterize the polysorbate samples down to the individual components. The system was applied to study the degradation of several polysorbate standards which occurred by enzymatic digestion or long-term storage. The system provided detailed information on the mechanism of degradation without the need for additional orthogonal analytical techniques.

Electrospray Ionization Efficiency Predictions and Analytical Standard Free Quantification for SFC/ESI/HRMS.

Supercritical fluid chromatography (SFC) is a promising, sustainable, and complementary alternative to liquid chromatography (LC) and has often been coupled with high resolution mass spectrometry (HRMS) for nontarget screening (NTS). Recent developments in predicting the ionization efficiency for LC/ESI/HRMS have enabled quantification of chemicals detected in NTS even if the analytical standards of the detected and tentatively identified chemicals are unavailable. This poses the question of whether analytical standard free quantification can also be applied in SFC/ES/HRMS. We evaluate both the possibility to transfer an ionization efficiency predictions model, previously trained on LC/ESI/HRMS data, to SFC/ESI/HRMS as well as training a new predictive model on SFC/ESI/HRMS data for 127 chemicals. The response factors of these chemicals ranged over 4 orders of magnitude in spite of a postcolumn makeup flow, expectedly enhancing the ionization of the analytes. The ionization efficiency values were predicted based on a random forest regression model from PaDEL descriptors and predicted values showed statistically significant correlation with the measured response factors (p < 0.05) with Spearman's rho of 0.584 and 0.669 for SFC and LC data, respectively. Moreover, the most significant descriptors showed similarities independent of the chromatography used for collecting the training data. We also investigated the possibility to quantify the detected chemicals based on predicted ionization efficiency values. The model trained on SFC data showed very high prediction accuracy with median prediction error of 2.20×, while the model pretrained on LC/ESI/HRMS data yielded median prediction error of 5.11×. This is expected, as the training and test data for SFC/ESI/HRMS have been collected on the same instrument with the same chromatography. Still, the correlation observed between response factors measured with SFC/ESI/HRMS and predicted with a model trained on LC data hints that more abundant LC/ESI/HRMS data prove useful in understanding and predicting the ionization behavior in SFC/ESI/HRMS.

A reduced order model for triethylene glycol natural gas dehydration system

In natural gas processing plants, glycol dehydration is commonly used to remove water from the gas streams, to avoid pipeline blockage and equipment breakdown due to hydrates formation. This paper proposed a reduced order model developed based on integrated simulation-optimization approach for the glycol dehydration system, with the aim to minimize its operating cost while satisfying pipeline quality specifications. Steady-state process simulation software was used to identify important operating parameters for the glycol dehydration process; these include reboiler temperature and flow ratio of the regeneration column, and solvent flowrate. The identified parameters are built into a non-linear programming model, which was developed as a reduced order model for ease of implementation in the plant. The studied parameters are reboiler duties, hot oil, condenser, and pump, as well as TEG make-up flow rate and CO2 equivalent (CO2-eq) emissions. The Pareto Front is developed to identify the minimum operating cost at different levels of water dew point specification. The work has resulted in the annual savings of more than 34.6%.

A calcium looping system powered by renewable electricity for long-term thermochemical energy storage, residential heat supply and carbon capture

Affordable long-term energy storage and CO2 capture technique are imperative to developing the future carbon–neutral energy system. This paper proposed a calcium looping system powered by excess renewable electricity to achieve long-term thermochemical energy storage, residential heat supply and carbon capture. The system can be constructed with industrial equipment and exploit natural, abundant and cheap limestone as the circulating material. The electrical calciner is designed to charge the energy and produce the high-pressured CO2 during the valley of the electricity grid, while the carbonator side is primarily driven by the discharged thermochemical energy and on-demand coordinates the heat network along with the carbon capture. The HEATSEP framework is adopted to optimize heat transfer processes and operating conditions for deriving the most favorable system configuration in the energy and economic aspects. Compared to the reference plant with electric boilers and short-term heat storage tanks, an energy penalty of around 32.3% total energy percentage points should be paid to drive long-term energy storage and carbon capture. The energy penalty is attributed to thermal losses in the calciner and heat exchanger networks, chemical energy lost by the make-up flow of fresh limestone, CO2 compression for storage and auxiliary power for solid/fluid circulations. Due to a comparable total plant investment cost to the reference plant, the proposed system achieves the CO2 avoidance cost of 111.1 RMB/tonCO2, showing some competitiveness over current post-combustion carbon capture techniques. The techno-economic competitiveness of the proposed system can be further strengthened by replacing natural or synthetic Ca-based materials with high cycling performance instead of limestone, as well as extending applicable occasions to achieve almost year-round operation.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Optimization and Control for Separation of Ethyl Benzene from C8 Aromatic Hydrocarbons with Extractive Distillation

Extractive distillation has great significance for the separation of ethylbenzene from C8 aromatic hydrocarbons. Herein, a distillation process for the separation of ethylbenzene was designed using methyl phenylacetate as an extractant. A genetic algorithm (GA) was used to evaluate the economic and environmental factors of the process, and Aspen Dynamic was used to assess the dynamic performance. The sequential optimization method was used to obtain the initial process parameters. Then, the total annual cost and CO2 emissions were minimized by NSGA-III to increase the economic and environmental benefits. To enhance the search performance of GA, the mutation probability and crossover probability were studied and adjusted. The optimal total annual cost and CO2 emissions were 11.7% and 23.7% lower than those of the initial process. Based on a steady process, two control strategies, which were the flow rate of the recycling solvent controlled by entrainer makeup flow rate (CS1) and the bottom flow rate of the extractant recovery column (CS2), were designed. The results showed that the temperature deviation of CS2 was smaller than that of CS1, and the temperature of the process was more stable under the control of CS2.

Supercritical Fluid Nanospray Mass Spectrometry.

Supercritical fluids are typically electrosprayed using an organic solvent makeup flow to facilitate continuous electrical connection and enhancement of electrospray stability. This results in sample dilution, loss in sensitivity, and potential phase separation. Premixing the supercritical fluid with organic solvent has shown substantial benefits to electrospray efficiency and increased analyte charge state. Presented here is a nanospray mass spectrometry system for supercritical fluids (nSF-MS). This split flow system used small i.d. capillaries, heated interface, inline frit, and submicron emitter tips to electrospray quaternary alkyl amines solvated in supercritical CO2 with a 10% methanol modifier. Analyte signal response was evaluated as a function of total system flow rate (0.5-1.5 mL/min) that is split to nanospray a supercritical fluid with linear flow rates between 0.07 and 0.42 cm/sec and pressure ranges (15-25 MPa). The nSF system showed mass-sensitive detection based on increased signal intensity for increasing capillary i.d. and analyte injection volume. These effects indicate efficient solvent evaporation for the analysis of quaternary amines. Carrier additives generally decreased signal intensity. Comparison of the nSF-MS system to the conventional SF makeup flow ESI showed 10-fold signal intensity enhancement across all the capillary i.d.s. The nSF-MS system likely achieves rapid solvent evaporation of the SF at the emitter point. The developed system combined the benefits of the nanoemitters, sCO2, and the low modifier percentage which gave rise to enhancement in MS detection sensitivity.

Control of the azeotropic distillation process for separation of acetonitrile and water with and without heat integration

A novel acetonitrile/water azeotropic distillation scheme shows obvious economic advantages. It is of great significance to study the dynamic controllability of this process. In this work, the dynamic control structures of the non heat-integration and full heat-integration azeotropic distillation are studied. The control ability of the proposed control structures is tested by adding flow disturbance and composition disturbance. For the non heat-integration scheme, the conventional PID control structure and composition controller is used to achieve effective control when a disturbance occurs. For the full heat-integration scheme, the temperature difference control scheme and the composition controller are used to handle disturbances. This control structure reduces the stabilization time and makes the purity of the product return to the set value. The effect of different makeup flow on the purity of acetonitrile under different disturbances is discussed. Moreover, two control strategies are synthetically analyzed and compared by calculating integral of squared error. Considering economy and controllability, it is concluded that control strategy of heat-integrated azeotropic distillation has more advantages. This study has certain guiding value for the research of chemical dynamic control.

Comparison of existing strategies for keeping symmetrical peaks in on-line Hydrophilic Interaction Liquid Chromatography x Reversed-Phase Liquid Chromatography despite solvent strength mismatch

In two-dimensional liquid chromatography, the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) is very attractive due to the complementarity of their separation mechanisms. On-line comprehensive HILIC x RPLC is well-known to give rise to a large retention space coverage when dealing with ionisable compounds. However, method development in on-line HILIC x RPLC is challenging due to the reversed solvent strength between both dimensions, which can greatly affect the peak shapes in the second RPLC dimension, and thus the separation quality and the method sensitivity. In the present contribution, we compared four strategies designed to avoid this problem: (1) flow splitting, which consists in reducing the injection volume in the second dimension (2D), (2) on-line dilution with a make-up flow and (3) on-line dilution with Active Solvent Modulation (ASM), which both consist in reducing the solvent strength of the injected fractions, and (4) Total Breakthrough Strategy, which we recently proposed. Unlike the three preceding strategies, this latter one consists in injecting large volumes of strong solvent in 2D. The performance of each strategy was evaluated for sub-hour separations of a tryptic digest in on-line HILIC x RPLC. In this work, we considered the critical case for which the same column internal diameters (i.e. 2.1 mm here) are used in both dimensions. Peak capacity, peak shapes and peak intensities were considered for this evaluation. The highest peak capacity could be achieved with Total Breakthrough Strategy while the lowest one with on-line dilution using ASM. Peak intensities were usually higher with on-line dilution approaches (make-up flow and ASM). However, despite the presence of breakthrough, peak intensities were approximately 7-fold higher with Total Breakthrough Strategy than with flow splitting.

Study on impedance size optimization of a one-way surge tank in a long-distance water supply system

Abstract The size of the impedance hole of the one-way surge tank will affect its protection against water hammer. In this paper, a mathematical model of one-way surge tank with impedance is established based on the characteristic line method. The pressure-reducing penetration formula of the one-way surge tank including impedance loss and the calculation formula of make-up water flow are deduced. Based on these formulas, the influence of impedance hole diameter ratio (ratio of impedance hole of one-way surge tank to diameter of water pipeline) on the water hammer protection effect is analyzed, and the reasonable value range of impedance hole diameter ratio is given. The correctness of the theoretical analysis results is verified by an engineering example. The results show that the pressure and flow relationship derived from the formula are consistent with the numerical simulation results. The penetration pressure drop of the one-way surge tank is inversely proportional to the impedance size. When the impedance hole diameter ratio is less than 0.2, the penetration pressure drop will cause serious negative pressure. The make-up water flow is proportional to the size of the impedance hole, and the make-up water volume should be reduced while ensuring that the pipeline has no negative pressure.

Hyphenation of supercritical fluid chromatography with different detection methods for identification and quantification of liamocin biosurfactants

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.

A Detailed One-Dimensional Hydrodynamic and Kinetic Model for Sorption Enhanced Gasification

Increased installation of renewable electricity generators requires different technologies to compensate for the associated fast and high load gradients. In this work, sorption enhanced gasification (SEG) in a dual fluidized bed gasification system is considered as a promising and flexible technology for the tailored syngas production for use in chemical manufacturing or electricity generation. To study different operational strategies, as defined by gasification temperature or fuel input, a simulation model is developed. This model considers the hydrodynamics in a bubbling fluidized bed gasifier and the kinetics of gasification reactions and CO2 capture. The CO2 capture rate is defined by the number of carbonation/calcination cycles and the make-up of fresh limestone. A parametric study of the make-up flow rate (0.2, 6.6, and 15 kg/h) reveals its strong influence on the syngas composition, especially at low gasification temperatures (600–650 °C). Our results show good agreement with the experimental data of a 200 kW pilot plant, as demonstrated by deviations of syngas composition (5–34%), lower heating value (LHV) (5–7%), and M module (23–32%). Studying the fuel feeding rate (22–40 kg/h), an operational range with a good mixing of solids in the fluidized bed is identified. The achieved results are summarized in a reactor performance diagram, which gives the syngas power depending on the gasification temperature and the fuel feeding rate.

Modelling of a fluidized bed carbonator reactor for post-combustion CO2 capture considering bed hydrodynamics and sorbent characteristics

This manuscript presents a general model for the fluidized bed carbonator reactor of the Calcium Looping (CaL) process based on bed hydrodynamics, kinetics and sorbent properties. The model proposed relies on the Kunii–Levenspiel theory for circulating fluidized beds in the fast fluidization regime and considers recent findings on the CO2 capture properties of CaO, including the effects of the diffusion-controlled carbonation stage and CaO modification by means of additives. By performing a sensitivity analysis, it is found that the solids inventory in the carbonator and superficial gas velocity are the main operational and hydrodynamic factors affecting the CO2 capture efficiency. Decay constant of the fraction of solids in the lean region and fraction of solids in the dense region are also identified as two important model parameters. Model predictions are shown to agree with experimental results retrieved from pilot-scale tests. The main novelty of the model is the consideration of a CaO/Al2O3 composite either as main CO2 sorbent used in the carbonator or as a make-up flow fed into the calciner. Using CaO/Al2O3 as the main CO2 sorbent leads to a 14.17% loss of capture efficiency after 100 cycles compared to 72.81% when using limestone derived CaO. It is demonstrated that feeding a very low ratio (~0.1) of the CaO/Al2O3 composite or a rather high ratio (~5) of limestone as makeup flow, leads to a significant improvement of the capture efficiency. The results obtained may be relevant for optimizing CaL operation conditions to be used in power plants.

Experimental investigation of the carbonation reactor in a tail-end Calcium Looping configuration for CO2 capture from cement plants

The calcium looping process is a high temperature post-combustion CO2 capture technology that is expected to be especially suitable for cement plants. Both processes rely on CaCO3 as common feedstock, enabling the reutilisation of purged calcium looping sorbent in the cement clinker manufacturing process. Thus, setting new boundary conditions for the calcium looping process. So far, calcium looping CO2 capture has been demonstrated at semi industrial scale for fossil fuelled power plants but not yet for cement plants. In this work, results obtained from University of Stuttgart's 200 kW calcium looping pilot plant investigating the so called tail-end calcium looping cement plant integration are presented focusing on the carbonation reactor. Different integration levels between the cement plant and the calcium looping process were assessed investigating make-up ratios up to 0.9 mol mol−1 and CO2 concentrations up to 0.33 m3m−3. Operation at high sorbent make-up rates or high integration levels enhanced the sorbent's CO2 carrying capacity whereas for low integration levels the sorbent activity converged towards its residual CO2 carrying capacity. For high sorbent make-up flows, CO2 capture was limited by the carbonation equilibrium yielding CO2 capture rates as high as 98% in the carbonator at carbonation temperatures around 600 ∘C, whereas for low integration CO2 capture was limited by the active amount of CaO being fed to the carbonator. The active space time carbonator model was applied with satisfactory agreement to the conducted experiments indicating the model's validity for calcium looping CO2 capture from cement plant. The obtained results emphasise the suitability of calcium looping CO2 capture for the decarbonisation of the cement sector.

Machine learning-based leakage fault detection for district heating networks

This study proposes a machine learning-based leakage fault detection (LFD) method that locates leakage faults using variation rates of observation data from installed flowmeters and pressure sensors. In the proposed LFD method, a delayed alert triggering algorithm periodically checks the time series of make-up flow rate to identify if the network leaks. When a district heating system is in normal conditions, a hydraulic simulation model built based on the results of impedance identification is used to simulate all possible leakage faults in the network, and the resulting change rates of observation data constitute a leakage data set. An LFD model is then trained using the leakage data set. When the delayed alert triggering algorithm sends a leakage signal, a variation rate vector of observations of this leakage condition is collected. According to the vector, the LFD model can output the name of the leakage pipe. The proposed LFD method is validated in a case study, where the mean values of accuracy and macro-F1 score of the LFD results are 85.85% and 0.99786, respectively. The results show that the proposed LFD method provides a feasible and convenient solution for heat providers to timely and accurately detect leakage faults in networks.

Online coupling of analytical hydrophobic interaction chromatography with native mass spectrometry for the characterization of monoclonal antibodies and related products

Native mass spectrometry (native MS) has seen tremendous development and an increase in application over the past decade for the study of proteins and protein complexes. Although conventionally performed using a static nanospray emitter in an offline fashion, native MS has been increasingly applied in hyphenated methods, where a wide variety of separation techniques are directly coupled to online native MS detection. Those new developments have greatly expanded the utility of native MS in protein biopharmaceutical characterization. Analytical hydrophobic interaction chromatography (HIC) method, although frequently used for the characterization of monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs), has rarely been explored for online coupling with native MS. This is largely due to the high salt concentrations used in HIC analysis that are not compatible with direct MS detection. In this study, we overcame this challenge via an innovative makeup and splitting flow design and successfully achieved online coupling of analytical HIC separation with native MS detection. The development and experimental setup of this HIC-MS method is outlined in detail to elucidate how this design could tackle the high salt concentrations used in HIC separation and ultimately achieve both good chromatographic resolution and MS data quality. Subsequently, the utility of this HIC-MS method was demonstrated in three different applications, where a mAb mixture, mAb molecular variants resulting from PTMs, and a Cys-based ADC mimic were all readily characterized in detail. Unlike previously reported HIC-MS methods, this newly developed method utilizes an analytical scale HIC column with conventional ligand so that the achieved separation profile is highly comparable to those obtained by a standard HIC-UV method. As a result, this HIC-MS method not only provides an alternative approach for in-depth characterization of mAbs and related products during their development but could also be readily applied to assist peak assignment and identity elucidation for the HIC-UV method used in quality control.

Application of open port sampling interface mass spectrometry (OPSI-MS) to deuterium exchange as an aid for structural elucidation.

Deuterium exchange has been demonstrated to provide additional information to accurate mass measurement and collision-induced dissociation on unknown chemical structures. An enhanced method for rapid deuterium exchange could make this technique more routine for structural elucidation. Open port sampling interface mass spectrometry (OPSI-MS) with an aprotic solvent offers a rapid method for performing deuterium incorporation. Samples of standard drug molecules have been analysed by OPSI-MS directly from solids using a make-up flow of acetonitrile + 0.1% trifluoroacetic acid. The resultant spectra were compared with those obtained by OPSI-MS analysis of the samples dissolved in deuterium oxide (D2 O). Solutions of these molecules in acetonitrile/D2 O were analysed using an Atmospheric Solids Analysis Probe (ASAP) at different temperatures to compare the suitability of this technique. The number of exchangeable hydrogens was obtained through deuterium exchange using the OPSI source, although there was some incomplete exchange or back-exchange observed. Molecules with one to five exchangeable hydrogens were successfully analysed. ASAP analysis produced more complicated spectra with higher levels of incomplete or back-exchanged ions; this was more pronounced at higher temperatures. The use of OPSI provides a method for the rapid determination of the number of exchangeable hydrogens within a molecule. This yields useful information as an aid to the structural elucidation of unknowns. ASAP produces incomplete exchange and cannot be used for incorporation studies.