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Spatial comprehensive three-dimensional chromatography (3D-LC) offers an innovative approach to achieve unprecedented resolving power in terms of peak capacity and sample throughput. This advanced technique separates components within a 3D separation space, where orthogonal retention mechanisms are incorporated. The parallel development of the second- and third-dimension stages effectively overcomes the inherent limitation of conventional multidimensional approaches, where sampled fractions are analyzed sequentially. This review focuses on the design aspects of the microchip for spatial 3D-LC and the selection of orthogonal separation modes to enable the analysis of intact proteins. The design considerations for the flow distributor and channel layout are discussed, along with various approaches to confine the flow during the subsequent development stages. Additionally, the integration of stationary phases into the microchip is addressed, and interfacing to mass spectrometry detection is discussed. According to Pareto optimality, the integration of isoelectric focusing, size-exclusion chromatography, and reversed-phase chromatography in a spatial 3D-LC approach is predicted to achieve an exceptional peak capacity of over 30,000 within a 1-h analysis, setting a new benchmark in chromatographic performance.

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Ion mobility-mass spectrometry (IM-MS) is an ever-evolving tool to separate ions in the gas phase according to electrophoretic mobility with subsequent mass determination. CE is rarely coupled to IM-MS, possibly due to similar separation mechanisms based on electrophoretic mobility. Here, we investigate the orthogonality of CE and ion mobility (IM) by analyzing a complex peptide mixture (tryptic digest of HeLa proteins) with trapped ion mobility mass spectrometry (TIMS-MS). Using the nanoCEasy interface, excellent sensitivity was achieved by identifying thousands of peptides and achieving a peak capacity of 7500 (CE: 203-323 in a 150 cm long capillary, IM: 27-31). Plotting CE versus mass and CE versus (inverse) mobility, a clear grouping in curved striped patterns is observed according to the charge-to-size and mass-to-charge ratios. The peptide charge in the acidic background electrolyte can be estimated from the number of basic amino acids, with a few exceptions where neighboring effects reduce the positive charge. A surprisingly high orthogonality of CE and IM is observed, which is obviously caused by solvation effects leading to different charges and sizes in the liquid phase compared to the gas phase. A high orthogonality of CE and ion mobility is expected to be observed for other peptide samples as well as other substance classes, making CE-IM-MS a promising tool for various applications.

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The objective of this work was to provide an unbiased comparison of one-dimensional reversed-phase liquid chromatography (1D-RPLC) and comprehensive two-dimensional RPLC (RPLC × RPLC), through calculations and experimental verifications. For this purpose, various quality descriptors were evaluated, including peak capacity, analysis time, dilution factor, number of runs in the second dimension, and injection volume. The same strategy was applied to small pharmaceuticals and peptides. Whatever the analysis time between 30 and 200 min, short columns of only 30 × 2.1 mm packed with sub-2-µm particles should be selected in both dimensions of the 2D-LC setup to obtain the best compromise in terms of peak capacity and sensitivity. The peak capacity in RPLC × RPLC vs. RPLC was significantly improved for analysis times beyond 5 min. However, extra-column volume located after the second-dimension column was found to be particularly critical for peptides, and up to 50% lower peak capacity was observed with MS vs. UV detection. Contrary to common belief, higher dilution is not always observed in RPLC × RPLC. With adequate analytical conditions, better sensitivity (in theory fivefold and in practice three- to fivefold) could be achieved in RPLC × RPLC compared to 1D-RPLC, regardless of the analysis time.

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Reversed-phase peptide separation in hydrogen deuterium exchange (HDX) mass spectrometry (MS) must be done with conditions where the back exchange is the slowest possible, the so-called quench conditions of low pH and low temperature. To retain maximum deuterium, separation must also be done as quickly as possible. The low temperature (0 °C) of quench conditions complicates the separation and leads primarily to a reduction in separation quality and an increase in chromatographic backpressure. To improve the separation in HDX MS, one could use a longer gradient, smaller particles, a different separation mechanism (for example, capillary electrophoresis), or multi-dimensional separations such as combining ion mobility separation with reversed-phase separation. Another way to improve separations under HDX MS quench conditions is to use a higher flow rate where separation efficiency at 0 °C is more ideal. Higher flow rates, however, require chromatographic systems (both pumps and fittings) with higher backpressure limits. We tested what improvements could be realized with a commercial UPLC/UHPLC system capable of ∼20,000 psi backpressure. We found that a maximum flow rate of 225 µL/min (using a 1 × 50 mm column packed with 1.8 µm particles) was possible and that higher flow rate clearly led to higher peak capacity. HDX MS analysis of both simple and particularly complex samples improved, permitting both shorter separation time, if desired, and providing more deuterium recovery.

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In this research, we developed an online comprehensive two-dimensional liquid chromatographic (LC × LC) method hyphenated with high-resolution mass spectrometry (HRMS) for the non-targeted identification of poly- and perfluorinated compounds (PFASs) in fire-fighting aqueous-film forming foams (AFFFs). The method exploited the combination of mixed-mode weak anion exchange-reversed phase with a octadecyl stationary phase, separating PFASs according to ionic classes and chain length. To develop and optimize the LC × LC method we used a reference training set of twenty-four anionic PFASs, representing the main classes of compounds occurring in AFFFs and covering a wide range of physicochemical properties. In particular, we investigated different modulation approaches to reduce injection band broadening and breakthrough in the second dimension separation. Active solvent and stationary phase assisted modulations were compared, with the best results obtained with the last approach. In the optimal conditions, the predicted peak capacity corrected for undersampling was higher than three-hundred in a separation space of about 60 min. Subsequently, the developed method was applied to the non-targeted analysis of two AFFF samples for the identification of homologous series of PFASs, in which it was possible to identify up to thirty-nine potential compounds of interest utilizing Kendrick mass defect analysis. Even within the samples, the features considered potential PFAS by mass defect analysis elute in the chromatographic regions discriminating for the ionic group and/or the chain length, thus confirming the applicability of the method presented for the analysis of AFFF mixtures and, to a further extent, of environmental matrices affected by the AFFF.

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Retention prediction of monoamine neurotransmitters has been compared for the generally applied linear solvent-strength model and quadratic polynomial three-parameter model. The design of experiments protocol has been applied to plan linear gradients within the experimental space with altered gradient time, mobile phase flow rate, and column temperature. Relative prediction errors increased at elevated temperature, which is more significant for the linear solvent-strength model when compared to the polynomial model. On the other hand, the predefined design of experiments space controls the retention time errors, as predictions for LC conditions that are outside of the plan are much less accurate and should be avoided. The final part of the work deals with the effect of extracolumn band dispersion on the peak capacity of linear gradients at various gradient times, mobile phase flow rates, and column temperature. The peak capacity determined for corrected experimental data were consistent with the published results dealing with the optimization of peak capacity in gradient elution.

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The application area of supercritical fluid chromatography expanded tremendously over the last years and more polar analytes such as biomolecules have become accessible. The growing interest in biopharmaceuticals and associated regulatory requirements demand alternative analytical tools. The orthogonal nature of supercritical fluid chromatography compared to reversed-phase liquid chromatography meets these needs and makes it a useful option during research and development. In this study, we present a systematic approach for the development of a supercritical fluid chromatography method for fingerprinting of tyrothricin, a complex therapeutic peptide covering a mass range from 1200 to 1900 Da. The substance was chosen due to the presence of cyclic and linear peptides and isomeric or highly similar amino acid sequences. Different column chemistries covering neutral, basic, and zwitterionic functionalities in combination with acidic, basic, and neutral additives were screened. Subsequently, Design-of-Experiments principles were utilized to perform optimization of the chromatographic parameters. The final mass spectrometry-compatible gradient method using a diol stationary phase, carbon dioxide, and a modifier consisting of methanol/water/methanesulfonic acid (100:2:0.1, v:v:v) was found to provide orthogonality and superior resolution to other methods published. Isomeric peptide compounds coeluting in reversed-phase liquid chromatography were resolved by applying the final method.

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The definition of a chromatographic analysis speed based simply on analysis time is an outdated concept to define conventional chromatography, fast chromatography, and emerging high-resolution techniques such as comprehensive two-dimensional and comprehensive three-dimensional gas chromatography. Here, the metric average theoretical peak time (ATPT) is proposed for separation speed, considering conventional and multidimensional separations. ATPT can be defined as the time (in ms per peak) needed to elute a theoretical peak in a chromatographic system. Using this metric, it is possible to define ranges, proposed for a normal speed (ATPT higher than 4000 ms/peak), high speed (ATPT range from 600 to 4000 ms/peak), very high speed (ATPT range from 200 to 600 ms/peak), hyper speed (ATPT range from 3.3 to 200 ms/peak) and ultra high speed chromatography (ATPT lower than 3.3 ms/peak), that combines time and efficiency metrics. This metric was applied in several contexts to demonstrate its robustness to evaluate chromatographic separations for different techniques and analytical conditions. Applications also demonstrate the advantages of the use of ATPT as a method development metric tool.

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We performed a systematic study of thirteen alkylamines used as ion-pairing reagents for ion-pair reversed-phase liquid chromatography (IP RP LC) separations of oligonucleotides on a C18 column. We proposed a method to classify the hydrophobicity of alkylamines by their retention in RP LC. The IP reagent hydrophobicity correlated with the retention and resolution of oligonucleotides in the corresponding IP mobile phases. The baseline resolution was achieved up to 30 mer for hydrophilic, or up to 50 mer for hydrophobic IP reagents. Hydrophobic alkylamines permitted useful oligonucleotide separations at relatively low buffer concentrations, such as 5-10 mM alkylamine-acetate IP systems. These buffers were compatible with mass spectrometry detection, however, replacement of acetic acid with hexafluoroisopropanol in the mobile phase improved the MS signal by 2-3 orders of magnitude. Experiments with native and chemically modified oligonucleotides highlighted the mixed-mode nature of IP RP LC. When using hydrophobic IP reagents, the ionic retention mechanism of oligonucleotides is enhanced while hydrophobic retention is diminished.

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A general and deterministic model is derived from the fundamentals of liquid chromatography to calculate retention time, peak width, peak capacity, and density of peak capacity in gradient liquid chromatography. The calculation of these chromatographic properties accounts for 1) the presence of initial (separation of the earliest eluters) and final (column wash) isocratic steps before and after the linear gradient, respectively, 2) the pre- (flow through needle and preheater tubes) and post-column (outlet and emitter tubes before MS detection) dispersion, 3) the compression of the chromatographic band, and 4) the retention of the organic modifier onto the RPLC column. The multiple and variable method parameters may include the column dimensions, particle size, flow rate, temperature, initial and final isocratic hold times, gradient time, gradient steepness, column conditioning/sample load time, and the pre- and post-column tube dimensions. The model enables the users to perform robust multi-dimensional optimization of UHPLC-MS methods and offers the possibility to predict the expected MS feature density for increased method performance. Method optimization can be further improved by matching the observed MS feature density (number of metabolites detected as function of time) to the predicted density of peak capacity. It is directly applied to the optimization of high-throughput RPLC separation methods specifically designed for large-scale urinary metabolic phenotyping.

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Structures for Lossless Ion Manipulations (SLIM) is a powerful variant of traveling wave ion mobility spectrometry (TW-IMS) that uses a serpentine pattern of microelectrodes deposited onto printed circuit boards to achieve ultralong ion path lengths (13.5 m). Ions are propelled through SLIM platforms via arrays of TW electrodes while RF and DC electrodes provide radial confinement, establishing near lossless transmission. The recent ability to cycle ions multiple times through a SLIM has allowed ion path lengths to exceed 1000 m, providing unprecedented separation power and the ability to observe ion structural conformations unobtainable with other IMS technologies. The combination of high separation power, high signal intensity, and the ability to couple with mass spectrometry places SLIM in the unique position of being able to address longstanding proteomics and metabolomics challenges by allowing the characterization of isomeric mixtures containing low abundance analytes.

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Eicosanoids - oxidative derivatives from arachidonic acid - represent biologically active lipid mediators in inflammatory processes. Different analytical methods treat eicosanoid analysis. Among which, reverse phase liquid chromatography figures as the appropriate method for eicosanoid profiling. RP-HPLC for eicosanoid analysis is often conducted on C18 columns. Some studies focused on profiling one family of eicosanoids; others considered all eicosanoid families. In both cases, co-elution remained a major issue and detection in mass spectrometry partially resolves this problem. In fact, the mass transitions used to monitor eicosanoid species are not specific enough and many isobars can be listed. For this, optimizing the RP-HPLC separation remains important. Based on the parameter Fs - deriving from the hydrophobic-subtraction model - and radar plots, we chose columns with different selectivities. The hydrophobic-subtraction model guided our interpretation of molecular interactions between eicosanoids and stationary phases. We founded our approach for selectivity optimization on peak capacity per minute and time needed values. Herein, we screened seven stationary phases and evaluated their chromatographic performances in RP-HPLC. Stationary phases presented different chemistry, type of silica, length, and particle size. Superficially porous particle columns registered better chromatographic profiles than classical stationary phases; and columns with embedded polar group did not serve our purpose. The stationary phase Accucore C30 - even being the least retentive - revealed the best selectivity and efficiency, and recorded the shorter duration for eicosanoid analysis.

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Bioactive peptides are increasingly used in clinical practice. Reversed-phase chromatography using formic or trifluoroacetic acid in the mobile phase is the most widely used technique for their analytical control. However, sometimes it does not prove sufficient to solve challenging chromatographic problems. In the search for alternative elution modes, the dynamic electrostatic repulsion reversed-phase was evaluated to separate eight probe peptides characterised by different molecular weights and isoelectric points. This technique, which involves TBAHSO4 in the mobile phase, provided the lowest asymmetry and peak width at half height values and the highest in peak capacity (about 200 for a gradient of 30 min) and resolution concerning the classic reversed-phase. All analyses were performed using cutting-edge columns developed for peptide separation, and the comparison of the chromatograms obtained shows how the dynamic electrostatic repulsion reversed-phase is an attractive alternative to the classic reversed-phase.

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This study reports on the assessment of the separation performance of hydrophobic interaction chromatography for intact protein analysis using non-porous butyl polymethacrylate phases. The maximum peak capacity in inverse gradient mode was reached at a volumetric flow rate which was significantly (10-20 times) higher than the flow rate yielding the minimum plate height in isocratic mode, as the gradient volume dominates the peak-capacity generation. The flow rate yielding the maximum peak capacity increased with decreasing gradient volume, i.e., steeper gradients, and also depends on the magnitude of the mass-transfer contribution to peak dispersion (affected by particle size and molecular diffusion coefficient of proteins) at these high flow rates. The maximum peak capacity using a 100 mm long column packed with 4 µm particles for steep 7.5 min gradients was determined to be 60. Increasing the column length by coupling columns leads to better gradient performance than increasing the gradient duration for gradients of 60 min and longer. Using a coupled column system (2 × 100 mm long columns packed with 4 µm particles), the maximum peak capacity was determined to be 105, which was 33% higher compared to that of a single column while applying a similar gradient volume. Decreasing the particle size to 2.3 µm leads to higher peak capacities even though the column was operated at lower volumetric flow rate. The maximum peak capacity obtained with the 2.3 µm column was 128% higher than was obtained with the coupled column. Even at suboptimal conditions, the 2.3 µm column yields a higher peak capacity (14%) than when using two coupled columns packed with 4 µm at optimal conditions (gradient time of 120 min and a flow rate of 0.5 mL/min).

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In a previous work, we explored zone broadening and the achievable plate numbers in linear drift tube ion mobility-mass spectrometry through developing a plate-height model [1]. On the basis of these findings, the present theoretical study extends the model by exploring peak-to-peak resolution and peak capacity in ion mobility separations. The first part provides a critical overview of chromatography-influenced resolution equations, including refinement of existing formulae. Furthermore, we present exact resolution equations for drift tube ion mobility spectrometry based on first principles. Upon implementing simple modifications, these exact formulae could be readily extended to traveling wave ion mobility separations and to cases when ion mobility spectrometry is coupled to mass spectrometry. The second part focuses on peak capacity. The well-known assumptions of constant plate number and constant peak width form the basis of existing approximate solutions. To overcome their limitations, an exact peak capacity equation is derived for drift tube ion mobility spectrometry. This exact solution is rooted in a suitable physical model of peak broadening, accounting for the finite injection pulse and subsequent diffusional spreading. By borrowing concepts from the theoretical toolbox of chromatography, we believe that the present study will help in integrating ion mobility spectrometry into the unified language of separation science.

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Mixed-mode chromatography open tubular column has been developed for peptide separation in electrochromatography. A column with 92 cm effective length and 50 µm internal diameter is fabricated internally with a copolymer sheet of restricted thickness. Catalyst facilitated binding of the coupling agent 3,5-bis (trifluoromethyl) phenyl isocyanate has been carried out at the interior surface of the column. The initiator sodium diethyldithiocarbamate was bound to the coupling agent. A small amount of N-[2-(acryloylamino) phenyl] acrylamide was used along with methacrylic acid and styrene in the monomer mixture to induce a little polar character in the stationary phase fabricated inside the column. Twenty-three peptides have been separated from a chemically digested protein mixture present in cytochrome C in capillary electrochromatography, in addition to the separation of six commercial peptides. We achieved an average plate count of over 1.5 million/m with the column of current study both for the digested protein components and commercial peptides using 70/30% v/v (acetonitrile/20 mM ammonium formate) at pH 6.5. In addition, the column resulted in baseline separation of all the peptides with very good resolution, enhanced peak capacity, and better retention time span.

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Cryogenic methods - those that employ cryogenic fluids/gases but also other approaches to generate reduced temperature - are versatile, functional and relatively easily implemented as part of a total gas chromatographic method. The general utility of a cold region is almost invariably as a trapping or focussing step, to collect analyte into a sharp zone. The success in effectively trapping analyte depends on analyte volatility and the temperature of the cold region. Analytes collection into a sorbent phase supported by cryotrapping usually provide a greater capacity trapping for the sorption step. Stripping analyte from a sample into a cryogenic trap, with subsequent introduction to GC as in a purge-and-trap method, sample introduction into an injector with incorporation of a cooling zone, manipulation and management of chromatographic bands during chromatography elution such as employed in multidimensional gas chromatography, and focussing analyte just prior to the detector, all have the same goal of concentrating the band, reducing its dispersion, and maximising response. This review summarises various approaches that demonstrate how cryogenic methods have been incorporated into gas chromatographic analysis.

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The detection, identification and quantification of drug metabolites plays a key role in drug discovery and development. Liquid chromatography (LC) coupled to mass spectrometry (MS) has become the primary technology for these studies due to its sensitivity and specificity. However, the presence of transition metals in the chromatography system and columns can result in non-specific and unwanted interactions with the drug and/or its metabolites, via electron-pair donation, leading to poor chromatography and analyte loss. The use of a hybrid organic/inorganic surface applied to the metal surfaces of the chromatography system and column has been demonstrated to reduce or eliminate these effects. When employed for the analysis of mouse urine, derived from the oral dosing of mice with the EGFR inhibitor gefitinib, we observed more symmetrical LC peaks. This resulted in a 33 % improvement in peak capacity for a 10 min reversed - phase gradient separation, a two-fold increase in MS response, cleaner MS spectra and improved peak response reproducibility. This hybrid surface barrier appears to offer significant advantages in the analysis of low-concentration metabolites, potentially facilitating the accurate determination of the elimination phase of the pharmacokinetic (PK) curve and detection of drug metabolites in microdosing or microsampling studies.

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In this study, thin-layer chromatography was applied for selective extraction of volatile compounds in perfume prior to analysis with solid phase microextraction and gas chromatography-mass spectrometry. The standard compounds were desorbed from the thin-layer chromatography plate and extracted at 80°C for 15 min showing good linearity of the calibration curves (R2  > 0.98) and acceptable recovery range (65-81%). The plate after the separation was cut into four smaller parts followed by solid phase microextraction/gas chromatography-mass spectrometry analysis, which revealed different compound profile in each part with the correlation between log P of the standard compounds and their positions along the thin-layer chromatography plate (R2  = 0.65). This approach was applied to analyze perfume compounds in the sample with strong matrix interference from the synthetic agarwood. Terpene hydrocarbons (woody-based odors), ketones/esters, aldehydes, ethers, and alcohols were mostly observed at 8 ± 1, 6 ± 1, 5 ± 2, 4 ± 2, and 3 ± 2 cm, respectively, from the bottom of the thin-layer chromatography plate. While, the conventional solid phase microextraction/gas chromatography-mass spectrometry analysis of this sample solution revealed only 62 compounds (including 35 perfume compounds), the four-piece approach resulted in 109 compounds (62 perfume compounds). Furthermore, the capability of thin-layer chromatography-Gas Analyzer approach to analyze the isomers in this complex sample was demonstrated.