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Temperature Responsive Liquid Chromatography (TRLC) offers an alternative and environmentally friendly way to perform reversed-phase like separations. Its use of temperature responsive polymers to control retention based on column temperature, instead of the fraction of organic modifier in the mobile phase mobile, eliminates the need for solvent composition gradients and allows, for example, for purely aqueous separations. In principle this temperature induced retention should allow for gradient elutions to be performed using downward temperature gradients to control retention and refocus the analyte peaks. Yet, the unavailability of dedicated commercial temperature controlling systems allowing suitable temperature control in TRLC limits implementations thereof often to isothermal or step gradient applications. In this work we study the potential of 1) a simple yet programmable water bath and of 2) a modified HPLC system allowing column temperature programming through controlled mixing of a warm and cold mobile phase streams. The performance of both systems was evaluated under both isocratic and gradient applications, resulting in a more thorough understanding of the influence of temperature gradients in TRLC. This knowledge is then applied to a sample of phenolic solutes, illustrating that, although both systems have some flaws, both are able to impose temperature gradients in TRLC resulting in significantly reduced retention and enhanced refocusing of the analyte peak., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

To allow an enhanced understanding of the order in packed HPLC columns, in this work a methodology for immobilizing native polar silica particles is developed based on the polymerization of a methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) as a cross-linker in the interstitial pores of HPLC columns. Subsequent mechanical cutting then allows scanning electron microscopy (SEM) based imagery of cross-sections of the packed bed. In this way, the packing efficiency of home-made and commercial HPLC columns with 4.6 mm inner diameter and 150 mm length comprising the same packing material of 5 µm silica particles are compared. The methodology is developed for native silica used in e.g. hydrophilic interaction liquid chromatography (HILIC) and in normal phase LC. In order to confirm the feasibility of the developed methodology, the conventional methods for the evaluation of column, efficiency and porosity, are also employed. The obtained porosity information is compared and showed the same trend with the external porosity measurements obtained via inverse size exclusion approach, illustrating its potential application to study the micro-heterogeneity of packed HPLC columns and to guide the optimization of the packing process of HPLC columns., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chiral Stationary-Phase Optimized Selectivity Liquid Chromatography (SOSLC) is proposed as a tool to optimally separate mixtures of enantiomers on a set of commercially available coupled chiral columns. This approach allows for the prediction of the separation profiles on any possible combination of the chiral stationary phases based on a limited number of preliminary analyses, followed by automated selection of the optimal column combination. Both the isocratic and gradient SOSLC approach were implemented for prediction of the retention times for a mixture of 4 chiral pairs on all possible combinations of the 5 commercial chiral columns. Predictions in isocratic and gradient mode were performed with a commercially available and with an in-house developed Microsoft visual basic algorithm, respectively. Optimal predictions in the isocratic mode required the coupling of 4 columns whereby relative deviations between the predicted and experimental retention times ranged between 2 and 7%. Gradient predictions led to the coupling of 3 chiral columns allowing baseline separation of all solutes, whereby differences between predictions and experiments ranged between 0 and 12%. The methodology is a novel tool allowing optimizing the separation of mixtures of optical isomers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable "heteroelement" (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH 2 -groups. The presence of a known number of I atoms in both the drug molecule and its metabolites enabled a cross-validation of the newly developed derivatization procedure and quantification based on monitoring of the introduced Br. The formation of the derivatives was quantitative, providing a 4:1 stoichiometric Br/NH 2 ratio. The derivatives were separated via reversed-phase HPLC with gradient elution. Bromine was determined via ICPMS at a mass-to-charge ratio of 79 using H 2 as a reaction gas to ensure interference-free detection, and iodine was determined at a mass-to-charge ratio of 127 for cross-validation purposes. The method developed shows a fit-for-purpose accuracy (recovery between 85% and 115%) and precision (repeatability <15% RSD). The limit of quantification (LoQ) for Br was approximately 100 μg/L.

A fast, accurate and precise method for the separation and determination of the total contents of drug-related Cl and Br in human blood plasma, based on high performance liquid chromatography - inductively coupled plasma - tandem mass spectrometry (HPLC-ICP-MS/MS), has been developed. The novel approach was proved to be a suitable alternative to the presently used standard methodology (i.e. based on a radiolabelled version of the drug molecule and radiodetection), while eliminating the disadvantages of the latter. Interference-free determination of (35)Cl has been accomplished via ICP-MS/MS using H2 as reaction gas and monitoring the (35)ClH2(+) reaction product at mass-to-charge ratio of 37. Br could be measured "on mass" at a mass-to-charge of 79. HPLC was relied on for the separation of the drug-related entities from the substantial amount of inorganic Cl. The method developed was found to be sufficiently precise (repeatability <10% RSD) and accurate (recovery between 95 and 105%) and shows a linear dynamic range (R(2)>0.990) from the limit of quantification (0.05 and 0.01 mg/L for Cl and Br in blood plasma, respectively) to at least 5 and 1mg/L for Cl and Br, respectively. Quantification via either external or internal standard calibration provides reliable results for both elements. As a proof-of-concept, human blood plasma samples from a clinical study involving a newly developed Cl- and Br-containing active pharmaceutical ingredient were analysed and the total drug exposure was successfully described. Cross-validation was achieved by comparing the results obtained on Cl- and on Br-basis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Capillary electrochromatography (CEC) is in essence a highly efficient and fast separation technique but practical constraints limit the current performance, robustness and routine implementation of the technique. In this work the kinetic performance limit (KPL) curve was used to evaluate commercial packed column CEC; this firstly in order to assess the broader applicability of the kinetic plot approach in electrodriven chromatographic techniques, and secondly to allow a more general unbiased comparison with HPLC performance. Evaluations were performed with a mixture of well retained and electrophoretically neutral phenones, to allow the observation of only chromatographic processes. Initial CEC retention time irreproducibility issues were solved by applying high acetonitrile content (80%) in the mobile phase, and solute retention was increased by increasing the phenone chain length. Comparison was performed with HPLC, with a column packed with an identical stationary phase to allow measurement of the performance under optimal conditions, and not with μ-LC on the CEC column as extra column peak broadening phenomena would thereby negatively affect the μ-LC performance. This comparison demonstrated that current HPLC performance largely outcompetes what is achievable with contemporary packed column CEC. Interestingly, significantly improved CEC performance could be obtained at lower temperatures (10°C) indicating a persistent degree of joule heating phenomena taking place in the contemporary packed column (100μm) CEC approach. Effective suppression of the latter opens possibilities for increasing the applicable voltage and outperforming HPLC and UHPLC., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Accurate quantitative analysis of amino acids forms an important part of targeted metabolic profiling as deviations from normal amino acid profiles can be indicative of several inborn metabolic disorders. A variable-length method development strategy for HPLC analysis on superficially porous particle columns using UV detection is proposed for the separation of 21 primary amino acids. This strategy consists of the evaluation of a large set of gradient and mobile phase compositions on short columns with different stationary phase properties to rapidly determine the best separation conditions leading to the highest number of separated peaks. These conditions are subsequently used to optimize the separation on a longer column length benefitting from its higher separation performance. A minimum critical resolution of 2.0 and a maximum analysis time of 10min were imposed to allow for accurate, robust and rapid quantitation of all amino acids over a large linear range. For amino acids playing a key role in commonly encountered amino acid and urea cycle disorders, a minimum critical resolution of 2.5 was pursued. Mass spectrometry was used for fast peak tracking during the method development procedure. It was demonstrated that the retention behavior of the studied amino acids is more pressure than temperature dependent. This allowed performing the entire method development strategy at the same maximum pressure on all column lengths, while preserving the retention profile, resulting in a significant time profit for the initial scouting runs. The applicability of the method was demonstrated by analyzing serum samples of several neonates diagnosed with metabolic disorders such as maple syrup urine disorder, phenylketonuria and citrullinemia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Chronic kidney disease (CKD) is a devastating illness characterized by accumulation of uremic retention solutes in the body. The objective of this study was to develop and validate a simple, rapid, and robust UPLC-MS-MS method for simultaneous determination, in serum, of seven organic acid uremic retention toxins, namely uric acid (UA), hippuric acid (HA), indoxylsulfate (IS), p-cresylglucuronide (pCG), p-cresylsulfate (pCS), indole-3-acetic acid (IAA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF). Isotopically labeled internal standards (d(5)-HA; 1,3-(15)N(2)-UA, and d(5)-IAA) were used to correct for variations in sample preparation and system performance. Separation on a C18 column was followed by negative electrospray ionization and tandem mass spectrometric detection. Accuracy was below the 15 % threshold. Within-day precision varied from 0.60 to 4.54 % and between-day precision was below 13.33 % for all compounds. The applicability of the method was evaluated by analyzing 78 serum samples originating both from healthy controls and from patients at different stages of CKD. These results were compared with those obtained by use of conventional HPLC-PDA-FLD methods. A good correlation was obtained between both methods for all compounds.

The present contribution investigates the quantitation aspects of mass-sensitive detectors with nebulizing interface (ESI-MSD, ELSD, CAD) in the constant pressure gradient elution mode. In this operation mode, the pressure is controlled and maintained at a set value and the liquid flow rate will vary according to the inverse mobile phase viscosity. As the pressure is continuously kept at the allowable maximum during the entire gradient run, the average liquid flow rate is higher compared to that in the conventional constant flow rate operation mode, thus shortening the analysis time. The following three mass-sensitive detectors were investigated: mass spectrometry detector (MS), evaporative light scattering detector (ELSD) and charged aerosol detector (CAD) and a wide variety of samples (phenones, polyaromatic hydrocarbons, wine, cocoa butter) has been considered. It was found that the nebulizing efficiency of the LC-interfaces of the three detectors under consideration changes with the increasing liquid flow rate. For the MS, the increasing flow rate leads to a lower peak area whereas for the ELSD the peak area increases compared to the constant flow rate mode. The peak area obtained with a CAD is rather insensitive to the liquid flow rate. The reproducibility of the peak area remains similar in both modes, although variation in system permeability compromises the 'long-term' reproducibility. This problem can however be overcome by running a flow rate program with an optimized flow rate and composition profile obtained from the constant pressure mode. In this case, the quantification remains reproducibile, despite any occuring variations of the system permeability. Furthermore, the same fragmentation pattern (MS) has been found in the constant pressure mode compared to the customary constant flow rate mode., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Supercritical fluid chromatography (SFC) is attributed many advantages over high performance liquid chromatography (HPLC). Next to the fact that SFC is greener than HPLC, which is especially important for preparative separations, SFC is claimed to be able to deliver faster separations at higher efficiencies (N) than HPLC. This is due to the higher diffusitivity of analytes in supercritical fluids compared to liquids (higher optimum mobile phase velocity) and to the lower viscosity of the mobile phases in SFC compared to HPLC, which results in smaller pressure drops allowing the use of longer columns and/or columns packed with smaller particles at higher velocities. In order to quantify this claimed kinetic performance advantage, it is essential to construct unbiased kinetic plots to make the comparison between HPLC and SFC. The high compressibility of the mobile phase in SFC however makes this problematic. A variable column length (L) kinetic plot method is therefore developed in this work. Because the pressure history in the column is kept constant for every data point in this method, this way of working definitely delivers exact values for the kinetic performance limits in SFC. It is shown that the traditional way of measuring the performance as a function of flow rate (fixed back pressure and column length) cannot deliver the same correct results as this variable L method. However, the isopycnic way of working on a fixed column length has also been proven to be a good alternative for the expensive and time consuming variable L method. Finally, isopycnic kinetic plots are used to compare SFC and HPLC performance in a quantitative way., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A comprehensive two-dimensional HPLC approach with a high degree of orthogonality was developed for analysis of di- to deca-oligonucleotides (ONs). Hydrophilic interaction liquid chromatography (HILIC) was used in the first dimension, and ion-pair reversed-phase liquid chromatography (IP-RPLC) was employed in the second dimension. The two dimensions were connected via a ten-port valve interface equipped with octadecyl silica (ODS) traps to immobilize and focus the ONs eluting from the first dimension prior to IP-RPLC separation. An aqueous make-up flow was used for effective trapping. The comprehensive two-dimensional HPLC system was optimized with a mixture consisting of 27 oligonucleotide standards. An overall chromatographic peak capacity of 500 was obtained. The use of the volatile buffer triethylamine acetate in the second dimension allowed straightforward coupling to electrospray ionization mass spectrometry (ESI-MS) and detection of each ON in the negative ionization mode., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This contribution discusses the difference in chromatographic performance when switching from the customary employed constant flow rate gradient elution mode to the recently re-introduced constant pressure gradient elution mode. In this mode, the inlet pressure is maintained at a set value even when the mobile phase viscosity becomes lower than the maximum mobile phase viscosity encountered during the gradient program. This leads to a higher average flow rate compared to the constant flow rate mode and results in a shorter analysis time. When both modes carry out the same mobile phase gradient program in volumetric units, normally identical selectivities are obtained. However, small deviations in selectivity are found due to the differences in pressure and viscous heating effects. These selectivity differences are of the same type as those observed when switching from HPLC to UHPLC and are inevitable when speeding up the analysis by applying a higher pressure. It was also found that, when using concentration-sensitive detectors, the constant pressure elution mode leads to identical peak areas as the constant flow rate mode. Also the linearity is maintained. In addition, the repeatability of the peak area and retention time remains the same when switching between both elution modes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We report on a series of explorative experiments wherein a non-porous pillar array column (NP-PAC) is coupled to a commercial capillary LC instrument. The performance of the system was evaluated by both non-retained and retained experiments using several types of samples. In order to minimize interfacing related dispersion, relatively large pillars (d(p)=11 μm) were defined so that a considerable depth could be achieved (50 μm), resulting in an equivalent cylindrical internal diameter of 252 μm. Connecting 20 channel tracks of 1mm wide and 7 cm long by previously developed distributor-based turns, a large channel length (1.4m) and volume (28μl) could be achieved without compromising the separation performance excessively. Establishing a van Deemter curve under non-retained conditions with off-chip injection and detection, a minimal plate height of 13 μm was established, resulting in some 100,000 plates obtained in 30 min. To demonstrate the practical applicability of the NP-PAC, high pressure operation was applied to perform a number of example separations (parabens, phenones, sulfonamides, steroids and BSA digest) during a continuous operation period of 3 months wherein some 500 injections were performed. In the gradient mode, the NP-PAC approach allowed to achieve good to reasonable peak capacities (n(p)=100-140 in 50-70 min) and symmetries for both large and small solutes and for both gradient and isocratic separation modes, with figures of merit for the quantification of the peaks in the ppm range, opening more perspectives for microfluidics-based small molecule analysis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The analysis of anthocyanins in natural products is of significant relevance in recent times due to the recognised health benefits associated with their consumption. In red grapes and wines in particular, anthocyanins are known to contribute important properties to the sensory (colour and taste), anti-oxidant- and ageing characteristics. However, the detailed investigation of the alteration of these compounds during wine ageing is hampered by the challenges associated with the separation of grape-derived anthocyanins and their derived products. High performance liquid chromatography (HPLC) is primarily used for this purpose, often in combination with mass spectrometric (MS) detection, although conventional HPLC methods provide incomplete resolution. We have previously demonstrated how on-column inter-conversion reactions are responsible for poor chromatographic efficiency in the HPLC analysis of anthocyanins, and how an increase in temperature and decrease in particle size may improve the chromatographic performance. In the current contribution an experimental configuration for the high efficiency analysis of anthocyanins is derived using the kinetic plot method (KPM). Further, it is shown how analysis under optimal conditions, in combination with MS detection, delivers much improved separation and identification of red wine anthocyanins and their derived products. This improved analytical performance holds promise for the in-depth investigation of these influential compounds in wine during ageing., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The present study investigates how strong the kinetic plot method is influenced by the changes in plate height, retention factor and apparent column permeability that arise under conditions of very high pressure. More precisely, the study investigates how well a set of performance measurements conducted on a single short column can be used to predict the performance of a long sequence of coupled columns. This has been investigated for the two practically most relevant thermal conditions, i.e., that of a forced-air oven and that of a still-air oven. Measuring column performance data for acetophenone and benzene on a series of coupled 3.5 microm columns that could be operated up to 1000 bar, it was found that the kinetic plot method provides accurate predictions of time versus efficiency for the still-air oven systems, over the entire range of investigated pressures and column lengths (up to 60 cm), provided k' and K(v0) are evaluated at the maximal pressure. For the forced-air oven which leads to worse performances than the still-air oven, the kinetic plot prediction is less accurate, partly because the thermal conditions (near-isothermal) tend to vary if the number of coupled columns increases. The fact that the thermal conditions of the column wall might vary with the column length is an additional complexity making very-high pressure separations less predictable and harder to interpret and model.

The complex anthocyanin fraction of red wines poses a demanding analytical challenge. We have found that anthocyanins are characterised by extremely low optimal chromatographic velocities, and as a consequence generic HPLC methods suffer from limited resolving power. Slow on-column inter-conversion reactions, particularly between carbinol and flavylium species, are shown to occur on the same time scale as chromatographic separation, leading to increased plate heights at normal chromatographic velocities. In order to improve current routine HPLC separations, the use of small (1.7 microm) particles and high temperature liquid chromatography (HTLC) were investigated. 1.7 microm particles provide better efficiency and higher optimal linear velocities, although column lengths of approximately 20 cm should be used to avoid the detrimental effects of conversion reactions. More importantly, operation at temperatures up to 50 degrees C increases the kinetics of inter-conversion reactions, and implies significantly improved efficiency under relatively mild analysis conditions. It is further demonstrated using relevant kinetic data that no on-column thermal degradation of these thermally labile compounds is observed at 50 degrees C and analysis times of <2h.

To contribute to the current debate about the "ideal" particle size range (sub-2 microm vs. supra-2 microm), the present study compares the kinetic performance of some commercially available sub-2 microm and 3.5 microm particles used under quasi-adiabatic conditions via the kinetic plot method. Under the adopted assumption that viscous heating effects can be neglected (which is uncertain in a pressure range above 400 bar), the obtained kinetic plots show that, provided each particle size is used in a column with properly optimized length, the gain in separation speed that sub-2 microm particle columns might have over maximally performing 2.5 microm particle columns is very small. Sub-2 microm particle columns can only yield a gain in separation speed in the range of high-speed/low-resolution-separations (total time based on k=10 below 5 or 10 min). And even in this range, the actual gain that can be expected is only marginally small (only a few %). The present study hence suggests that the development and the use of particles in the 2-3 microm range should deserve more attention than it did in the past few years. However, to be competitive, this 2-3 microm material should be packed in relatively long columns, with a packing quality matching that of the current best performing 3.5 microm particle columns. The supra-2 microm particles should also be able to withstand the same pressures as the sub-2 microm particle material one is comparing it to.

The pressure-drop characteristics of six sub-2 microm columns of three different manufacturers but with the same surface chemistry (C(18)) have been studied using the recently introduced total pore blocking method for the determination of the external porosity and by using scanning electron microscopy pictures to measure the actual size of the particles. Having used the Sauter-mean particle size to correctly account for the particle size spread, and having corrected for the influence of the intra-particle porosity, it was found that all columns yielded Kozeny-Carman constant (f(KC)) values close to 180, in agreement with the theory. This agreement could subsequently be used to quantify how the different system parameters such as mean particle size, packing density and intra-particle porosity (all tending to vary significantly from manufacturer to manufacturer) each contributes to the observed total bed permeability. Small (upward) deviations from the f(KC)=180-value could be correlated to a larger width of the particle size distribution, and more notably to the existence of a high size ratio of the largest to the smallest particles present in the particle batches.

Quantitative analysis of triacylglycerols (TGs) in plant oils and animal fats by normalization of peak areas can lead to erroneous results due to the large response differences with common HPLC detectors between the various TGs. The charged aerosol detector (CAD), that generates an almost universal response for non-volatile compounds, was combined with non-aqueous reversed-phase HPLC (NARP-HPLC) to develop a simple quantitative method, without need for RFs, for the analysis of complex natural TG mixtures from plant oils. Two 25 cm Hypersil ODS columns, connected in series, and a mobile phase gradient composed of acetonitrile, 2-propanol and hexane were used. Mobile phase compensation was applied, by mixing of the column effluent with the inversed gradient delivered by a second HPLC pump, for the suppression of the response dependency of the analytes on the mobile phase composition. Calibration curves of 16 saturated (from C7:0 to C22:0) and 3 unsaturated (C18:1, C18:2, C18:3) single-acid TG standards were measured and their RFs were compared with a previously described method using atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The variation in response of the most common TGs (containing fatty acid chains from 12 to 19 carbons) could be reduced to less than 5% making the combination of NARP-HPLC with CAD and mobile phase compensation an adequate tool for fast quantitative analysis of TGs in common plant oils.