This study represents the first extensive residue analysis of prehistoric pottery from northern Belgium. It examines pottery use and culinary practices across the Mesolithic-Neolithic transition, from the late 6th to the early 4th millennium cal BC. Residue analyses were performed on more than 200 samples from nine archaeological sites, representing different cultural groups from this transitional phase. This includes the analysis of charred food residues encrusted on the vessel surfaces by elemental analysis-isotope ratio mass spectrometry (EA-IRMS), gas chromatography-mass spectrometry (GC-MS), stereomicroscopic analysis and Scanning Electron Microscopy (SEM), as well as the analysis of absorbed lipids by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). This study provides the first evidence of ruminant dairy fats in Early Neolithic Limburg pottery, supporting the hypothesis that this pottery was produced and used by LBK farmers rather than hunter-gatherer populations. The first indigenous pottery of the Swifterbant culture was frequently used to process freshwater fish (often together with plant foods) and ruminant meat, although several of the studied vessels likely contained mixtures of resources which could also include porcine products. Ruminant dairy is nearly absent from this pottery. Similar results were obtained for pottery of the subsequent Michelsberg culture/Group of Spiere of the late 5th and early 4th millennium cal BC. The limited presence of ruminant dairy fats in this pottery contrasts with the findings for Middle Neolithic pottery from neighbouring regions, providing further evidence for the existence of regional variations in pottery use or culinary practices throughout prehistoric NW Europe. However, our current view of pottery use during the Mesolithic-Neolithic transition in northern Belgium might be biased by the difficulties in distinguishing between wild and domesticated ruminant adipose fats as well as in detecting plant foods through lipid residue analysis.

In theory, comprehensive two-dimensional liquid chromatography (2D-LC) allows for significant enhanced peak capacity compared to one-dimensional high performance LC (1D-HPLC). However, reaching such a separation performance while also obtaining robust, easily implementable, and sensitive methods proves challenging. Because it can hinder the broader use of 2D-LC, there is a need for developing easier, more trouble-free approaches that feature the benefits of LC×LC while not compromising with what can be done with 1D-HPLC. Commercial 2D-LC interfaces are based on two-position multiport valves composed of two loops, which are alternatingly used either to collect the effluent from the first dimension (1D), or to inject their content to the second dimension (2D). This design implies that if large sampling volumes transferred to the second column need to be avoided, a comparatively (much) higher flow rate and broader column i.d. is required for the second column. However, doing so can lead to a loss in sensitivity because of dilution and impractical analytical chromatography as a result of the high flow rates involved. In most LC×LC column combinations, this problem is exacerbated thanks to the high eluotropic strength of transferred loop volumes. However, when the elution strength of the transferred solvent is very small, refocusing the analytes can be obtained, which allows the user to overcome such issues. For example, this is the case when a purely aqueous separation mode is combined with reversed-phase LC (RPLC) in the 1D and 2D, respectively. Temperature responsive LC (TRLC), which is an emerging LC mode requiring only water as the mobile phase and whereby retention is controlled via temperature only, is promising in this context. In this second installment about TRLC, we illustrate the unique benefits of the combination of this separation mode with RPLC in comprehensive 2D-LC. The potential of the approach is shown through the analysis of representative standard mixtures, active pharmaceutical ingredients, synthetic impurities and phenolics in natural products.

Comprehensive two-dimensional liquid chromatography (LC×LC) can provide enhanced resolving power and higher peak capacities for the separation of complex samples. The transfer of fractions of too high eluotropic strength from the first dimension, however, can lead to peak broadening. This process is related to the column dimensions, the flow rates, mobile phase compositions, and stationary phase compatibility. Temperature-responsive LC (TRLC) uses a smart polymer coupled to silica (poly(N-isopropylacrylamide), pNIPAAm), that exhibits a change in polarity upon modest variations in column temperature. Retention is thus modulated by temperature and not by organic solvents, allowing for the use of purely aqueous mobile phases. As these aqueous mobile phases depict a very low eluotropic strength on a reversed-phase column, it facilitates band refocusing at the second-dimension column head in TRLC×RPLC. One of the remaining obstacles of TRLC is the long analysis time. In this research, the potential of this column combination in terms of analyte refocusing will be exploited. First, it is shown that upwards flow gradients can be implemented in the first dimension of TRLC×RPLC. As the flow in the second dimension is maintained at a constant level, a first-dimension flow gradient does not lead to impaired sensitivity and has no negative effects on the resulting peak size. Then, the novel combination of a downwards temperature gradient with an upwards flow gradient will be introduced to speed up the analyses further. Analysis time was, depending on the method used, reduced by 36-54%, as demonstrated by the analysis of mixtures of food additives, phenolic compounds, and small molecule pharmaceuticals mimicking impurity analysis at a 0.05% level.

In recent years, TRLC was introduced as a separation mode that offers interesting features when implemented in the first dimension (1D) of 2D-LC. The separation mode is based on the coupling of stimuli-responsive polymer to the silica supporting material. Commonly, poly(N-isopropyl acrylamide) is used, which depicts increasing retention of hydrophobic compounds for rising temperatures. One of the major benefits of this type of stationary phase is that these effects occur in water, thus allowing for the usage of purely aqueous mobile phases. In combination with RPLC, this establishes a great advantage in terms of robust modulation, as full solute refocusing at the 2D column head is obtained without methodical complexity for solutes which can be retained in RPLC. With more commonly used LC×LC combinations, this transfer of 1D effluent is often hampered due to the peak distorting effects of transferring high organic content. It was shown in TRLC×RPLC, that the volume transferred can be chosen freely, tested for transfer volumes of 10-2000 µL without peak broadening. The combination of the analyte refocusing and the resulting almost unlimited transferable injection volumes in 2D also paves the way for the implementation of flow rate gradients in the 1D. In this way, in this work, the possibilities offered by TRLC in 2D-LC are explored to reduce analysis times while maintaining or improving the overall method performance. This can be done via the usage of single-component type gradients (comprising either a decreasing temperature or increasing flow rate gradient) or combined dual-component gradients (combining temperature and flow rate gradients). It is shown that a change in flow rate (e.g. from 100 - 400 µL/min) and the consequent increase in modulation volume has little to no effect on the 2D separation. The possibilities of the approach are demonstrated through the separation of representative mixtures of small pharmaceuticals and food additives.

Refractive index detection (RID) is attractive because it allows approaching the benefits of universal detection with liquid chromatography, by which ideally standard independent calibration and hence compound independent quantification becomes possible. Nevertheless, the implementation of RID has remained limited as it offers poor detection sensitivity while only being compatible with isocratic mobile phases. The implementation of compositional solvent gradients has remained prohibitively challenging in commercial HPLC-RID systems due to the resulting drastic alterations in refractive index and extreme baseline drift. While the refractive index is also highly dependent on temperature, more leeway appears possible to mitigate the problem, particularly when the used temperature gradients can be limited. Temperature-responsive liquid chromatography (TRLC) allows obtaining isocratic reversed phase type of separations, whereby retention is modulated via temperature changes similar to 15 degrees C-20 degrees C above and below the polymer conversion temperature. Elution profiles, reminiscent of what can be obtained with solvent gradients in conventional RPLC, can then be obtained by enacting downwards temperature gradients on the columns. This work comprises a proof-of-principle to illustrate the possibilities of combining thermal gradient TRLC with RID. The observed baseline drift appeared thereby very minor (<5 nRIU min(-1)), and hence easily controllable. Short chain fatty acids are used as representative compounds to assess this new approach. Overlapping calibration lines are accordingly obtained for all fatty acids between butyric and decanoic acid.

Temperature-responsive liquid chromatography (TRLC) is an emerging green high performance liquid chromatography (HPLC) mode allowing reversed phase-type separations while necessitating only water as the mobile phase. The columns therein are typically packed with silica particles to which stimuli-responsive polymers are anchored. In hydrophobic interaction TRLC, such polymers depict a loss of water solubility when increasing the temperature above a characteristic conversion temperature, causing large changes in retention over quite narrow and mild temperature ranges (~5–55 °C). TRLC circumvents the concerns about analyte or column degradation that can occur when implementing high temperatures (>80 °C) on conventional reversed- phase columns. It allows for high performance liquid chromatography (HPLC) using only water often spiked with the additives typically used in reversed-phase LC. Therefore, this separation mode allows for greener, cheaper, and isocratic analyses under non-denaturing conditions. The absence of compositional solvent gradients also allows for the exploitation of temperature gradients in combination with refractive index detection. Purely aqueous hydrophobic interaction TRLC is mostly applicable for solutes depicting a 1 < LogP < 5, yet these ranges can be expanded through implementation of combined aqueous or organic mobiles phases, while preserving the temperature-responsive effects. In this first TRLC installment, our recent developments, new possibilities, and current limitations of the use of 1-D TRLC are discussed, while the column performance is described with respect to the fundamentals of HPLC.

Achievable sensitivity of online 2D-LC systems is still hampered compared to conventional (U)HPLC, as sensitivity is often traded off to increase peak capacity. This limitation is mostly imposed by the modulation problem. To maximize orthogonality, less compatible separation modes are combined. Thus, solvents of high elution-strength are transferred between the two dimensions. A possible solution is the use of narrow columns in the 1D, maintained at low flow rates to naturally decrease the transferred volumes, but consequently, sample-loading capacities are also lowered. To minimize these effects and allow for fast gradients, the second dimension (2D) column then often must be rather broad, operated at high flow rates. The combination of a 1 mm column in the 1D paired with a 4.6 mm column in the 2D, however, leads to a >20-fold loss in sensitivity for concentration-sensitive detectors due to the dilution of the sample in the 2D flow when assuming comparable flow velocities. The latter is in reality even further exacerbated in LC×LC due to the shorter residence times in the fast flow operated second dimension. RPLC×RPLC allows for some more freedom in column choices, nowadays more often a 2 mm ID column is combined with a 3 mm ID column, also lowering detection sensitivity. Rarely, 2 mm columns are implemented in the 2D, as this is mostly only applicable if aqueous mobile phases are used in the 1D, e.g in IEX×RPLC. Nevertheless, it was shown that 2D-LC is promising, also for the determination of low abundant impurities at a 0.05% level in pharmaceutical quality control if methodical parameters are optimized for high sensitivity. Here, temperature-responsive liquid chromatography (TRLC) is paired with RPLC. Such stationary phases allow for separation in a purely aqueous mobile phase, which solves some of the methodical complexities discussed above. Because full solute refocusing at the 2D column head is obtained in TRLC×RPLC, the transferred volume of 1D effluent is not a determining factor. Thus, the dilution problem experienced with all concentration-sensitive detectors in comprehensive 2D-LC offers the prospect to be overcome, as columns of the same diameter in both dimensions can be used (2.1×2.1 mm I.D.). Optimally, the detrimental dilution problem can even be inversed towards a refocusing based sensitivity enhancement, by combining a broader 1D column with a narrower 2D column (2.1×1 mm I.D.). In this work these aspects are demonstrated through comparative LOD determinations for various column I.D. combinations in comprehensive TRLC×RPLC-UV. Addiontally, split-less transfer of the 2D effluent to the MS from the 1 mm column is introduced. This strategy allows more facile and more sensitive 2D-LC-MS/UV implementations.

Phenolic compounds are an interesting class of natural products because of their proposed contribution to health benefits of foods and beverages and as a bio-source of organic (aromatic) building blocks. Phenolic extracts from natural products are often highly complex and contain compounds covering a broad range in molecular properties. While many 1D-LC and mass spectrometric approaches have been proposed for the analysis of phenolics, this complexity inevitably leads to challenging identification and purification. New insights into the composition of phenolic extracts can be obtained through online comprehensive two-dimensional liquid chromatography (LC × LC) coupled to photodiode array and mass spectrometric detection. However, several practical hurdles must be overcome to achieve high peak capacities and to obtain robust methods with this technique. In many LC × LC configurations, refocusing of analytes at the head of the 2D column is hindered by the high eluotropic strength of the solvent transferred from the 1D to the 2D, leading to peak breakthrough or broadening. LC × LC combinations whereby a purely aqueous mobile phase is used in the 1D and RPLC is used in the 2D are unaffected by these phenomena, leading to more robust methods. In this contribution, the combination of temperature-responsive liquid chromatography (TRLC) with RPLC is used for the first time for the analysis of phenolic extracts of natural origin to illustrate the potential of this alternative combination for natural product analyses. The possibilities of the combination are investigated through analysis of wine extracts by TRLC × RPLC-DAD and TRLC × RPLC-ESI-MS.

In comprehensive two-dimensional liquid chromatography (LC × LC), solvents of high eluotropic strength are frequently used in the first dimension (1D), which lead to peak broadening in the second dimension (2D). In the majority of the current LC × LC column combinations, analytes are less than optimally refocused upon transfer to the second column, which negatively affects sensitivity. Furthermore, the typical combination of 1 or 2.1 mm columns in the 1D paired with a 3 mm (or broader) column in the 2D leads to at least a 9- or 4-fold dilution and a corresponding loss of sensitivity when using concentration-sensitive detectors. This occurs due to the enhanced radial dilution of the analytes in a broader column, while the sensitivity problem is further exacerbated in LC × LC due to the high flow operated 2D. In this paper, we introduce a solution to neutralize and inverse this dilution problem through a reconcentrating solution using temperature-responsive liquid chromatography (TRLC) in the 1D, which is a purely aqueous separation mode. Full solute refocusing at the 2D column head is thereby obtained when TRLC is combined with reversed-phase liquid chromatography (RPLC). This is shown for the combination of a 2.1 mm I.D. TRLC column with decreasing RPLC column diameters (3-2.1-1 mm) operated at the same linear velocities, hence a resulting decrease in dilution, respectively. Ultraviolet (UV) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) detection were used to determine the experimental detection limits. Sensitivity improvements with UV detection were somewhat lower than expected, but represent ∼1.5- and 3-fold sensitivity enhancement when using a 1 mm I.D. column compared to 2.1 or 3 mm I.D. columns in the 2D, respectively. This is attributed to extra-column dispersion and the poorer performance of 1 mm I.D. columns. A major benefit of the use of 1 mm I.D. columns in the 2D is that it allows split-free coupling of 2D effluent with ESI-MS (at 450 μL/min), making the coupling robust and simple. When using ESI-MS even better, albeit more variable, sensitivity enhancements were obtained on the narrower columns. The benefits of the methodology are demonstrated for paraben test solutes and for phenolic compounds in a blueberry extract by TRLC × RPLC-UV-ESI-TOF-MS.

Temperature-responsive liquid chromatography (TRLC) allows for extensive retention and selectivity tuning through temperature in HPLC. This is mainly achieved through the use of a stationary phases comprising of a temperature-responsive polymer which undergoes a reversible change from hydrophilic to hydrophobic behaviour upon increasing the temperature. The approach can allow for reversed phase type separations to be achieved with purely aqueous mobile phases, whereby the retention is controlled through temperature instead of mobile phase composition. Despite the promising nature of such form of retention control under isocratic mobile phase conditions, TRLC can suffer from excessive retention of highly apolar solutes even at lower column temperatures whereby the polymer is considered hydrophilic. This is related both to a residual apolarity of the polymer chain and due to the high log P's and low water solubility of higly apolar compounds. While it was known that elution in TRLC doesn't necessarily has to be performed under purely aqueous conditions and that the use of organic co-solvents to the water is possible, the impact thereof on the temperature responsive behaviour itself had not yet been investigated in a systematic way. Therefore in this work the advantages and drawbacks of the use of the organic co-solvents methanol and acetonitrile in TRLC is assessed on two types of temperature reponsive phases: poly-N-N-propylacrylamide (PNNPAAm) and poly-N-isopropylacrylamide (PNIPAAm). The influence of organic co-solvents is investigated with two representative test mixtures (comprising 4 parabens and 5 apolar steroids).

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.

A comprehensive two-dimensional liquid chromatography-based biomimetic platform (LCxLC) has been developed and validated for drug diffusion studies. Human serum albumin (HSA) and immobilized artificial membrane (IAM) were thereby used in the first (D-1) and second (D-2) separation dimension, respectively. While the former was meant to emulate the blood, the latter was instead intended to mimic the intestinal mucosa epithelium. Therefore, the experimental conditions, i.e. pH, temperature and buffer composition, were modulated to reflect faithfully in vivo conditions. 30 compounds, whose effective intestinal permeability (Peff) assayed in situ on humans by a validated technique was known from the literature, were used as model drugs. A good and orthogonal separation was achieved for the whole dataset, although for a better distribution of the most polar compounds in the elution window a segmented gradient elution program had to be employed. Interestingly, the passively uptaken compounds having the most favourable P-eff populated a specific area of the 2D plots, implying that the affinity for HSA and IAM has to lie in specific ranges in order for a compound to be satisfactorily absorbed from the intestinal lumen. Although these results should be regarded as preliminary, this work paves an entirely new and unprecedented way to profile pharmaceutically relevant compounds for their in vivo absorption and distribution potential. (C) 2020 Elsevier B.V. All rights reserved.

Temperature responsive liquid chromatography (TRLC) allows for separation of organic solutes in purely aqueous mobile phases whereby retention is controlled through temperature. The vast majority of the work has thus far been performed on poly[N-isopropylacrylamide] (PNIPAAm)-based columns, while the performance of other temperature responsive polymers has rarely been compared under identical conditions. Therefore, in this work, two novel TRLC phases based on poly[N-n-propylacrylamide] (PNNPAAm) and poly[N,N-diethylacrylamide] (PDEAAm) are reported and compared to the state of the art PNIPAAm based column. Optimal comparison is thereby obtained by the use of controlled radical polymerizations, identical molecular weights, and by maximizing carbon loads on the silica supporting material. Analysis of identical test mixtures of homologue series and pharmaceutical samples revealed that PNNPAAm performs in a similar way as PNIPAAm while offering enhanced retention and a shift of the useable temperature range toward lower temperatures. PDEAAm offers a range of novel possibilities as it depicts a different selectivity, allowing for enhanced resolution in TRLC in, for example, coupled column systems. Reduced plate heights of 3 could be obtained on the homemade columns, offering the promise for reasonable column efficiencies in TRLC despite the use of bulky polymers as stationary phases in HPLC.

In this study, the possibilities of temperature responsive × reversed phase liquid chromatography (TRLC × RPLC) are assessed in terms of pharmaceutical impurity analysis. Due to the increased peak capacity per unit time they offer, two-dimensional LC approaches are gaining relevance for the analysis of complex drug formulations. Because the latter depicts a larger predisposition for the occurrence of an increased number of impurities, current 1D-HPLC approaches often prove insufficient. Since many LC × LC methods are limited by modulation, solvent compatibility, orthogonality, and sensitivity issues, the combination of TRLC × RPLC is explored in this work for pharmaceutical impurity analysis. As this combination of a purely aqueous separation with RPLC allows for systematic and optimization-free refocusing in the second dimension, it opens possibilities for generic LC × LC requiring minimal to no method development, in this way overcoming a major perceived contemporary hurdle of LC × LC. The approach is demonstrated with a representative mixture of 17 solutes comprising 11 corticosteroids and 6 progestogens. Orthogonality and peak capacities were assessed on three RP core-shell column selectivities (Poroshell EC-C18, phenyl-hexyl and PFP). Although the TRLC × EC-C18 combination offered somewhat better orthogonality, the combination with the PFP column proved the best for the separation at hand. Depending on the composition of the mixture, the use of full, shifted, or segmented gradients allowed facile optimization of the separation. The developed platform allowed detection of the impurities at the 0.05% level compared to a selected main compound, while also opening up possibilities for analysis of formulations comprising two active ingredients.

Three new amidation approaches are evaluated to incorporate tyramine on methyl ester functional poly(2-oxazolines).

Due to the increasing complexity of pharmaceutical drug formulations, as e.g. used in combination therapies, assessment of the active ingredients and of the thereby related impurities is becoming an ever more challenging task by conventional 1-dimensional chromatographic separation techniques. This because baseline separation of all solutes in complex samples can thereby not be obtained anymore, while the impurities typically arising at very low concentrations require also sensitive detection to enable their identification and quantification following current regulations.1 In order to address limited peak capacities, comprehensive 2-dimensional LCxLC approaches are increasingly used to analyse natural and synthetic samples of high complexity. Although the possibilities of LCxLC have been incrementally improving, overall many of the current approaches are limited in their robustness, characterized by modulation problems, too low orthogonality and undersampling.2 Especially the modulation issue often becomes visible in form of solvent immiscibility and excessive eluting strength issues, leading to e.g. peak broadening, decreased sensitivity and repeatability issues. Only a few comprehensive LCxLC modes, all using purely aqueous separation modes in the first dimension, are today exempted from these concerns. One of those is the recently introduced combination of temperature-responsive chromatography with reversed phase liquid chromatography (TRLCxRPLC).3 Such temperature responsive phases thereby depict an adaptable hydrophobicity and hence retention characteristics as a function of temperature. A main benefit of the approach is that it forgoes the need for the addition of organic solvents to the mobile phase. This new separation technique inherently reduces the methodical complexity of multidimensional LC, by allowing problem free modulation when using a conventional loop based 10-port valve and a purely aqueous mobile phase in the first dimension. This on the one hand allows for the transfer of high sample volumes from the first to the second dimension with near perfect peak refocussing, and on the other hand can facilitate more sensitive detection compared to conventional LCxLC approaches, which often require miniaturization of the first dimension. The possibilities offered by TRLCxRPLC in terms of selectivity, sensitivity, peak capacities and quantitative potential are assessed for improved separation of pharmaceutical mixtures. Therefore, synthetic mixtures of structurally similar pharmaceutical compounds are investigated, while optimizing the first and second dimension to optimally use the given separation space and the added selectivity. Additionally, several column (core-shell) chemistries are assessed in the second dimension.