Incorporating a liquid-core-waveguide cell in recycling liquid chromatography for detailed studies of photodegradation reactions.

In this work, a microfluidic photoreactor was embedded in a recycling liquid-chromatography system. Mixtures were separated on an analytical column and compounds of interest were subsequently introduced into the light-reactor cell. After degradation, the content of the light-reactor cell was reinjected onto the same column to separate the parent compound from its degradation products. A separated degradation product could be re-introduced into the photoreactor and irradiated again. The next generation of degradation products could again be separated on the same analytical column. This recycling procedure proved an excellent tool to elucidate degradation pathways. This was demonstrated using riboflavin, better known as vitamin B2. By degrading it in the first cycle, degradation products were isolated and subjected to a second degradation in the light-reactor cell. This allows pinpointing secondary products and connect these with primary degradation products. Compared to previous work, this configuration is simpler, cheaper, and more user-friendly, while offering the unique possibility to easily connect degradation products to the initial compounds in a mixture.

Assessing the feasibility of stationary-phase-assisted modulation for two-dimensional liquid-chromatography separations.

Two-dimensional liquid chromatography (2DLC) offers great separation power for complex mixtures. The frequently encountered incompatibility of two orthogonal separation systems, however, makes its application complicated. Active-modulation strategies can reduce such incompatibility issues considerably. Stationary-phase-assisted modulation (SPAM) is the most-common of these techniques, but also the least robust due to the major disadvantage that analytes may elute prematurely. The range of liquid chromatography (LC) applications continues to expand towards ever more complex mixtures. Retention modelling is increasingly indispensable to comprehend and develop LC separations. In this research, a tool was designed to assess the feasibility of applying SPAM in 2DLC. Several parameters were investigated to accurately predict isocratic retention of analytes on trap columns under dilution-flow conditions. Model parameters were derived from scanning-gradient experiments performed on analytical columns. The trap-to-trap repeatability was found to be similar to the prediction error. Dead volumes for the trap columns could not be accurately determined through direct experimentation. Instead, they were extrapolated from dead-volume measurements on analytical columns. Several known retention models were evaluated. Better predictions were found using the quadratic model than with the log-linear ("linear-solvent-strength") model. Steep scanning gradients were found to result in inaccurate predictions. The impact of the dilution flow on the retention of analytes proved less straightforward than anticipated. Under certain conditions dilution with a weaker eluent was found to be counter productive. A tool was developed to quantify the effect of the dilution flow and to predict whether SPAM could be applied in specific situations. For nine different analytes under 36 different sets of conditions and with three different modulation times, the SPAM tool yielded a correct assessment in more than 95% of all cases (less than 5% false positives plus false negatives).

Combining Photodegradation in a Liquid-Core-Waveguide Cell with Multiple-Heart-Cut Two-Dimensional Liquid Chromatography.

Photodegradation greatly affects everyday life. It poses challenges when food deteriorates or when objects of cultural heritage fade, but it can also create opportunities applied in advanced oxidation processes in water purification. Studying photodegradation, however, can be difficult because of the time needed for degradation, the inaccessibility of pure compounds, and the need to handle samples manually. A novel light-exposure cell, based on liquid-core-waveguide (LCW) technology, was embedded in a multiple-heart-cut two-dimensional liquid chromatography system by coupling the LCW cell to the multiple-heart-cut valve. The sample was flushed from the heart-cut loops into the cell by an isocratic pump. Samples were then irradiated using different time intervals and subsequently transferred by the same isocratic pump to a second-dimension sample loop. The mixture containing the transformation products was then subjected to the second-dimension separation. In the current setup, about 30-40% of the selected fraction was transferred. Multiple degradation products could be monitored. Degradation was found to be faster when a smaller sample amount was introduced (0.3 µg as compared to 1.5 µg). The system was tested with three applications, that is, fuchsin, a 19th-century synthetic organic colorant, annatto, a lipophilic food dye, and vitamin B complex.

Liquid Core Waveguide Cell with In Situ Absorbance Spectroscopy and Coupled to Liquid Chromatography for Studying Light-Induced Degradation.

In many areas, studying photostability or the mechanism of photodegradation is of high importance. Conventional methods to do so can be rather time-consuming, laborious, and prone to experimental errors. In this paper we evaluate an integrated and fully automated system for the study of light-induced degradation, comprising a liquid handler, an irradiation source and exposure cell with dedicated optics and spectrograph, and a liquid chromatography (LC) system. A liquid core waveguide (LCW) was used as an exposure cell, allowing efficient illumination of the sample over a 12 cm path length. This cell was coupled to a spectrograph, allowing in situ absorbance monitoring of the exposed sample during irradiation. The LCW is gas-permeable, permitting diffusion of air into the cell during light exposure. This unit was coupled online to LC with diode array detection for immediate and automated analysis of the composition of the light-exposed samples. The analytical performance of the new system was established by assessing linearity, limit of detection, and repeatability of the in-cell detection, sample recovery and carryover, and overall repeatability of light-induced degradation monitoring, using riboflavin as the test compound. The applicability of the system was demonstrated by recording a photodegradation time profile of riboflavin.

Characterization of a liquid-core waveguide cell for studying the chemistry of light-induced degradation.

Many organic compounds undergo changes under the influence of light. This might be beneficial in, for example, water purification, but undesirable when cultural-heritage objects fade or when food ingredients (e.g., vitamins) degrade. It is often challenging to establish a strong link between photodegradation products and their parent molecules due to the complexity of the sample. To allow effective study of light-induced degradation (LID), a low-volume exposure cell was created in which solutes are efficiently illuminated (especially at low concentrations) while simultaneously analysed by absorbance spectroscopy. The new LID cell encompasses a gas-permeable liquid-core waveguide (LCW) connected to a spectrograph allowing collection of spectral data in real-time. The aim of the current study was to evaluate the overall performance of the LID cell by assessing its transmission characteristics, the absolute photon flux achieved in the LCW, and its capacity to study solute degradation in presence of oxygen. The potential of the LID set-up for light-exposure studies was successfully demonstrated by monitoring the degradation of the dyes eosin Y and crystal violet.

Measuring and using scanning-gradient data for use in method optimization for liquid chromatography.

The use of scanning gradients can significantly reduce method-development time in reversed-phase liquid chromatography. However, there is no consensus on how they can best be used. In the present work we set out to systematically investigate various factors and to formulate guidelines. Scanning gradients are used to establish retention models for individual analytes. Different retention models were compared by computing the Akaike information criterion and the prediction accuracy. The measurement uncertainty was found to influence the optimum choice of model. The use of a third parameter to account for non-linear relationships was consistently found not to be statistically significant. The duration (slope) of the scanning gradients was not found to influence the accuracy of prediction. The prediction error may be reduced by repeating scanning experiments or - preferably - by reducing the measurement uncertainty. It is commonly assumed that the gradient-slope factor, i.e. the ratio between slopes of the fastest and the slowest scanning gradients, should be at least three. However, in the present work we found this factor less important than the proximity of the slope of the predicted gradient to that of the scanning gradients. Also, interpolation to a slope between that of the fastest and the slowest scanning gradient is preferable to extrapolation. For comprehensive two-dimensional liquid chromatography (LC × LC) our results suggest that data obtained from fast second-dimension gradients cannot be used to predict retention in much slower first-dimension gradients.

Recent applications of retention modelling in liquid chromatography.

Recent applications of retention modelling in liquid chromatography (2015-2020) are comprehensively reviewed. The fundamentals of the field, which date back much longer, are summarized. Retention modeling is used in retention-mechanism studies, for determining physical parameters, such as lipophilicity, and for various more-practical purposes, including method development and optimization, method transfer, and stationary-phase characterization and comparison. The review focusses on the effects of mobile-phase composition on retention, but other variables and novel models to describe their effects are also considered. The five most-common models are addressed in detail, i.e. the log-linear (linear-solvent-strength) model, the quadratic model, the log-log (adsorption) model, the mixed-mode model, and the Neue-Kuss model. Isocratic and gradient-elution methods are considered for determining model parameters and the evaluation and validation of fitted models is discussed. Strategies in which retention models are applied for developing and optimizing one- and two-dimensional liquid chromatographic separations are discussed. The review culminates in some overall conclusions and several concrete recommendations.

Development and validation of an analytical protocol for the sampling and quantitative analysis of ions on the surface of unstable historic glass in museum collections using ion-exchange chromatography.

The early identification of unstable glass objects in museum collections is essential for their conservation, but as yet cannot be accomplished straightforwardly. Accordingly, this paper describes the development and validation of a simple protocol for quantitative determination of ions characteristic of the chemical decay of historic glass, using surface swabbing combined with ion-exchange chromatography. The establishment of a robust protocol is an important step in the development of an early warning system for the chemical deterioration of unstable glass. Using a model system, the protocol was validated for specificity, linearity, accuracy, precision, limits of detection, and limits of quantification for 10 anionic species (fluoride, acetate, formate, chloride, nitrite, bromide, nitrate, carbonate, sulfate and phosphate) and 6 cationic species (lithium, sodium, ammonium, potassium, magnesium and calcium). Good validation parameters (R2 > 0.995; RSD < 5%; Recovery 90-100%) were obtained for acetate, formate, nitrite, nitrate, phosphate, lithium, sodium, potassium, magnesium and calcium. Chloride (R2 = 0.934; RSD = 13.6%; recovery 71.4%) and carbonate (R2 = 0.993; RSD = 10.3%; recovery 120%) had poor validation parameters. Sulfate had low recovery (78.2%), but high reproducibility (RSD = 4.32%) with R2 = 0.997. Limits of quantification were below 1 mg/L for all analytes, which is satisfactory for the study of unstable glass in museum collections. The validated sampling protocol was trialled using artificially aged unstable glass fragments, which resulted in a high relative standard deviation (between 1 and 30%). The ability to achieve improved care of historic glass by application of the validated protocol in museum collections is discussed in the context of a pilot study undertaken at the Rijksmuseum, Amsterdam.

Characterization of Dye Extracts from Historical Cultural-Heritage Objects Using State-of-the-Art Comprehensive Two-Dimensional Liquid Chromatography and Mass Spectrometry with Active Modulation and Optimized Shifting Gradients.

Unbiased characterization of dyes and their degradation products in cultural-heritage objects requires an analytical method which provides universal separation power regardless of dye classes. Dyes are small molecules that vary widely in chemical structure and properties, which renders their characterization by a single method challenging. We have developed a comprehensive two-dimensional liquid chromatography method hyphenated with mass spectrometry and UV-vis detection. We use stationary-phase-assisted modulation to enhance the method in terms of detection limits and solvent compatibility and to reduce the analysis time. The PIOTR program was used to optimize an assembly of shifting second-dimension gradients, which resulted in a high degree of orthogonality (80% in terms of the asterisk concept). The resulting method is universally applicable to all classes of dyes extracted from cultural-heritage objects. Thanks to the high peak capacity and orthogonality, dye components can be separated from chemically similar impurities and degradation products, providing a detailed fingerprint of the dyes mixture in a specific sample. The method was applied to a number of challenging dye extracts from 17th- and 19th-century cultural-heritage objects.

Surface Acoustic Wave Nebulisation Mass Spectrometry for the Fast and Highly Sensitive Characterisation of Synthetic Dyes in Textile Samples.

Surface acoustic wave nebulisation (SAWN) mass spectrometry (MS) is a method to generate gaseous ions compatible with direct MS of minute samples at femtomole sensitivity. To perform SAWN, acoustic waves are propagated through a LiNbO3 sampling chip, and are conducted to the liquid sample, which ultimately leads to the generation of a fine mist containing droplets of nanometre to micrometre diameter. Through fission and evaporation, the droplets undergo a phase change from liquid to gaseous analyte ions in a non-destructive manner. We have developed SAWN technology for the characterisation of organic colourants in textiles. It generates electrospray-ionisation-like ions in a non-destructive manner during ionisation, as can be observed by the unmodified chemical structure. The sample size is decreased by tenfold to 1000-fold when compared with currently used liquid chromatography-MS methods, with equal or better sensitivity. This work underscores SAWN-MS as an ideal tool for molecular analysis of art objects as it is non-destructive, is rapid, involves minimally invasive sampling and is more sensitive than current MS-based methods. Graphical Abstract ᅟ.

Characterisation of chemical components for identifying historical Chinese textile dyes by ultra high performance liquid chromatography - photodiode array - electrospray ionisation mass spectrometer.

This research makes the first attempt to apply Ultra High Performance Liquid Chromatography (UHPLC) coupled to both Photodiode Array detection (PDA) and Electrospray Ionisation Mass Spectrometer (ESI-MS) to the chemical characterisation of common textile dyes in ancient China. Three different extraction methods, respectively involving dimethyl sulfoxide (DMSO)-oxalic acid, DMSO and hydrochloric acid, are unprecedentedly applied together to achieve an in-depth understanding of the chemical composition of these dyes. The first LC-PDA-MS database of the chemical composition of common dyes in ancient China has been established. The phenomena of esterification and isomerisation of the dye constituents of gallnut, gardenia and saffron, and the dye composition of acorn cup dyed silk are clarified for the first time. 6-Hydroxyrubiadin and its glycosides are first reported on a dyed sample with Rubia cordifolia from China. UHPLC-PDA-ESI-MS with a C18 BEH shield column shows significant advantages in the separation and identification of similar dye constituents, particularly in the cases of analysing pagoda bud and turmeric dyed sample extracts.

Characterization of synthetic dyes by comprehensive two-dimensional liquid chromatography combining ion-exchange chromatography and fast ion-pair reversed-phase chromatography.

In the late 19th century, newly invented synthetic dyes rapidly replaced the natural dyes on the market. The characterization of mixtures of these so-called early synthetic dyes is complicated through the occurrence of many impurities and degradation products. Conventional one-dimensional liquid chromatography does not suffice to obtain fingerprints with sufficient resolution and baseline integrity. Comprehensive two-dimensional liquid chromatography (LC×LC) is employed in this study, with ion-exchange chromatography in the first dimension and fast ion-pair liquid chromatography in the second. Retention in the first dimension is largely determined by the number of charges, while the selection of a small ion-pair reagent (tetramethylammonium hydroxide) in the second dimension causes retention to be largely determined by the molecular structure of the dye. As a result, there is a high degree of orthogonality of the two dimensions, similar to the values typically encountered in GC×GC. The proposed LC×LC method shows a theroretical peak capacity of about 2000 in an analysis time of about three hours. Clear, informative fingerprints are obtained that open a way to a more efficient characterization of dyes used in objects of cultural heritage.

Investigation of crimson-dyed fibres for a new approach on the characterization of cochineal and kermes dyes in historical textiles.

The colorant behaviour of cochineal and kermes insect dyes in 141 experimentally-dyed and 28 artificially-aged samples of silk and wool was investigated using ultra-high performance liquid chromatography coupled to photodiode array detector (UHPLC-PDA), liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) and image scanning electron microscopy - energy dispersive X-ray spectroscopy (SEM-EDX). Partial-least squares discriminant analysis (PLS-DA) was then used to model the acquired UHPLC-PDA data and assess the possibility of discriminating cochineal insect species, as well as their correspondent dyed and aged reference fibres. The resulting models helped to characterize a set of 117 red samples from 95 historical textiles, in which UHPLC-PDA analyses have reported the presence of cochineal and kermes insect dyes. Analytical investigation of the experimentally-dyed and artificially-aged fibres has demonstrated that the ratio of compounds in the insects dye composition can change, depending on the dyeing conditions applied and the type of fibres used. Similarities were observed when comparing the UHPLC-MS and SEM-EDX results from the dyed and aged references with the historical samples. This was verified with PLS-DA models of the chromatographic data, facilitating the classification of the cochineal species present in the historical samples. The majority of these samples were identified to contain American cochineal, which is in agreement with historical and dye identification literature that describe the impact of this dyestuff into European and Asian dyeing practices, after the Iberian Expansion in the 16th century. The analytical results emphasize the importance of using statistical data interpretation for the discrimination of cochineal dyes, besides qualitative and quantitative evaluation of chromatograms. Hence, the combination of UHPLC-PDA with a statistical classification method, such as PLS-DA, has been demonstrated to be an advisable approach in future investigations to assess closely related species of natural dyes in historical textile samples. This is particularly important when aiming to achieve more accurate interpretations about the history of works of art, or the application of natural dyes in old textile production.

Evaluation between ultrahigh pressure liquid chromatography and high-performance liquid chromatography analytical methods for characterizing natural dyestuffs.

An evaluation was undertaken of ultrahigh pressure liquid chromatography (UHPLC) in comparison to high-performance liquid chromatography (HPLC) for characterizing natural dyes in cultural heritage objects. A new UHPLC method was optimized by testing several analytical parameters adapted from prior UHPLC studies developed in diverse fields of research. Different gradient elution programs were tested on seven UHPLC columns with different dimensions and stationary phase compositions by applying several mobile phases, flow rates, temperatures, and runtimes. The UHPLC method successfully provided more improved data than that achieved by the HPLC method. Indeed, even though carminic acid has shown circa 146% higher resolution with HPLC, UHPLC resulted in an increase of 41-61% resolution and a decrease of 91-422% limit of detection, depending on the dye compound. The optimized method was subsequently assigned to analyse 59 natural reference materials, in which 85 different components were ascribed with different physicochemical properties, in order to create a spectral database for future characterization of dyes in cultural heritage objects. The majority of these reference samples could be successfully distinguished with one single method through the examination of these compounds' retention times and their spectra acquired with a photodiode array detector. These results demonstrate that UHPLC analyses are extremely valuable for the acquisition of more precise chromatographic information concerning natural dyes with complex mixtures of different and/or closely related physicochemical properties, essential for distinguishing similar species of plants and animals used to colour cultural heritage objects.

High-performance liquid chromatography and non-destructive three-dimensional fluorescence analysis of early synthetic dyes.

Chromatographic and spectroscopic techniques are evaluated for the analysis of early synthetic dyes. The research focuses on the analysis of dyestuffs that were developed and used in the period 1850-1900, which has not been well investigated so far. The aim of this study was to develop and evaluate techniques that can be applied in the field of culture and art. A selection of 65 synthetic dyestuffs was chosen to investigate the usefulness of the analytical techniques applied. As a case study three embroideries, designed by the well-known 19th century French painter Emile Bernard, were investigated to obtain more information about the use and behaviour of synthetic dyes.