The development of a generic analysis method for natural and synthetic dyes by ultra-high-pressure liquid chromatography with photo-diode-array detection and triethylamine as an ion-pairing agent.

In cultural heritage the characterization of organic colorants is a challenging task. Currently, different chromatographic techniques are used to analyze natural and synthetic dyes separately, since the classes differ significantly in chemical properties and, therefore, chromatographic behavior. To save time, costs and sample material, we developed a method suitable for a wide variety of organic colorants using ultra-high-performance liquid chromatography coupled to a photo-diode-array detector. Gradient elution was performed with a mobile phase consisting of water and methanol with 5 mM triethylamine added as an ion-pairing agent at a pH of 3. Both linear and step gradients were optimized using the 'Program for Interpretive Optimization of Two-dimensional Resolution' (PIOTR) Pirok et al. [22]. Two optimized linear gradients and two step gradients were evaluated experimentally. The method was applied on a complex dye mixture containing nearly 130 natural- and synthetic-dye reference compounds. More than 100 of these compounds could be identified in a single experiment. The feasibility of the method was demonstrated by analyzing samples of several precious objects that were found in the Texel shipwreck Vos et al. [2] and of two embroideries of Emile Bernard, the results of which are described in this paper.

Heterogeneity analysis of polymeric carboxylic acid functionality by selective derivatization followed by size exclusion chromatography.

Waterborne polymers are increasingly applied in our society, replacing traditional solvent-borne coatings and thus reducing environmental impact of coatings. The majority of waterborne dispersions are stabilized by the incorporation of neutralizable carboxylic acid functionality. The characterization of synthetic waterborne polymer systems can be performed by a wide variety of chromatographic and spectroscopic techniques. However, none of these approaches is able to determine the acid functionality distribution over the molecular-weight distribution directly. In this research, an innovative approach is developed which enables this analysis. The approach is based on the specific and complete derivatization of carboxylic acid functionality with phenacylbromide. Size exclusion chromatography (SEC) analysis of the derivatized polymers is performed followed by ultraviolet- (UV) and refractive index (RI) detection, enabling the quantitative determination of the acid content per molecular weight fraction. The applicability of the developed protocol is shown for various polymer systems.

Acid monomer analysis in waterborne polymer systems by targeted labeling of carboxylic acid functionality, followed by pyrolysis - gas chromatography.

Pyrolysis - gas chromatography - (PyGC) is a common method to analyse the composition of natural and synthetic resins. The analysis of acid functionality in, for example, waterborne polyacrylates and polyurethanes polymers has proven to be difficult due to solubility issues, inter- and intramolecular interaction effects, lack of detectability in chromatographic analysis, and lack of thermal stability. Conventional analytical techniques, such as PyGC, cannot be used for the direct detection and identification of acidic monomers, due to thermal rearrangements that take place during pyrolysis. To circumvent this, the carboxylic acid groups are protected prior to thermal treatment by reaction with 2-bromoacetophenone. Reaction conditions are investigated and optimised wrt. conversion measurements. The aproach is applied to waterborne polyacryalates and the results are discussed. This approach enables identification and (semi)quantitative analysis of different acid functionalities in waterborne polymers by PyGC.

Switching solvent and enhancing analyte concentrations in small effluent fractions using in-column focusing.

A novel approach to achieve solvent switching and focusing of sub-column-volume analyte fractions in liquid chromatography is presented. By altering the temperature between loading and elution in back-flush mode, solvent transfer of analytes and focusing occurs, provided that the analytes exhibit temperature dependent retention on a given trap column. When retention on the trap decreases with increasing temperature, which is almost always the case, the loading of the trap-column takes place at a higher temperature than the elution. This principle is demonstrated using three small aromatic molecules (toluene, p-xylene and benzophenone) on a capillary monolithic column. On this column, the analytes show a traditional van't Hoff dependence on temperature with enthalpy effects of, -15, -16 and -18 kJ mol(-1), respectively, for a mobile phase of 25% acetonitrile in water. The column was loaded at 110 °C, cooled in an ice bath and eluted in back-flush mode at 0 °C. When operated in this way, the analytes are transferred from the loading solvent to the elution solvent, achieving solvent switching. Substantial focusing can also be obtained if the desorption solvent is stronger than the loading solvent.

Optimization and evaluation of radially interconnected versus bifurcating flow distributors using computational fluid dynamics modelling.

Two main groups of flow distributors, viz. "bifurcating distributors" (BF) and "radially interconnected distributors" (RI), as well as some hybrid distributors were investigated. Computational fluid dynamics was used to evaluate the performance of the distributors and to establish the design yielding the most uniform velocity field and the smallest variance for the bands emerging from the distributor. A minimum channel width of 100 µm was considered to allow the use of micro-milling techniques for chip fabrication. The main factors that influenced the values of band variances were identified. The performance of the distributors was found to correlate most strongly with the volume of the flow distributors. The separation bed should be positioned immediately after, but not against the flow distributor. It was concluded that BF distributors perform best in terms of band variance. The values of band variances for the BF distributor decreased with increasing angle between bifurcation branches and the lowest value of about 0.01 mm(2) was found for α=175°. Both BF and RI flow distributors were found to perform reasonably well when imperfections were present in the structure. However, severe blockages (exceeding 75% of the cross-sectional area and length) of channels in, especially, BF flow distributors may jeopardize their performance.

Methods for the chemical analysis of degradable synthetic polymeric biomaterials.

The performance of biodegradable polymeric systems strongly depends on their physical as well as on their chemical properties. Therefore, detailed chemical analysis of such systems is essential. Enzymatic and chemical hydrolysis are the primary biodegradation mechanisms for these materials. This review provides an overview of the strategies and analytical methods used for the structural and compositional chemical analysis of nondegraded, partially degraded, and fully degraded synthetic polymeric biomaterials with an emphasis on modern solution-based techniques that yield large amounts of information. The degradation methods that facilitate the study of polymeric networks are also described.

A versatile system for studying the enzymatic degradation of multi-block poly(ester amide)s.

The suitability of biomaterials for specific biomedical applications can be investigated through their in vitro biodegradability with selected enzymes and through their degradation kinetics. A system was developed for studying the enzymatic degradation of poly(ester amide)s (PEAs) coatings under sink conditions, with on-stream analysis of degradation products by liquid chromatography coupled to time-of-flight mass spectrometry (LC-ToF-MS). A coated capillary was treated by an enzyme solution in pulses (pulse-feed mode) or continuously (continuous-feed mode) with different flow rates. The water-soluble products resulting from the interaction of enzyme with the PEA coating were deposited on-line on a reversed-phase LC column, separated by gradient-elution LC, ionized by electrospray ionization (ESI), and identified based on ToF-MS data. The experiments underline the benefits of the experimental set-up, which requires only small amounts of coating and enzyme and produces detailed results rapidly. The system was investigated using different injection volumes (pulses) of an α-chymotrypsin solution in varying concentrations, different flow rates, and different lengths of coated capillary. The versatility of the system makes it easy to follow the course of degradation and to differentiate between primary and secondary degradation products. The system was applied to study the degradation of a di-block and a tri-block PEA. Specific degradation products showed different time profiles than the (more gradual) overall weight loss. Continuous-feed-mode analysis allowed the convenient determination of highly stable amide-bond-containing fragments, while pulse-feed-mode analysis revealed benzyl ester-containing products as primary degradation products.

Monitoring the in vitro enzyme-mediated degradation of degradable poly(ester amide) for controlled drug delivery by LC-ToF-MS.

To scrutinize materials for specific biomedical applications, we need sensitive and selective analytical methods that can give more insight into the process of their biodegradation. In the present study, the enzymatic degradation of multiblock poly(ester amide) based on natural amino acids, such as lysine and leucine, was performed with serine proteases (α-chymotrypsin (α-CT) and proteinase K (PK)) in phosphate-buffered saline solution at 37 °C for 4 weeks. Fully and partially degraded water-soluble products were analyzed by liquid chromatography hyphenated with time-of-flight mass spectrometry using an electrospray interface (LC-ESI-ToF-MS). Tracking the release of monomeric and oligomeric products into the enzyme media during the course of enzymatic degradation revealed the preferences of α-CT and PK toward ester and amide bonds: both α-CT and PK showed esterase and amidase activity. Although within the experimental time frame up to 30 and 15% weight loss was observed in case of α-CT and PK, respectively, analysis by size exclusion chromatography showed no change in the characteristic molecular-weight averages of the remaining polymer. This suggests that the enzymatic degradation occurs at the surface of this biomaterial. A sustained and linear degradation over a period of 4 weeks supports the potential of this class of poly(ester amide)s for drug delivery applications.

Fast in vitro hydrolytic degradation of polyester urethane acrylate biomaterials: structure elucidation, separation and quantification of degradation products.

Synthetic biomaterials have evoked extensive interest for applications in the field of health care. Prior to administration to the body a quantitative study is necessary to evaluate their composition. An in vitro method was developed for the quick hydrolytic degradation of poly-2-hydroxyethyl methacrylate (pHEMA), poly(lactide-co-glycolide50/50)1550-diol (PLGA(50:50)(1550)-diol), PLGA(50:50)(1550)-diol(HEMA)(2) and PLGA(50:50)(1550)-diol(etLDI-HEMA)(2) containing ethyl ester lysine diisocyanate (etLDI) linkers using a microwave instrument. Hydrolysis time and temperature were optimized while monitoring the degree of hydrolysis by (1)H NMR spectroscopy. Complete hydrolytic degradation was achieved at 120°C and 3 bar pressure after 24 h. Chemical structure elucidations of the degradation products were carried out using (1)H and (13)C NMR spectroscopy. The molecular weight (MW) of the polymethacrylic backbone was estimated via size-exclusion chromatography coupled to refractive index detection (SEC-dRI). A bimodal MW distribution was found experimentally, also in the pHEMA starting material. The number average molecular weights (M(n)) of the PLGA-links (PLGA(50:50)(1550)-diol) were calculated by high pressure liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS) and (1)H NMR. The amounts of the high and low MW degradation products were determined by SEC-dRI and, HPLC-TOF-MS, respectively. The main hydrolysis products poly (methacrylic acid) (PMAA), ethylene glycol (EG), diethylene glycol (DEG), lactic acid (LA), glycolic acid (GA) and lysine were recovered almost quantitatively. The current method leads to the complete hydrolytic degradation of these materials and will be helpful to study the degradation behavior of these novel cross-linked polymeric biomaterials.

Comprehensive study on the optimization of online two-dimensional liquid chromatographic systems considering losses in theoretical peak capacity in first- and second-dimensions: a Pareto-optimality approach.

A method to optimize different objectives (total analysis time, total peak capacity, and total dilution) has been applied to comprehensive two-dimensional liquid chromatography. The approach is based on Pareto-optimality, and it yields optimal parameters (column particle sizes, column diameters, and modulation times). Losses in the peak capacities in the first dimension (due to undersampling) and in the second dimension (due to high injection volumes) have been taken into account. The first effect (detection band broadening) reduces the original peak capacity by about a half, the second effect can reduce the total peak capacity by an additional half. Thus, the total loss in peak capacity may be 75% of its theoretical value. Analytical expressions to calculate these effects under gradient and isocratic conditions are derived. Of particular interest is the study of the optimal modulation times, which corresponded to between 2 and 3 two-dimensional runs per one-dimensional peak. The effects of using gradient or isocratic elution, conventional (40 MPa) or "ultra-high" (100 MPa) pressures, and focusing between the first and second dimensions were also studied. A trade-off between total peak capacity, total analysis time, and total dilution can be established.

Selection of adequate optimization criteria in chromatographic separations.

Computer-assisted optimization of chromatographic separations is still a fruitful activity. In fact, advances in computerized data handling should make the application of systematic optimization strategies much easier. However, in most contemporary applications, the optimization criterion is not considered to be a key issue (Vanbel, J Pharm Biomed, 21:603-610, 1999). In this paper, an update of the importance of selecting adequate criteria in chromatographic separation is presented.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene-diene rubber using gas chromatography and mass spectrometry II. Addition and combination reactions.

The dicumyl-peroxide-initiated addition and combination reactions of mixtures of alkanes (n-octane, n-decane) and alkenes [5,6-dihydrodicyclopentadiene (DCPDH), 5-ethylidene-2-norbornane (ENBH) and 5-vinylidene-2-norbornane (VNBH)] were studied to mimic the peroxide cross-linking reactions of terpolymerised ethylene, propylene and a diene monomer (EPDM). The reaction products of the mixtures were separated by both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GCxGC). The separated compounds were identified from their mass spectra and their GC and GCxGC elution pattern. Quantification of the various alkyl/alkyl, alkyl/allyl and allyl/allyl combination products shows that allylic-radicals comprise approximately 60% of the substrate radicals formed. The total concentration of the products formed by combination is found to be independent of the concentration and the type of alkene. The total concentration of the products formed by addition to the alkene increases with increasing concentration of alkene. In addition, the total concentration of the formed addition products depends strongly on the type of the alkene used, viz. VNBH>ENBH approximately DCPDH, which is a consequence of differences in steric hindrance of the unsaturation. The peroxide curing efficiency, defined as the number of moles of cross-linked products formed per mol of peroxide, is 173% using 9% (w/w) 5-vinylidene-2-norbornane (VNBH). This indicates that the addition reaction is recurrent. All these findings are consistent with experimental studies on peroxide curing of EPDM rubber. In addition, the present results provide more-detailed structural information, increasing the understanding of the mechanism of peroxide curing of EPDM. The described approach to use low-molecular-weight model compounds followed by GC-mass spectrometry (MS) and GCxGC-MS analysis is proven to be a very powerful tool to study the cross-linking of EPDM.

Branched-polymer separations using comprehensive two-dimensional molecular-topology fractionation x size-exclusion chromatography.

Branching has a strong influence on the processability and properties of polymers. However, the accurate characterization of branched polymers is genuinely difficult. Branched molecules of a certain molecular weight exhibit the same hydrodynamic volumes as linear molecules of substantially lower weights. Therefore, separation by size-exclusion chromatography (SEC), will result in the co-elution of molecules with different molecular weights and branching characteristics. Chromatographic separation of the polymer molecules in sub-microm channels, known as molecular-topology fractionation (MTF), may provide a better separation based on topological differences among sample molecules. MTF elution volumes depend on both the topology and molar mass. Therefore co-elution of branched molecules with linear molecules of lower molar mass may also occur in this separation. Because SEC and MTF exhibit significantly different selectivity, the best and clearest separations can be achieved by combining the two techniques in a comprehensive two-dimensional (MTFxSEC) separation system. In this work such a system has been used to demonstrate branching-selective separations of star branched polymers and of randomly long-chain-branched polymers. Star-shaped polymers were separated from linear polymers above a column-dependent molecular weight or size.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene (-diene) rubber using gas chromatography and mass spectrometry I. Combination reactions of alkanes.

The combination reaction of linear and branched alkanes, initiated by dicumylperoxide, has been studied as a model for the combination cross-linking reaction of peroxide-cured terpolymerised ethylene, propylene and diene monomer. Both gas chromatography-mass spectrometry (GC-MS) and comprehensive two-dimensional GC-MS (GCxGC-MS) analyses have been employed to analyse the isomeric reaction products. The identification of these products based on their MS fragmentation patterns is quite complex, due to the high tendency of random rearrangements. Careful elucidation of the high-mass ions at optimised ionisation energy (55eV) has resulted in proposed structures for the different isomeric reaction products. The structure assignment by MS is in agreement with the GCxGC elution pattern and with the result of a theoretical model to predict the boiling points and, thus, the GC retention times. In addition, a model that provided a direct correlation between chemical structure and retention times was developed and this was found to provide a useful fit. Quantification of the identified reaction products by GC separation and flame ionization detection allows classification according to the hydrogen abstraction sites for the alkanes by dicumylperoxide. The selectivity for hydrogen abstraction generally follows the expected order, but a higher reactivity was observed for the methylene group next to a primary methyl group, while a reduced reactivity of the methylene group next to ethyl and to methyl groups was observed. The used approach proved to be a very powerful tool to enhance our understanding of the mechanism of peroxide cross-linking of (branched) alkanes.

Towards unsupervised analysis of second-order chromatographic data: automated selection of number of components in multivariate curve-resolution methods.

A method to apply multivariate curve-resolution unattendedly is presented. The algorithm is suitable to perform deconvolution of two-way data (e.g. retrieving the individual elution profiles and spectra of co-eluting compounds from signals obtained from a chromatograph equipped with multiple-channel detection: LC-DAD or GC-MS). The method is especially adequate to achieve the advantages of deconvolution approaches when huge amounts of data are present and manual application of multivariate techniques is too time-consuming. The philosophy of the algorithm is to mimic the reactions of an expert user when applying the orthogonal projection approach--multivariate curve-resolution techniques. Basically, the method establishes a way to check the number of significant components in the data matrix. The performance of the method was superior to the Malinowski F-test. The algorithm was tested with HPLC-DAD signals.

Experimental study on band dispersion in channels structured with micropillars.

The band dispersion in channels with an orderly pillar structure with a pressure-driven liquid flow was determined. Several channels with different geometries were etched in a silicon wafer and enclosed by a glass wafer. The microchannels obtained had the same depth, pillar disposition, and overall porosity, but different pillar diameters and channel widths. The broadening of narrow bands of a fluorescent sample solution flowing through the channels was measured using a fluorescence microscope. It was shown that the peak dispersion occurring in the channels can be much lower than in conventional packed columns, as a consequence of the higher degree of order of the solid structure. Reduced plate heights of approximately 0.2 could be obtained for (nonretained) bands. No correlation was found between the aspect ratio (the ratio of the channel width and the pillar diameter) and band dispersion. The geometrical construction of the sidewall region was shown to play a critical role for channel performance. A good agreement was found with predictions for the optimal sidewall geometry obtained previously with simulation studies.