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.

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.

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.

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.

Characterisation of UV-cured acrylate networks by means of hydrolysis followed by aqueous size-exclusion combined with reversed-phase chromatography.

UV-cured networks prepared from mixtures of di-functional (polyethylene-glycol di-acrylate) and mono-functional (2-ethylhexyl acrylate) acrylates were analysed after hydrolysis, by aqueous size-exclusion chromatography coupled to on-line reversed-phase liquid-chromatography. The mean network density and the fraction of dangling chain ends of these networks were varied by changing the concentration of mono-functional acrylate. The amount and the molar-mass distribution of the polyethylene-glycol chains between cross-links (M(XL)) and polyacrylic acid (PAA) backbone chains (the so-called kinetic chain length (kcl)) in the different acrylate networks were determined quantitatively. The molar-mass distribution of kcl revealed an almost linear dependence on the concentration of mono-functional acrylate. Analysis of the starting materials showed a significant concentration of mono-functional polyethylene-glycol acrylate. In combination with the analysis of the extractables of the UV-cured networks (polymers not attached to the network, impurities that originate from the photo-initiator and unreacted monomers), more insight in the total network structure was obtained. It was shown that the UV-cured networks contain only small fractions of residual compounds. With these results, the chemical network structure for the different UV-cured acrylate polymers was expressed in network parameters such as the number of PAA units which are cross-linked, the degree of cross-linking, and the network density, which is the molar concentration of effective network chains between cross-links per volume of the polymers. The mean molar mass of chains between chemical network junctions (M(C)) was calculated and compared with results obtained from solid-state NMR and DMA. The mean molar mass of chains between network junctions as determined by these methods was similar.

On-line determination of carboxylic acids, aldehydes and ketones by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionisation mass spectrometry after derivatization with 2-nitrophenylhydrazine.

2-Nitrophenylhydrazine (2-NPH) is widely used for the derivatization of carboxylic acids, aldehydes and ketones, in industrial and biological samples. These compounds react with 2-NPH to form derivatives, which are separated by high-performance liquid chromatography (HPLC) and detected with diode array detection (DAD). The UV spectra give information about the functionality of the compounds: carboxylic acid or ketone/aldehyde. Most of the eluting compounds in "known" samples are well characterised by the retention time (comparison with those of standards) of the 2-NPH derivative and their UV spectrum. The identification of different unknown 2-NPH derivatives of carboxylic acids, ketones and/or aldehydes, in industrial or biological samples, based on retention time and/or UV spectrum is not sufficient. These unknown 2-NPH compounds can be identified with on-line atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) based on the molecular mass or/and the fragmentation of the derivative. A novel and specific on-line HPLC-DAD-APCI(-)-MS method is described for the determination of carboxylic acids, ketones and aldehydes, after on-line pre-column derivatization with 2-NHP. The fragmentation of different 2-NPH derivatives were investigated and the possibilities of APCI(-)-MS detection were demonstrated by the on-line identification of an unknown derivative, which turned out to be a side product between 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 2-NPH in the presence of high concentrations of a cyclic amide in the sample solution.

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. III. Separation mechanism and gradient optimization.

The first six linear and cyclic oligomers of polyamide-6 can be quantitatively determined in the polymer using HPLC with the sandwich injection method and an aqueous acetonitrile gradient. In this final part of the triptych concerning the determination of the oligomers in polyamide-6, the irregular elution behavior of the cyclic monomer compared to the cyclic oligomers was investigated. We also optimized the separation of the involved polyamide oligomers, with respect to gradient steepness, stationary phase, column temperature and mobile phase pH. The irregular elution behavior of the cyclic monomer could be caused by its relatively large exposed/accessible hydrophobic surface, which permits relatively easy penetration into the hydrophobic stationary phase giving extra retention. The dipole moment of the different oligomers was used as a measure for this exposed/accessible hydrophobic area to correlate the retention factors using quantitative structure-retention relationships. We also studied the retention behavior of the polyamide, which is injected each run directly onto the column and modifies the stationary phase. Using a 250-microl post gradient injection zone of formic acid on a 250x3 mm Zorbax SB-C18 column, the polyamide could be effectively removed from the stationary phase after each separation. The linear solvent strength (LSS) model was used to optimize the separation of the first six linear and cyclic oligomers. As the LSS model assumes a linear correlation between the modifier concentration and the logarithm of the retention factor and the cyclic monomer and dimer show extreme curvation of this relation in the eluting region, we investigated different models to predict gradient elution from isocratic data. A direct translation of the isocratic data to gradient retention times did not yield adequate retention times using the LSS model. It was found that the LSS model worked acceptably if gradient retention times were used as input data. Even for fast non-linearly eluting components, an average error of 0.4 resolution units of 4sigma was obtained. Using the LSS model in combination with different column temperatures and mobile phase pH values, a separation of the first six linear and cyclic oligomers was accomplished.

Quantitation of functionality of poly(methyl methacrylate) by liquid chromatography under critical conditions followed by evaporative light-scattering detection. Comparison with NMR and titration.

Atom transfer radical polymerisation (ATRP) is a versatile 'living' controlled polymerisation technique for the synthesis of well-defined architectures such as block copolymers, gradient copolymers, hyperbranched polymers and telechelic polymers. ATRP provides control over molecular mass and molecular mass distribution and is suitable for the polymerisation of a wide variety of monomers, including methyl methacrylate. A chromatographic method was developed for an endgroup-based separation of low-molecular-mass poly(methyl methacrylate) (PMMA), based on liquid chromatography under critical conditions. With this method the PMMA, irrespective of its low-molecular-mass, is separated according to endgroups (functionality) due to interactions of the polar endgroups with the non-modified silica based stationary phase. The different series were identified using on-line atmospheric pressure ionisation electrospray mass spectrometry and quantified by evaporative light scattering detection. These results were compared with those obtained by NMR and titration.

Endgroup-based separation and quantitation of polyamide-6,6 by means of critical chromatography.

Polyamide-6,6 is a polycondensation product from the two monomers adipic acid and 1,6-hexamethylenediamine. Depending on the reacted amount of these monomers, different ratios of amine and carboxylic acid endgroups can be formed. Besides linear chains, cyclic polyamides will also be formed. Using critical chromatography, polyamide-6,6 can be separated independently of molar mass. Retention is based solely on endgroup functionality. It is demonstrated that high-molecular-mass polyamide-6,6 (Mw approximately 20,000-30,000) can be separated using this approach. The separation was optimized by using different parameters, such as percentage modifier, temperature and pressure. The concentration of phosphoric acid was used for selective retention of the different endgroup functionalities. Using this property, a new method called critical gradient chromatography was performed where the mobile phase changes from a weak to a strong solvent with respect to the endgroup functionality, while retaining the critical conditions of the backbone unit. Quantification using UV detection is discussed.