Investigation of the effects of solvent-mismatch and immiscibility in normal-phase × aqueous reversed-phase liquid chromatography.

Comprehensive two-dimensional liquid chromatography (LC × LC) is an attractive separation technique that allows achieving high peak capacities and information on chemical correlations. Unfortunately, its application in industrial practice is still not widespread due to limiting factors such as complex method development, tedious method optimization and solvent-incompatibility (such as solvent-strength mismatch or immiscibility experienced during fraction transfer). A severe case of solvent-incompatibility is encountered in the comprehensive coupling of normal-phase LC and reversed-phase LC (NPLC × RPLC). NPLC × RPLC is considered a desirable LC × LC system, especially for the characterization of synthetic polymers, due to the high orthogonality of the two retention mechanisms. However, its experimental realization often suffers from solvent-injection effects in the RPLC dimension, such as peak-deformation, peak-splitting, or even unretained elution ("breakthrough") of sample components. Such a decrease in performance or loss of retention is highly dependent on the types of solvents used. To explore the boundaries of solvent compatibility, we applied large-volume injections (LVI) of reference analytes (e.g. alkyl benzenes; ethoxylate and propoxylate polymers) dissolved in water-immiscible sample solvents, such as dichloromethane, n-hexane, and isooctane in fast water-based gradient RPLC separations (using methanol or acetonitrile as eluent). It was found that, when using highly aqueous initial gradient conditions, hydrophobic sample diluents were retained and eluted during the applied gradient. Depending on the relative retention of the retained diluent and the sample analytes, good chromatograms for LVI of immiscible solvents were obtained, comparable with injections under ideal conditions. The conclusions from injection experiments in aqueous RPLC were verified by coupling an NPLC system with a gradient from isooctane to tetrahydrofuran and an RPLC system with a gradient from water to acetonitrile in an online comprehensive NPLC × RPLC separation of a mixture of propoxylate polymers. The separation provided separation of the polymers based on their number of hydroxyl end-groups (NPLC) and oligomer chain-length (RPLC), without suffering from significant band-broadening effects due to solvent-mismatch upon injection in the second-dimension RPLC system.

Fast determination of functionality-type × molecular-weight distribution of propoxylates with varying numbers of hydroxyl end-groups using gradient-normal-phase liquid chromatography × ultra-high pressure size-exclusion chromatography.

Understanding the relation between chemical characteristics and properties of synthetic polymers is one of the challenges faced by analytical chemists in industry. This is a complex task, as polymers are not synthesized as single molecule, but are populations of chemically similar compounds with distributions over several properties. The latter include, for example, molecular weight, nature of end-groups (functionality), and chemical composition. In this paper, comprehensive two-dimensional liquid chromatography was used to determine the combined functionality-type and molecular-weight distributions of hydroxy­functionalized propoxylates. Propoxylates derived from different initiators (one up to eight terminal hydroxyl groups) were separated in the first dimension using a gradient normal-phase LC separation (NPLC). In the second dimension ultra-high pressure size-exclusion chromatography separation (UHPSEC), further speciating distributions based on molecular size. The developed NPLC × SEC method with evaporative light-scattering detection can be used for the fast screening (< 30 min) of mutually dependent functionality-type and molecular-weight distributions of unknown propoxylates.

Perspectives on the future of multi-dimensional platforms.

Two-dimensional liquid chromatography (2D-LC) formats have emerged to help address separation problems that are too complex for conventional one-dimensional LC. There are a number of obstacles to the proliferation of 2D-LC that are gradually being removed. Reliable commercial instrumentation has become available and data analysis software is being improved. Detector-sensitivity and phase-system compatibility issues can largely be solved by using active-modulation strategies. The remaining challenge, developing good and fast 2D-LC methods within a reasonable time, may be solved with smart algorithms. The technology platform that has been developed for 2D-LC also creates a number of other possibilities. Between the two separation stages, all kinds of physical (e.g. dissolution) or chemical (e.g. enzymatic or light-induced degradation) processes can be made to take place, allowing a wide variety of experiments to be performed within a single, efficient and automated analysis. All these developments are discussed in this paper and a number of critical issues are identified. A practical example, the characterization of polysorbates by high-resolution comprehensive two-dimensional liquid chromatography in combination with high-resolution mass spectrometry, is described as a culmination of recent developments in 2D-LC and as an illustration of the current state of the art.

Characterization of complex polyether polyols using comprehensive two-dimensional liquid chromatography hyphenated to high-resolution mass spectrometry.

Polyether polyols are often used in formulated systems, but their complete characterization is challenging, because of simultaneous heterogeneities in chemical composition, molecular weight and functionality. One-dimensional liquid chromatography-mass spectrometry is commonly used to characterize polyether polyols. However, the separation power of this technique is not sufficient to resolve the complexity of such samples entirely. In this study, comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry (LC × LC-HRMS) was used for the characterization of (i) castor oil ethoxylates (COEs) reacted with different mole equivalents of ethylene oxide and (ii) a blended formulation consisting of glycerol ethoxylate, glycerol propoxylate and glycerol ethoxylate-random-propoxylate copolymers. Retention in the first (hydrophilic-interaction-chromatography) dimension was mainly governed by degree of ethoxylation, while the second reversed-phase dimension resolved the samples based on degree of propoxylation (blended formulation) or alkyl chain length (COEs). For different COE samples, we observed the separation of isomer distributions of various di-, tri- and tetra-esters, and such positional isomers were studied by tandem mass spectrometry (LC-MS/MS). This revealed characteristic fragmentation patterns, which allowed discrimination of the isomers based on terminal or internal positioning of the fatty-acid moieties and provided insight in the LC × LC retention behavior of such species.

Preparation of cyanoacrylate derivatives and comparison of dual action cyanoacrylate formulations.

The use of cyanoacrylate followed by fluorescent reagents is well known in the field of fingermark visualisation. Treatment with fluorescent reagents that stain the cyanoacrylate deposits will not only enhance the visibility of fingermarks previously thought unusable, but also reveal previously unseen marks. Downside of this approach is the exposure of the evidential material to large amounts of solvent, such as water, methanol and ethanol, thereby potentially destroying other forensic traces. New cyanoacrylate derivates with fluorescent and UV-active properties can be used for one step dual action visualisation of latent fingermarks. Increased optical properties can be achieved by addition of functional groups via the Steglich esterification of cyanoacetic acid with N-(3-dimetylaminopropyl)-N'-ethylcarbodiimide (EDC). The UV-active ester can be created via the Knoevenagel condensation with formaldehyde to form poly-cyanoacrylate. These poly-cyanoacrylates can be depolymerised to form monomer cyanoacrylates. In this paper we compare these ethylcyanoacrylate derivatives with commercially available cyanoacrylate formulations. We have shown that the use of poly-cyanoacrylate derivatives can yield fully developed fingerprints. The initiator of the polymerisation reaction towards novel reagents can be altered according to the need for particular optical properties.