Physical characterization using diffusion NMR spectroscopy.

NMR diffusion measurements (or dNMR) provide a powerful tool for analysis of solution organization and microgeometry of the environment by probing random molecular motion. Being a very versatile method, dNMR can be applied to a large variety of samples and systems. Here, a brief introduction into dNMR and a summary of recent advances in the field are presented. The research topics include restricted diffusion, anisotropic diffusion, polymer dynamics, solution structuring and dNMR method development. The dNMR studied systems include plants, cells (cell models), liquid crystals, polymer solutions, ionic liquids, supercooled solutions, untreated water, amino acid solutions and more. It is demonstrated how a variety of dNMR methods can be applied to a system to extract the data on particular structures present among, formed by or surrounding the diffusing particles. It is also demonstrated how dNMR methods can be developed to allow probing larger geometries, low sample concentrations and faster processes. Copyright © 2016 John Wiley & Sons, Ltd.

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection.

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

Post column derivatisation analyses review. Is post-column derivatisation incompatible with modern HPLC columns?

Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 µm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 µL. A second part of this review illustrated briefly the effect of dead volume on column performance. The experiment evaluated the change in resolution and separation efficiency of some weak to moderately retained solutes on a 250 × 4.6 mm i.d. column packed with 5 µm particles. The data showed that reaction loops beyond 100 µL resulted in a very serious loss of performance. Our study concluded that practitioners of PCD methods largely avoid the use of UHPLC-type column formats, so yes, very much, PCD is incompatible with the modern HPLC column.

Assessing the performance of curtain flow first generation silica monoliths.

Analytical scale active flow technology first generation silica monolithic columns kitted out in curtain flow mode of operation were studied for the first time. A series of tests were undertaken assessing the column efficiency, peak asymmetry and detection sensitivity. Two curtain flow columns were tested, one with a fixed outlet ratio of 10% through the central exit port, the other with 30%. Tests were carried out using a wide range in inlet flow segmentation ratios. The performance of the curtain flow columns were compared to a conventional monolithic column. The gain in theoretical plates achieved in the curtain flow mode of operation was as much as 130%, with almost Gaussian bands being obtained. Detection sensitivity increased by as much as 250% under optimal detection conditions. The permeability advantage of the monolithic structure together with the active flow technology makes it a priceless tool for high throughput, sensitive, low detection volume analyses.

Investigating retention characteristics of a mixed-mode stationary phase and the enhancement of monolith selectivity for high-performance liquid chromatography.

The synthesis and chromatographic behavior of an analytical size mixed-mode bonded silica monolith was investigated. The monolith was functionalized by an in situ modification process of a bare silica rod with chloro(3-cyanopropyl)dimethyl silane and chlorodimethyl propyl phenyl silane solutions. These ligands were selected in order to combine both resonance and nonresonance π-type bonding within a single separation environment. Selectivity studies were undertaken using n-alkyl benzenes and polycyclic aromatic hydrocarbons in aqueous methanol and acetonitrile mobile phases to assess the methylene and aromatic selectivities of the column. The results fit with the linear solvent strength theory suggesting excellent selectivity of the column was achieved. Comparison studies were performed on monolithic columns that were functionalized separately with cyano and phenyl ligands, suggesting highly conjugated molecules were able to successfully exploit both of the π-type selectivities afforded by the two different ligands on the mixed-mode column.

Enhancing the separation performance of the first-generation silica monolith using active flow technology: parallel segmented flow mode of operation.

Active flow technology (AFT) columns are designed to minimise inefficient flow processes associated with the column wall and radial heterogeneity of the stationary phase bed. This study is the first to investigate AFT on an analytical scale 4.6mm internal diameter first-generation silica monolith. The performance was compared to a conventional first-generation silica monolith and it was observed that the AFT monolith had an increase in efficiency values that ranged from 15 to 111%; the trend demonstrating efficiency gains increasing as the volumetric flow to the detector was decreased, but with no loss in sensitivity.

Effect of parallel segmented flow chromatography on the height equivalent to a theoretical plate III--influence of the column length, particle diameter, and the molecular weight of the analyte on the efficiency gain.

The effects of column length on performance in segmented flow chromatography were tested. Column efficiencies were measured for 4.6mm I.D. 3, 5, 7.5 and 10 cm long columns packed with 3.0 µm Hypurity-C18 fully porous particles and of 4.6mm I.D. 5, 10, 15 and 25 cm long columns packed with 5 µm Hypersil GOLD C18 particles. For each column length and particle type, two different configurations were tested: (1) both the inlet and outlet column endfittings were standard and (2) the inlet endfitting was standard but the outlet endfitting allowed parallel segmentation of the exiting flow into a central and a peripheral coaxial region. The segmentation flow ratio was set at 45% (for 3 µm) and at 43% or 21% (for 5 µm). Four samples were used, naphthalene, toluene, butylbenzene, and insulin, which has a ten times smaller diffusion coefficient than the small molecules. The column performance for the low molecular weight compound is significantly improved at velocities above the optimum value when the outlet flow rate is segmented because longitudinal diffusion and mass transfer resistance of this compound in the stationary phase are negligible sources of band broadening at reduced linear velocities between 5 and 25. At high flow rate (4 mL/min), the long-range eddy dispersion terms are about 3.9, 3.2, 2.6, and 1.8h unit lower for the 3, 5, 7.5 and 10 cm long columns, respectively. The longer the column, the lower the efficiency improvement because the border effects are smaller. This result was not systematically observed for the columns packed with 5 µm particles because the transverse dispersion is larger. In contrast, the gain in column efficiency is marginal for insulin because the mass transfer mechanism of this compound is mostly controlled by the slow diffusivity of insulin across Hypurity-C18 particles.

Lipid order, saturation and surface property relationships: a study of human meibum saturation.

Tear film stability decreases with age however the cause(s) of the instability are speculative. Perhaps the more saturated meibum from infants may contribute to tear film stability. The meibum lipid phase transition temperature and lipid hydrocarbon chain order at physiological temperature (33 °C) decrease with increasing age. It is reasonable that stronger lipid-lipid interactions could stabilize the tear film since these interactions must be broken for tear break up to occur. In this study, meibum from a pool of adult donors was saturated catalytically. The influence of saturation on meibum hydrocarbon chain order was determined by infrared spectroscopy. Meibum is in an anhydrous state in the meibomian glands and on the surface of the eyelid. The influence of saturation on the surface properties of meibum was determined using Langmuir trough technology. Saturation of native human meibum did not change the minimum or maximum values of hydrocarbon chain order so at temperatures far above or below the phase transition of human meibum, saturation does not play a role in ordering or disordering the lipid hydrocarbon chains. Saturation did increase the phase transition temperature in human meibum by over 20 °C, a relatively high amount. Surface pressure-area studies showing the late take off and higher maximum surface pressure of saturated meibum compared to native meibum suggest that the saturated meibum film is quite molecularly ordered (stiff molecular arrangement) and elastic (molecules are able to rearrange during compression and expansion) compared with native meibum films which are more fluid agreeing with the infrared spectroscopic results of this study. In saturated meibum, the formation of compacted ordered islands of lipids above the surfactant layer would be expected to decrease the rate of evaporation compared to fluid and more loosely packed native meibum. Higher surface pressure observed with films of saturated meibum compared to native meibum suggests greater film stability especially under the high shear stress of a blink.

Selectivity in separation using π electron-rich stationary phases for the comprehensive two-dimensional analysis of café espresso.

The multidimensional high-performance liquid chromatography separations of the complex sample matrix found in café espresso coffee were completed on the propyl phenyl and butyl phenyl columns that contain 3 and 4 carbon atoms in the spacer chain, respectively. Phenyl type stationary phases are able to undergo unique π-π interactions with aromatic compounds. Previous works have found that there are differences in retention characteristics between these chain lengths and this was explored further here. It was found that when analysing the separations by quadrants, using a geometric approach to factor analysis and by measuring the normalised mean radius, subtle differences in the separations were observed and the butyl phenyl phase was more selective for the high to medium polarity species. However, there was very little difference in separation behaviour for the hydrophobic components within the coffee sample. Overall, the analysis of the entire separation showed very little difference.

Cyano bonded silica monolith--development of an in situ modification method for analytical scale columns.

This study investigates the synthesis and chromatographic behaviour of an analytical size cyanopropyl "cyano" bonded silica monolith. Surface modification was undertaken by treating a neat silica monolith with chloro(3-cyanopropyl)dimethyl silane in dry heptane over a two day period. The resulting monolith showed stability over the duration of the testing program that involved flushing the column with more than 2000 column volumes of mobile phase. Efficiency measurements before and after sylation verified that the integrity of the silica monolith itself was not affected by the modification process, the highest number of theoretical plates (N/m) using anisole was 81,650. A brief selectivity test was then undertaken to assess methylene selectivity and phenyl selectivity. Elemental analysis was used to determine the homogeneity of the carbon load throughout the monolithic bed, and was compared to two commercial C18 and one 'self' modified C18 silica monoliths. The development of the in situ modification is also discussed.

Pi-selective stationary phases: (I) Influence of the spacer chain length of phenyl type phases on the aromatic and methylene selectivity of aromatic compounds in reversed phase high performance liquid chromatography.

Phenyl type stationary phases of increasing spacer chain length (phenyl, methyl phenyl, ethyl phenyl, propyl phenyl and butyl phenyl, with 0-4 carbon atoms in the spacer chain, respectively) were synthesised and packed in house to determine the impact that the spacer chain length has on the retention process. Two trends in the aromatic selectivity, q(aromatic), were observed, depending on whether the number of carbon atoms in the spacer chain is even or odd. Linear log k' vs phi plots were obtained for each stationary phase and the S coefficient was determined from the gradient of these plots. For the phenyl type phases, the S vs n(c) plots of the retention factors of linear polycyclic aromatic hydrocarbons vs the number of rings exhibit a distinct discontinuity that between 3 and 4 rings, which increases with increasing spacer chain length for even phases but decreases for odd phases. Accordingly, we suggest that the retention factors depend differently on the number of carbon atoms in the spacer chain depending on whether this number is even or odd and that this effect is caused by different orientations of the aromatic ring relative to the silica surface.

Pi-selective stationary phases: (II) Adsorption behaviour of substituted aromatic compounds on n-alkyl-phenyl stationary phases.

The frontal analysis method was used to measure the adsorption isotherms of phenol, 4-chlorophenol, p-cresol, 4-methoxyphenol and caffeine on a series of columns packed with home-made alkyl-phenyl bonded silica particles. These ligands consist of a phenyl ring tethered to the silica support via a carbon chain of length ranging from 0 to 4 atoms. The adsorption isotherm models that fit best to the data account for solute-solute interactions that are likely caused by pi-pi interactions occurring between aromatic compounds and the phenyl group of the ligand. These interactions are the dominant factor responsible for the separation of low molecular weight aromatic compounds on these phenyl-type stationary phases. The saturation capacities depend on whether the spacer of the ligands have an even or an odd number of carbon atoms, with the even alkyl chain lengths having a greater saturation capacity than the odd alkyl chain lengths. The trends in the adsorption equilibrium constant are also significantly different for the even and the odd chain length ligands.

Pi-selective stationary phases: (III) Influence of the propyl phenyl ligand density on the aromatic and methylene selectivity of aromatic compounds in reversed phase liquid chromatography.

The retention characteristics of phenyl type stationary phases for reversed phase high performance liquid chromatography are still largely unknown. This paper explores the retention process of these types of stationary phases by examining the retention behaviour of linear PAHs and n-alkylbenzenes on a series of propyl phenyl stationary phases that have changes in their ligand density (1.23, 1.31, 1.97, 2.50 micromol m(-2)). The aromatic and methylene selectivities increased with increasing ligand density until a point where a plateau was observed, overall the propyl phenyl phases had a higher degree of aromatic selectivity than methylene selectivity indicating that these columns are suitable for separations involving aromatic compounds. Also, retention characteristics relating to the size of the solute molecule were observed to be influenced by the ligand density. It is likely that the changing retention characteristics are caused by the different topologies of the stationary phases at different ligand densities. At high ligand densities, the partition coefficient became constant.

Phenyl-type and C1 stationary phases for environmentally friendlier chromatography.

C1 and phenyl-type stationary phases were assessed in terms of their environmental impact on separations using as test solutes polycyclic aromatic hydrocarbons. Methanol (MeOH) and acetonitrile (ACN) mobile-phase gradients were employed. These stationary phases were examined to determine if different physical and chemical properties possessed by these surfaces decreased the organic solvent consumption, and yet maintained peak capacity. The cumulative energy demand (CED) was used to gauge the environmental impact of the separations. The separation of the polycyclic aromatic hydrocarbon test mixture using current methodologies (i.e. a C18/ACN combination) had a CED of 1.13 MJ-eq, and a peak capacity of 27 peaks (resolving 7 of 12 peak pairs with R(s)>1). In comparison, a butyl phenyl stationary phase with a methanol mobile phase had a peak capacity of 26, but with a CED of 0.670 MJ-eq. Monolithic columns containing C18 and C1 phases were also tested. A monolithic C18 column with MeOH had the lowest CED at 0.675 MJ-eq, a peak capacity of 28 peaks and good resolving power (resolving ten peak pairs with R(s)>1), suggesting that this is a viable option with respect to reducing environmental impact for these types of analyses.

Community structure and antibiotic production of Streptomyces nodosus bioreactors cultured in liquid environments.

Immobilized bacteria are being assessed by industry for drug delivery, novel fermentation systems and the protection of organisms in harsh environments. Alginate bioreactors containing Streptomyces nodosus were examined for community structure, cell viability and amphotericin production under different growth conditions. When cell proliferation was encouraged, substrate hyphae were found inside the alginate matrix and within multicellular projections on the surface of the capsule. The periphery of these projections had erect and branched hyphae, morphologically identical to aerial hyphae. Antibiotic production from immobilized organisms was assessed using conditioned culture medium to eliminate the emergence of a free-dwelling population. These organisms sporulated with reduced antibiotic production compared with free-dwelling cultures. The commitment to sporulate was independent of a surface but dependent on community size and nutritional status. This is the first report of the sporulation of S. nodosus in liquid cultures and description of the multicellular community the organism adopts at a solid-liquid interface.

Retention of polycyclic aromatic hydrocarbons on propyl-phenyl stationary phases in reversed-phase high performance liquid chromatography.

A study on the retention of PAHs on three Propyl-phenyl stationary phases was conducted, assessing absolute retention, selectivity, retention correlation and thermodynamic behaviour. The chromatographic retention data revealed that each of the three Propyl-phenyl columns exhibited differences in absolute retention, however, comparison of the compensation temperatures derived from enthalpy-entropy compensation plots showed that the underlying processes responsible for the retention on these columns were the same.

An assessment of the retention behaviour of polycyclic aromatic hydrocarbons on reversed phase stationary phases--thermodynamic behaviour on C18 and phenyl-type surfaces.

The thermodynamic retention behaviour of a linear series of polycyclic aromatic hydrocarbons (PAHs) was investigated on C18 and selected phenyl-type reversed-phase stationary phases, namely C18, C18 Aqua, Propyl-phenyl and Synergi polar-RP stationary phases, using methanol mobile phases. The Propyl-phenyl stationary phase, despite having the lowest surface coverage, was found to exhibit significantly larger enthalpic interactions to the other Phenyl-type phase (Synergi polar-RP) even though this had a much higher surface coverage. This indicated that stronger interactions between the PAHs and the stationary phase ligands were occurring on the Propyl-phenyl phase. Evaluation of the elution band profile of the PAHs in the aqueous methanol mobile phase revealed fairly symmetrical bands for the C18, C18 Aqua and Synergi polar-RP, but severe peak tailing on the Propyl-phenyl phase. A change in mobile phase from methanol to acetonitrile improved the peak shape of the PAHs on the Propyl-phenyl phase, leading to the assumption that unfavourable pi-pi interactions were occurring between the electron-rich PAHs and the electron-rich phenyl rings of the Propyl-phenyl phase.

Visualising viscous fingering in chromatography columns: high viscosity solute plug.

The interface between two fluids that have different viscosities and are percolating through a porous bed is unstable. Sooner or later, a flow instability termed viscous fingering (VF) develops. This phenomenon is important in chromatography because the solute plug does not have the same viscosity as the mobile phase. Because the sample is often much more viscous than the mobile phase, it is the interface at the rear of the sample band that is usually unstable. This situation is frequent in many modes of chromatography, e.g., in preparative and in multidimensional chromatography, in size exclusion chromatography, in frontal analysis, and in other physicochemical measurements (e.g., determination of adsorption isotherms and of mass transfer parameters). When the solute plug is more viscous than the mobile phase, we observed that the solute band compressed. When the viscosity contrast increased up to 0.30 cP, fingers appeared to trail behind the solute plug. The development of fingers then became more substantial as the viscosity contrast increased. To avoid effects associated with VF, the mobile phase and the solute plug should have nearly the same viscosity.

Interactions of poly(tert-butyl acrylate)-poly(styrene) diblock copolymers with lipids at the air-water interface.

Diblock copolymers with hydrophilic poly(tert-butyl acrylate) (PtBA) and hydrophobic poly(styrene) (PS) blocks were synthesized with a view to use them as a surfactant in tear film for increasing the ocular comfort in dry eye syndrome. Interactions of six PtBA-PS copolymers with four important lipids found in the tear film, namely cholesterol, cholesteryl palmitate, dipalmitoyl phosphatidylcholine, and phosphatidylinositol, were studied at the air-water interface using a Langmuir trough. Thermodynamics of mixing of the copolymers and the lipids in the mixed monolayers was determined by calculating excess free energy of mixing. The diblock copolymers showed repulsive interactions with cholesteol and cholesteryl palmitate, near neutral interactions with dipalmitoyl phosphatidylcholine, and attractive interactions with phosphatidylinositol. The lipids interacted with the PS component of the copolymer. The results indicate that a copolymer with a small hydrophilic group and a big hydrophobic group can be a likely candidate for forming stable interactions with the lipids present in the tear film and hence increase the ocular comfort.

An assessment of the retention behaviour of polycyclic aromatic hydrocarbons on reversed phase stationary phases: selectivity and retention on C18 and phenyl-type surfaces.

In this manuscript the retention and selectivity of a set of linear and non-linear PAHs were evaluated on five different reversed-phase columns. These phases included C18 and C18 Aqua stationary phases, as well as three phenyl phases: Propyl-phenyl, Synergi polar-RP and Cosmosil 5PBB phase. Overall, the results revealed that the phenyl-type columns offered better separation performance for the linear PAHs, while the separation of the structural isomer PAHs was enhanced on the C18 columns. The Propyl-phenyl column was found to have the highest molecular-stationary phase interactions, as evidenced by the greatest rate of change in 'S' (0.71) as a function of the molecular weight in the PAH homologous series, despite having the lowest surface coverage (3% carbon load) (where S is the slope of a plot of logk versus the solvent composition). In contrast, the C18 Aqua column, having the highest surface coverage (15% carbon load) was found to have the second lowest molecular-stationary phase interactions (rate of change in S=0.61). Interestingly, the Synergi polar-RP column, which also is a phenyl stationary phase behaved more 'C18-like' than 'phenyl-like' in many of the tests undertaken. This is probably not unexpected since all five phases were reversed phase.