π-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 ϕ 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.

Contributions to high performance liquid chromatography

High performance liquid chromatography (HPLC) is an enabling science with application in all scientific disciplines requiring analysis or purification. The research described here details performance aspects of the chromatography column, which lead to a new design concept in the chromatography column. Studies were also undertaken to characterise selectivity leading to new stationary phases. Research on fluid dynamics in packed beds showed how a mismatch in solvent viscosities between the injection plug and mobile pahse influences the performance of on-line multidimentional HPLC. Selectivity id detection was also examined in an effort to better understand sample analysis. 

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.

DryLab® optimised two-dimensional high performance liquid chromatography for differentiation of ephedrine and pseudoephedrine based methamphetamine samples

In-silico optimised two-dimensional high performance liquid chromatographic (2D-HPLC) separations of a model methamphetamine seizure sample are described, where an excellent match between simulated and real separations was observed. Targeted separation of model compounds was completed with significantly reduced method development time. This separation was completed in the heart-cutting mode of 2D-HPLC where C18 columns were used in both dimensions taking advantage of the selectivity difference of methanol and acetonitrile as the mobile phases. This method development protocol is most significant when optimising the separation of chemically similar chemical compounds as it eliminates potentially hours of trial and error injections to identify the optimised experimental conditions. After only four screening injections the gradient profile for both 2D-HPLC dimensions could be optimised via simulations, ensuring the baseline resolution of diastereomers (ephedrine and pseudoephedrine) in 9.7 min. Depending on which diastereomer is present the potential synthetic pathway can be categorized.

Protocols for finding the most orthogonal dimensions for two-dimensional high performance liquid chromatography

The selection of two high performance liquid chromatography (HPLC) columns with vastly different retention mechanisms is vital for performing effective two-dimensional (2D-) HPLC. This paper reports on a systematic method to select a pair of HPLC columns that provide the most different separations for a given sample. This was completed with the aid of a HPLC simulator that predicted retention profiles on the basis of real experimental data, which is difficult when the contents of sample matrices are largely-or completely-unknown. Peaks from the same compounds must first be matched between chromatograms to compare the retention profiles and optimised 2D-HPLC column selection. In this work, two methods of matching peaks between chromatograms were explored and an optimal pair of chromatography columns was selected for 2D-HPLC. First, a series of 17 antioxidants were selected as an analogue for a coffee extract. The predicted orthogonality of the standards was 39%, according to the fractional surface coverage 'bins' method, which was close to the actual space utilisation of the standard mixture, 44%. Moreover, the orthogonality for the 2D-HPLC of coffee matched the predicted value of 38%. The second method employed a complex sample matrix of urine to optimise the column selections. Seven peaks were confidently matched between chromatograms by comparing relative peak areas of two detection strategies: UV absorbance and potassium permanganate chemiluminescence. It was found that the optimal combinations had an orthogonality of 35% while the actual value was closer to 30%.

Assessing the detectability of antioxidants in two-dimensional high-performance liquid chromatography

This paper explores the analytical figures of merit of two-dimensional high-performance liquid chromatography for the separation of antioxidant standards. The cumulative two-dimensional high-performance liquid chromatography peak area was calculated for 11 antioxidants by two different methods--the areas reported by the control software and by fitting the data with a Gaussian model; these methods were evaluated for precision and sensitivity. Both methods demonstrated excellent precision in regards to retention time in the second dimension (%RSD below 1.16%) and cumulative second dimension peak area (%RSD below 3.73% from the instrument software and 5.87% for the Gaussian method). Combining areas reported by the high-performance liquid chromatographic control software displayed superior limits of detection, in the order of 1 × 10(-6) M, almost an order of magnitude lower than the Gaussian method for some analytes. The introduction of the countergradient eliminated the strong solvent mismatch between dimensions, leading to a much improved peak shape and better detection limits for quantification.

Blind column selection protocol for two-dimensional high performance liquid chromatography

The selection of two orthogonal columns for two-dimensional high performance liquid chromatography (LC×LC) separation of natural product extracts can be a labour intensive and time consuming process and in many cases is an entirely trial-and-error approach. This paper introduces a blind optimisation method for column selection of a black box of constituent components. A data processing pipeline, created in the open source application OpenMS®, was developed to map the components within the mixture of equal mass across a library of HPLC columns; LC×LC separation space utilisation was compared by measuring the fractional surface coverage, fcoverage. It was found that for a test mixture from an opium poppy (Papaver somniferum) extract, the combination of diphenyl and C18 stationary phases provided a predicted fcoverage of 0.48 and was matched with an actual usage of 0.43. OpenMS®, in conjunction with algorithms designed in house, have allowed for a significantly quicker selection of two orthogonal columns, which have been optimised for a LC×LC separation of crude extractions of plant material.

Qualitative and quantitative analysis of lipid classes in fish oils by thin-layer chromatography with an Iatroscan flame Ionization detector (TLC-FID) and liquid chromatography with an evaporative light scattering detector (LC-ELSD)

Combined effects of hydrogen and air flow rates on the peak response of selected neutral lipid classes (triacylglycerol, diacylglycerol, monoacylglycerol, free fatty acids, and ethyl esters) were studied to optimize and calibrate the Iatroscan Mk-6s Chromarod system for the qualitative and quantitative analysis of lipid classes by thin-layer chromatography (TLC) with flame ionization detection in fish oil during the transesterification process. Air flow rate of 2 L/min, hydrogen flow rate of 150-160 mL/min, and scan rate of 30 s/rod were found to be the optimum conditions. All samples were also analyzed by high performance liquid chromatography (HPLC) with evaporative light scattering detection. Quantitative results obtained by TLC with the flame ionization detection method were comparable to those obtained from HPLC with evaporative light scattering detection.

π-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 μmol 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.

Determination of neurotransmitters and their metabolites using one- and two-dimensional liquid chromatography with acidic potassium permanganate chemiluminescence detection

High-performance liquid chromatography with chemiluminescence detection based on the reaction with acidic potassium permanganate and formaldehyde was explored for the determination of neurotransmitters and their metabolites. The neurotransmitters norepinephrine and dopamine were quantified in the left and right hemispheres of rat hippocampus, nucleus accumbens and prefrontal cortex, and the metabolites vanillylmandelic acid, 3,4-dihydrophenylacetic acid, 5-hydroxyindole-3-acetic acid and homovanillic acid were identified in human urine. Under optimised chemiluminescence reagent conditions, the limits of detection for these analytes ranged from 2.5 × 10−8 to 2.5 × 10−7 M. For the determination of neurotransmitter metabolites in urine, a two-dimensional high-performance liquid chromatography (2D-HPLC) separation operated in heart-cutting mode was developed to overcome the peak capacity limitations of the one-dimensional separation. This approach provided the greater separation power of 2D-HPLC with analysis times comparable to conventional one-dimensional separations.

Mixed cultures of food-grade probiotic bacteria and enteric bacteria demonstrate both synergism and inhibition on menaquinone production

The interaction between probiotic (Enterococcus spp., Lactobacillus spp., and Lactococcus spp.) and enteric (Bacteroides spp., Escherichia coli, and Salmonella spp.) bacteria with respect to menaquinone production was examined. Menaquinones were measured in cell pellets by high-pressure liquid chromatography and the main homologues produced were MIK_7–11. The growth of both Bacteroides and E. coli cultured with the 3 probiotics was significantly inhibited with concomitant reduction in menaquinone production. The vitamin K status of humans could be affected by consumption of probiotic dairy foods via the contribution made by gut microflora.