Search

To determine the clinical outcomes and evaluate the perspectives of children with Type 1 diabetes (T1D) and their parents managing their child on hybrid closed-loop (HCL) therapy.Children with T1D on HCL attending a tertiary diabetes centre between April 2019 and July 2021 were included. A retrospective analysis of glycaemic data was conducted to determine the clinical outcomes. Time spent in closed loop, time in target glucose range (TIR 3.9-10 mmol/L), hypoglycaemia and hyperglycaemia were collected at baseline, 4 weeks, 3 and 6 months post-HCL. User experience was assessed by questionnaires administered to parents of children with T1D.Seventy-one children, mean (SD) age of 12.2 (3.2) years were commenced on HCL. Ten (14%) discontinued HCL use, with 60% discontinuing within the first 6 months. Glycaemic outcomes were analysed in 52 children. Time spent in closed loop was 78 (21) % at 4 weeks, declined to 69 (28) % at 3 months (P = 0.037) and 63 (34) % at 6 months (P = 0.001). The mean %TIR increased from 59.8 at baseline to 67.6 at 3 months and 65.6 at 6 months with a mean adjusted difference of 7.8% points [95% CI 3.6, 11.9] and 5.5% points [95% CI 1.4, 9.5], respectively. There was a reduction in time 10 mmol/L and time 3.9 mmol/L from baseline to 6 months. Although families faced challenges with technology, better glucose control with reduced glycaemic fluctuations were reported.HCL therapy is associated with improved glycaemia; however, adequate support and education are required for best outcomes.

Objectives Glucose monitoring is vital in children with persistent hypoglycaemia to reduce the risk of adverse neuro-behavioural outcomes; especially in children with hyperinsulinism. The role of continuous glucose monitoring (CGM) systems in monitoring glucose levels in this cohort is limited. The objective of this study was to ascertain the effectiveness of CGM and to evaluate parents’ experience of using CGM for monitoring glucose levels in children with hypoglycaemia. Methods Retrospective analysis of sensor glucose (SG) values from Dexcom G4 CGM with paired finger-prick blood glucose (BG) values was performed to determine the accuracy of CGM. The parent experience of CGM was assessed using a questionnaire administered to families of children with congenital hyperinsulinism currently attending the clinic. Results SG data from 40 children (median age 6 months) with persistent hypoglycaemia (60% Hyperinsulinism) were analysed. The mean difference between 5,650 paired BG and SG values was 0.28 mmol/L. The sensitivity and specificity of CGM to identify severe hypoglycaemia (BG Conclusions Although the high number of false-positive readings precludes the routine use of CGM in the evaluation of hypoglycaemia, it avoids unnecessary BG testing during normoglycaemia. It is an acceptable tool for parents for monitoring their children who are at risk of hypoglycaemia. Newer CGM systems with improved accuracy at lower glucose levels have the potential to further improve monitoring.

Background Vulvodynia and pudendal neuralgia comprise significant contributors to vulvar-related pain and its impact on daily life. Aim A retrospective clinical audit was conducted at the Women's Health & Research Institute of Australia, Sydney, to determine the pattern of use and the efficacy of the application of topical amitriptyline 0.5% plus oestriol 0.03% in organogel (AOO), to the vulvar vestibule in reducing the impact of pain on daily life. Materials and methods There were 1174 patients who received a script from May 2017 until February 2020: 1054 patients agreed to be contacted and had a valid email address. Results There were 376 (35.7%) patients who replied. Pain with intercourse was the main indication for use. Treatment was rated effective by 51.2% (95% CI: 35.4-66.8%) of patients less than 30 years of age, 66.7% (95% CI: 57.3-74.9%) of patients 30-50 years of age, and 58.3% (95% CI: 50.9-65.4%) in patients over 50. Stinging at the site of application was the most commonly reported side effect. Conclusion Topical AOO is an effective and well-tolerated treatment for vulvar pain.

The use of three-dimensional printing in chemistry education has expanded greatly in the past 10 years. The technique has been used to demonstrate a range of concepts including molecular structure,...

Objectives There is a paucity of information regarding the profile of entire paediatric endocrine clinics and how they are changing. This study aimed to analyse the clinic profile of the Western Australian tertiary paediatric endocrine outpatient service over 10 years and compare to national and international data. Methods Retrospective analysis of the Western Australian Paediatric Endocrine Database (WAPED) between 2007 and 2017 looking at the frequency, proportion and longitudinal change of diagnosis categories, specific diagnoses, and gender breakdown. Results In total, 2,791 endocrine diagnoses were recorded for 2,312 patients. The most frequent reason for referral (22.1% of patients), was for evaluation of abnormalities in thyroid function. The most common diagnosis being hypothyroidism (76.7%). Evaluation of short stature was the reason for referral in 19.2% of patients, 14.6% of whom were diagnosed with growth hormone deficiency. Evaluation of puberty disorders, syndromes with endocrine features and disorders of calcium and phosphate metabolism were other common reasons for clinic referral, seen in 11.3, 9.8 and 8.2% of patients respectively. Between 2007 and 2017, the odds ratio of a thyroid diagnosis increased by 1.07 per year (95% CI: 1.02–1.12), whilst the odds ratio of a short stature diagnosis decreased by 0.91 per year (95% CI: 0.87–0.95). Conclusions The profile of the WAPED is similar to previously published national and international data. The analysis of the profile of diagnoses and its longitudinal change over a ten-year period offer a unique opportunity to guide clinic planning, resource allocation and future research.

Aim To determine the frequency of protracted bacterial bronchitis (PBB) in children referred to tertiary care with chronic cough and describe management prior to referral. Methods A retrospective cohort study of all new patients with a history of ≥4 weeks of cough seen at the only tertiary paediatric outpatient respiratory service in Western Australia between July 2018 and June 2019. Medical records were reviewed until a final diagnosis was documented or otherwise for a period of 18 months. Results PBB was the most common cause and comprised 37.9% of all children referred to tertiary respiratory care with chronic cough. In children with PBB, the median cough duration at the time of first specialist review was 5.1 months (IQR 2.1-12.0 months). The most common referral source of PBB was primary practice (40.9%) and the most common working diagnosis pre-referral was asthma (15.9%). Seventy-eight percent of children with PBB had an ongoing cough at their first respiratory review, and of these, only 13.5% had been prescribed 4 weeks of antibiotics prior to their respiratory review. Asthma treatment had been prescribed for 34.0% of children with PBB. Conclusion PBB is the most common cause of chronic cough in children referred to tertiary respiratory care and is frequently misdiagnosed and undertreated pre-referral. There is a need to facilitate diagnosis and optimal management of PBB in primary care, which could result in earlier symptom resolution and potentially limit disease progression to bronchiectasis.

Purpose Microperimetry measures differential light sensitivity (DLS) at specific retinal locations. The aim of this study is to examine the variation in DLS across the macula and the contribution to this variation of cone distribution metrics and retinal eccentricity. Methods Forty healthy eyes of 40 subjects were examined by microperimetry (MAIA) and adaptive optics imaging (rtx1). Retinal DLS was measured using the grid patterns: foveal (2°-3°), macular (3°-7°), and meridional (2°-8° on horizontal and vertical meridians). Cone density (CD), distribution regularity, and intercone distance (ICD) were calculated at the respective test loci coordinates. Linear mixed-effects regression was used to examine the association between cone distribution metrics and loci eccentricity, and retinal DLS. Results An eccentricity-dependent reduction in DLS was observed on all MAIA grids, which was greatest at the foveal-parafoveal junction (2°-3°) (-0.58 dB per degree, 95% confidence interval [CI]; -0.91 to -0.24 dB, P < 0.01). Retinal DLS across the meridional grid changed significantly with each 1000 cells/deg2 change in CD (0.85 dB, 95% CI; 0.10 to 1.61 dB, P = 0.03), but not with each arcmin change in ICD (1.36 dB, 95% CI; -2.93 to 0.20 dB, P = 0.09). Conclusions We demonstrate significant variation in DLS across the macula. Topographical change in cone separation is an important determinant of the variation in DLS at the foveal-parafoveal junction. We caution the extrapolation of changes in DLS measurements to cone distribution because the relationship between these variables is complex. Translational Relevance Cone density is an independent determinant of DLS in the foveal-parafoveal junction in healthy eyes.

Delays within the motor domain are often overlooked as an early surveillance marker for autism. The present study evaluated motor difficulties and its potential as an early predictive marker for later autism likelihood in a cohort of infants (N = 96) showing early behavioral signs of autism aged 9–14 months. The motor domain was evaluated using the motor subscales of the Mullen Scales of Early Learning at baseline, and at a 6-month follow-up. The Autism Diagnostic Observation Schedule – Toddler Module (ADOS-T) was completed at follow-up as a measure of autism likelihood. Motor difficulties were common at baseline, with 63/96 (65.6%) infants scoring very low or below average in the gross motor domain and 29/96 (30.2%) in the fine motor domain. At follow-up, gross motor difficulties had resolved for many, with 23/63 (36.5%) infants maintaining these difficulties. Fine motor difficulties resolved in fewer infants, with 20/29 (69.0%) continuing to present with fine motor delays at follow-up. Adjusted linear regression models suggested that fine motor scores at baseline (β = −0.12, SE = 0.04) and follow-up (β = −0.17, SE = 0.05) were associated with higher ADOS-T scores; with difficulties across both timepoints (β = 5.60, SE = 1.35) the strongest (largest in magnitude) association with ADOS-T scores of the predictors examined. Motor difficulties are prominent in children displaying emerging signs of autism, with persistent fine motor difficulties predictive of the developing autism phenotype. The findings indicate the potential clinical value of including evaluation of motor skills within early autism surveillance measures. Lay Summary: This prospective study evaluated motor development over a 6-month period in infants showing early behavioral signs of autism. Atypical motor development was a common feature of infants showing early signs of autism and persistent fine motor difficulties were predictive of the emerging autism phenotype.

Neutral silicon vacancy (SiV^{0}) centers in diamond are promising candidates for quantum networks because of their excellent optical properties and long spin coherence times. However, spin-dependent fluorescence in such defects has been elusive due to poor understanding of the excited state fine structure and limited off-resonant spin polarization. Here we report the realization of optically detected magnetic resonance and coherent control of SiV^{0} centers at cryogenic temperatures, enabled by efficient optical spin polarization via previously unreported higher-lying excited states. We assign these states as bound exciton states using group theory and density functional theory. These bound exciton states enable new control schemes for SiV^{0} as well as other emerging defect systems.

Adaptive optics flood illumination ophthalmoscopy (AO-FIO) is an established imaging tool in the investigation of retinal diseases. However, the clinical interpretation of AO-FIO images can be challenging due to varied image quality. Therefore, image quality assessment is essential before interpretation. An image assessment tool will also assist further work on improving the image quality, either during acquisition or post processing. In this paper, we describe, validate and compare two automated image quality assessment methods; the energy of Laplacian focus operator (LAPE; not commonly used but easily implemented) and convolutional neural network (CNN; effective but more complex approach). We also evaluate the effects of subject age, axial length, refractive error, fixation stability, disease status and retinal location on AO-FIO image quality. Based on analysis of 10,250 images of 50 × 50 μm size, at 41 retinal locations, from 50 subjects we demonstrate that CNN slightly outperforms LAPE in image quality assessment. CNN achieves accuracy of 89%, whereas LAPE metric achieves 73% and 80% (for a linear regression and random forest multiclass classifier methods, respectively) compared to ground truth. Furthermore, the retinal location, age and disease are factors that can influence the likelihood of poor image quality.

Background Birth order effects have been linked to variability in intelligence, educational attainment and sexual orientation. First- and later-born children have been linked to an increased likelihood of an Autism Spectrum Disorder (ASD) diagnosis, with a smaller body of evidence implicating decreases in cognitive functioning with increased birth order. The present study investigated the potential association between birth order and ASD diagnostic phenotypes in a large and representative population sample. Methods Data were obtained from an ongoing prospective diagnostic registry, collected between 1999 and 2017, including children (1-18 years of age, n = 5,404) diagnosed with ASD in the state of Western Australia. Children with ASD were ranked relative to sibling's birth to establish birth order within families at time of ASD diagnosis. Information reported to the registry by health professionals at the time of diagnostic evaluation included demographic and family characteristics, functional abilities and intellectual capacity. Results Adaptive functioning and intelligence scores decreased with increasing birth order, with later-born children more likely to have an intellectual disability. Compared to first-born children with siblings, first-born children without siblings at the time of diagnosis also exhibited decreased cognitive functioning. Conclusions These findings demonstrate for the first time an association between increasing birth order and variability in ASD clinical phenotypes at diagnosis, with potential evidence of reproductive curtailment in children without siblings. Taken together, these findings have significant implications for advancing understanding about the potential mechanisms that contribute to heterogeneity in ASD clinical presentations as a function of birth order and family size.

In recent years semiconducting qubits have undergone a remarkable evolution, making great strides in overcoming decoherence as well as in prospects for scalability, and have become one of the leading contenders for the development of large-scale quantum circuits. In this Review we describe the current state of the art in semiconductor charge and spin qubits based on gate-controlled semiconductor quantum dots, shallow dopants, and color centers in wide band gap materials. We frame the relative strengths of the different semiconductor qubit implementations in the context of quantum simulations, computing, sensing and networks. By highlighting the status and future perspectives of the basic types of semiconductor qubits, this Review aims to serve as a technical introduction for non-specialists as well as a forward-looking reference for scientists intending to work in this field., Review article, feedback welcome, 27 pages, 5 figures

This project looks at the intersection between gunshot damage to road signs and the use of geographic information systems (GIS) for forensic intelligence. The project has surveyed several distinct ...

This study further advances the development of a software program for the analysis of bloodstain patterns formed from a sledgehammer apparatus capable of creating reproducible blood spatter...

Optimising detergency at lower temperatures is of increasing interest due to environmental and economic factors, and requires a greater understanding of the effects of temperature on the adsorption of surfactant mixtures at interfaces. The adsorption properties of surfactant mixtures and biosurfactant/surfactant mixtures have been studied at room temperatures and at temperatures below ambient using surface tension and neutron reflectivity measurements. For the ternary surfactant mixture of octaethylene monododecyl ether, C12E8, sodium dodecyl 6-benzene sulfonate, LAS, and sodium dioxyethylene glycol monododecyl sulfate, SLES, the surface tension at the air-water interface increases with decreasing temperature. In contrast, there is a notable reduction in the increase in the surface tension with a decrease in temperature from 25 °C to 10 °C for the 5 component rhamnolipid/surfactant mixture of the mono-rhamnose, R1, and di-rhamnose, R2, with C12E8/LAS/SLES. The associated neutron reflectivity data for the ternary C12E8/LAS/SLES mixture and the significant observation is that the 3, 4, and 5-component mixtures containing rhamnolipids in conjunction with the other surfactants show changes in composition and adsorbed amounts of the individual components which are close to the experimental error. However the significant observation is that the neutron reflectivity data indicate that the improved surface tension tolerance at lower temperatures is associated with the dominance of the rhamnolipid adsorption in such mixtures. Hence the introduction of the rhamnolipids provides a tolerance to the adverse effects associated with reduced temperatures, and a potential for improved detergency at relatively low temperatures.

Utilizing a combination of time-resolved Infrared (IR) spectroscopies, we are able to probe the effects of a small biological molecule (cholesterol) on the dynamics of lipid membranes from picoseconds to milliseconds. By monitoring the ultrafast dynamics of the system with multi-dimensional IR spectroscopy, we are able to resolve the influence cholesterol has on the electric field fluctuations inside the membrane. We use temperature-jump (T-jump) spectroscopy to extend our experimentally-observable time window beyond the vibrational lifetime of our probe and find that the presence of cholesterol introduces significant inhomogeneity into the relaxation rates of the system. This approach provides new insight into the interaction between cholesterol and lipids, while also demonstrating the utility of this combined IR spectroscopic toolbox for studying multiscale dynamics.

Measures to control the expanding use of synthetic cannabinoids demand new analytical methodology to identify and determine compounds within this rapidly evolving class. Herein, we identify seven synthetic cannabinoids (AM-2201, UR-144, XLR-11, A796,260, 5F-AKB48, PB-22 and 5F-PB-22) present in eleven herbal products sold in Victoria, Australia, prior to their ban in 2014, using a combination of GC-MS, HPLC, ESI-MS, and NMR. In aid of this work, we synthesised the synthetic cannabinoids AM-2201 and 5F-AKB48. We then explore for the first time, the chemiluminescence detection of synthetic cannabinoids using three commonly used reagents: permanganate, manganese(IV), and tris(2,2′‐bipyridine)ruthenium(III). Using the permanganate reagent, no chemiluminescence signal was obtained, but the manganese(IV) and tris(2,2′‐bipyridine)ruthenium(III) reagents gave analytically useful responses with all synthetic cannabinoids under investigation except 5F-AKB48. Calibration curves for PB-22, 5F-PB-22, AM-2201 and 5F-AKB48 prepared using HPLC with UV absorbance and/or chemiluminescence detection were used to determine the total cannabinoid content extracted with methanol (1 mL) from of six of the herbal products (10 mg), which ranged from 0.072 to 0.77 mg.

Principal component analysis (PCA) loading plots were used to elucidate key differences between two-dimensional high performance liquid chromatography (2D-HPLC) fingerprint data from samples collected from stages along the Papaver somniferum industrial process chemistry workflow. Data reduction was completed using a 2D-HPLC peak picking approach as a precursor to chemometric analysis. Using comparisons of the final stages of product extraction as an example, PCA analysis of characteristic 2D-HPLC fingerprints accounted for 84.9% of variation between the two sample sets measured in triplicate, with 64.7% explained by PC1. Loadings plots of PC1 on each sample set identified where the significant changes were occurring and normalised bubble plots provided an indication of the relative importance of each of these changes. These findings highlight 2D-HPLC with appropriate chemometric analysis as a useful tool for the exploration of bioactive molecules within biomass.

Er3+ ions in crystals are promising for quantum networks. We demonstrate ion implantation of Er3+ into TiO2, a potential low-noise host material, with narrow optical and spin linewidths and high implantation yield.

Atomic and atom-like defects in the solid-state are widely explored for quantum computers, networks and sensors. Rare earth ions are an attractive class of atomic defects that feature narrow spin and optical transitions that are isolated from the host crystal, allowing incorporation into a wide range of materials. However, the realization of long electronic spin coherence times is hampered by magnetic noise from abundant nuclear spins in the most widely studied host crystals. Here, we demonstrate that Er$^{3+}$ ions can be introduced via ion implantation into TiO$_2$, a host crystal that has not been studied extensively for rare earth ions and has a low natural abundance of nuclear spins. We observe efficient incorporation of the implanted Er$^{3+}$ into the Ti$^{4+}$ site (40% yield), and measure narrow inhomogeneous spin and optical linewidths (20 and 460 MHz, respectively) that are comparable to bulk-doped crystalline hosts for Er$^{3+}$. This work demonstrates that ion implantation is a viable path to studying rare earth ions in new hosts, and is a significant step towards realizing individually addressed rare earth ions with long spin coherence times for quantum technologies.

Despite its important role in biology and nanotechnology, many questions remain regarding the molecular mechanism and dynamics by which oligonucleotides recognize and hybridize to their complementary sequence. The thermodynamics and kinetics of DNA oligonucleotide hybridization and dehybridization are often assumed to involve an all-or-nothing two-state dissociation pathway, but deviations from this behavior can be considerable even for short sequences. We introduce a new strategy to characterize the base-pair-specific thermal dissociation mechanism of DNA oligonucleotides through steady-state and time-resolved infrared spectroscopy. Experiments are interpreted with a lattice model to provide a structure-specific interpretation. This method is applied to a model set of self-complementary 10-base-pair sequences in which the placement of GC base pairs is varied in an otherwise AT strand. Through a combination of Fourier transform infrared and two-dimensional infrared spectroscopy, experiments reveal varying degrees of deviation from simple two-state behavior. As the temperature is increased, duplexes dissociate through a path in which the terminal bases fray, without any significant contribution from loop configurations. Transient temperature jump experiments reveal time scales of 70-100 ns for fraying and 10-30 μs for complete dissociation near the melting temperature. Whether or not frayed states are metastable intermediates or short-lived configurations during the full dissociation of the duplex is dictated by the nucleobase sequence.

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.

AIMS: To establish the ability of the rhamnolipids biosurfactants from Pseudomonas aeruginosa, in the presence and absence of caprylic acid and ascorbic acid, to disrupt bacterial biofilms, compared with the anionic alkyl sulphate surfactant Sodium dodecyl sulphate (SDS). METHODS AND RESULTS: Pseudomonas aeruginosa ATCC 15442 biofilms were disrupted by rhamnolipids at concentrations between 0·5 and 0·4 g l(-1) and with SDS at 0·8 g l(-1) . The combination of rhamnolipids 0·4 g l(-1) and caprylic acid at 0·1 g l(-1) showed a remarkable effect on biofilm disruption and cell killing. After 30 min of treatment most of the biofilm was disrupted and cell viability was significantly reduced. Neither caprylic acid nor ascorbic acid has any effect on biofilm disruption at 0·1 g l(-1) . SDS is an effective antimicrobial agent; however, in the presence of caprylic acid its effect was neutralized. CONCLUSIONS: The results show that rhamnolipids at low concentration in the presence of caprylic acid are promising molecules for inhibition/disruption of biofilms formed by Ps. aeruginosa ATCC 15442. SIGNIFICANCE AND IMPACT OF THE STUDY: The disruption of biofilms has major significance in many industrial and domestic cleaning applications and in medical situations.

The COVID-19 pandemic has had broad social implications for children around the world. While the initial government response has focused on public health strategies to contain the spread of the virus and the creation of sufficient capacity within hospitals to manage patients with acute medical complications, it was recognised that the social, economic and mental health consequences of COVID-19 would follow. In Western Australia (WA), we have had relatively few COVID-19 cases in the general population and even fewer in children. Similar to paediatric hospitals internationally,1 our emergency department presentations and overall hospital admissions have fallen significantly in 2020 (figure 1). However, since the commencement of the COVID-19 pandemic in Australia, we have observed a 104% increase (95% CI +56% to +166%, p

Color centers in diamond are attractive candidates for implementing single-atom quantum memories in a quantum network. Recently, we reported a color center that exhibits favorable optical properties and long spin coherence times. The integration of this color center, SiV0, into nanophotonic circuits could enable efficient entanglement distribution over long distances.

Australian clandestine drug laboratories are constantly utilising alternative methods to produce methamphetamine, in part as restrictions are placed by Government on, for example, chemicals such as phenyl-2-propanone (P2P) (in the early 1980s), or on pseudoephedrine-containing pharmaceuticals, from the mid-2000s. This paper discusses the nitro-aldol reaction occurring between nitroethane and benzaldehyde, which can be utilised in a number of differing routes, in the presence of different bases. The resulting products, namely phenyl-2-nitropropene (P2P pathway) and 2-nitro-1-phenyl-1-propanol (ephedrine pathway) are directly dependant on which base is used; as such, the base may be used to provide an indication of a possible manufacture pathway of methamphetamine at a clandestine laboratory.

This work describes a method for two-dimensional high performance liquid chromatography (2D-HPLC) that uses an isocratic mobile phase with a temperature gradient in the first dimension. Temperature programming was used to manipulate solvent elution strength in place of a mobile phase concentration gradient. This ensured that all eluent fractions transferred into the second dimension were of an identical solvent composition, i.e. the second dimension injection solvent did not increase during the course of the analysis. When applied to a complex natural product extract of coffee, the separation was completed in 35 min and had an orthogonality of 35% (calculated using the bins method) and a spreading angle of 52° as determined via a geometric approach to factor analysis. This approach, incorporating a temperature gradient in the first dimension, compared favourably to previously reported 2D-HPLC separations of coffee, with similar or shorter analysis times.

While data processing methods in metabolomic studies often work with 'n' number of dimensions, analytical techniques, with the notable exception of NMR, have mostly stuck only to one. Peak overlap continues to be a problem and there is an ever-present demand to maximize the number of metabolites that can be separated and identified in a single run. One method that might help to overcome these issues is multidimensional liquid chromatography, which uses two columns of different phases. A sequential collection of aliquots is made from the first column and reinjected onto a second, and the resulting data are then plotted in 2D or 3D space. The total peak capacity of such a system is the combined peak capacities of each column. The 'offline' version of this technique, using a fraction collector, was introduced over 30 years ago but with recent advances in instrumentation and software, particularly the 'online' approach using automated switching valves, has led to increasing interest in the technique. Both offline and online methods can be carried out as a comprehensive procedure, or via 'heart-cutting', in which only specific peaks are analysed in the second dimension. Past applications include proteomics, natural product chemistry, forensic science and pharmaceutical analysis. These successes are likely to be built on in the future as new column chemistries and bio-informatic approaches are developed. In this review an overview of the theory of twodimensional liquid chromatography is presented, its potential in the field of metabolomics is assessed and predictions for future research directions are made.

Widely known for its recreational use, the cannabis plant also has the potential to act as an antibacterial agent in the medicinal field. The analysis of cannabis plants/products in both pharmacological and forensic studies often requires the separation of compounds of interest and/or accurate identification of the whole cannabinoid profile. In order to provide a complete separation and detection of cannabinoids, a new two-dimensional liquid chromatography method has been developed using acidic potassium permanganate chemiluminescence detection, which has been shown to be selective for cannabinoids. This was carried out using a Luna 100 A CN column and a Poroshell 120 EC-C18 column in the first and second dimensions, respectively. The method has utilized a large amount of the available separation space with a spreading angle of 48.4° and a correlation of 0.66 allowing the determination of more than 120 constituents and mass spectral identification of ten cannabinoids in a single analytical run. The method has the potential to improve research involved in the characterization of sensitive, complex matrices.

The effect of ion binding in the selectivity filter of the potassium channel KcsA is investigated by combining amide I Fourier-transform infrared spectroscopy with structure-based spectral modeling. Experimental difference IR spectra between K(+)-bound KcsA and Na(+)-bound KcsA are in good qualitative agreement with spectra modeled from structural ensembles generated from molecular dynamics simulations. The molecular origins of the vibrational modes contributing to differences in these spectra are determined not only from structural differences in the selectivity filter but also from the pore helices surrounding this region. Furthermore, the coordination of K(+) or Na(+) to carbonyls in the selectivity filter effectively decouples the vibrations of those carbonyls from the rest of the protein, creating local probes of the electrostatic environment. The results suggest that it is necessary to include the influence of the surrounding helices in discussing selectivity and transport in KcsA and, on a more general level, that IR spectroscopy offers a nonperturbative route to studying the structure and dynamics of ion channels.

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.

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%.

As monolithic columns become more extensively used in separation based applications due to their good flow and high surface characteristics, there has arisen the need to establish simple, reliable fabrication methods for fluidic coupling and sealing. In particular, the problem of liquid tracking between a monolith's outer surface and the sealing wall, resulting in poor flow-through performance, needs to be addressed. This paper describes a novel resin-based encapsulation method that penetrates 0.3 mm into the outer surface of a 4 mm diameter monolith, removing the so-called wall-effect. Results based on the peak analysis from 1 µL of 0.4% thiourea injected into a 98 : 2 water : methanol mobile phase flowing at 1 mL min−1 indicate excellent flow conservation through the monolith. A comparison of peak shape and height equivalent to a theoretical plate (HETP) data between the reported resin-based method and the previously reported heat shrink tubing encapsulation methodology, for the same batch of monoliths, suggests the resin based method offers far superior flow characteristics. In addition to the improved flow properties, the resin casting method enables standard polyether ether ketone (PEEK) fittings to be moulded and subsequently unscrewed from the device offering simple reliable fluidic coupling to be achieved.

This paper describes a non-destructive “peak parking” protocol in order to assess the axial heterogeneity of an in situ modified monolithic column for high performance liquid chromatography; a “gradient stationary phase” was designed whereby the ligand density decreases along the length of the rod in the “forward flow” configuration. The results of multi-location peak parking demonstrated a consistent increase in peak variance from the 1 cm position of the column to the 9 cm location. This increase in band broadening supported the theory of a decreasing ligand density along the length of this gradient column. This is consistent with efficiency measurements performed in both the forward and reverse flow directions, with an improved efficiency (15% increase in N m−1) in the reverse direction. These results are consistent with theoretical investigations into stationary phase gradients.

Using temperature-jump infrared spectroscopy, we are able to trigger a gel-to-fluid phase transition in lipid vesicles and monitor in real time how a membrane protein responds to structural changes in the membrane. The melting of lipid domains in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles is observed to occur in as fast as 50 ns, with a temperature dependence characteristic of critical slowing. Gramicidin D (gD) added to the membrane responds primarily to the change in thickness of the membrane on a timescale coincident with the membrane melting. Using structure-based spectral modeling, we assign the conformational changes to compression and rotation of a partially dissociated gD dimer. Free energy calculations indicate that the high rate is a result of near-barrierless diffusion on a protein energy landscape that is radically reshaped by membrane thinning. The structural changes associated with the phase transition are similar to the fluctuation modes of fluid phase membranes, highlighting the importance of understanding the dynamic nature of the membrane environment around proteins.

The composition of the air–water adsorbed layer of the ternary surfactant mixture, octaethylene monododecyl ether, C12E8, sodium dodecyl 6-benzenesulfonate, LAS, and sodium dioxyethylene glycol monododecyl sulfate, SLES, and of each of the binary mixtures, with varying amounts of electrolyte, has been studied by neutron reflectivity. The measurements were made above the mixed critical micelle concentration. In the absence of electrolyte adsorption is dominated by the nonionic component C12E8 but addition of electrolyte gradually changes this so that SLES and LAS dominate at higher electrolyte concentrations. The composition of the adsorbed layer in both binary and ternary mixtures can be quantitatively described using the pseudo–phase approximation with quadratic and cubic interactions in the excess free energy of mixing (GE) at both the surface and in the micelles. A single set of parameters fits all the experimental data. A similar analysis is effective for a mixture in which SDS replaces SLES. Addition...

The composition of the adsorbed layer of a ternary surfactant mixture at the air-water interface has been studied by neutron reflectivity. The adsorption of the ternary mixture of octaethylene monododecyl ether (C12E8) sodium dodecyl 6-benzene sulfonate (LAS), and sodium dioxyethylene glycol monododecyl sulfate (SLES), as well as each of the binary mixtures, at solution concentrations greater than the mixed critical micelle concentration is highly nonideal. In the ternary mixture, the surface adsorption is dominated by C12E8 and LAS, and there is little SLES at the interface. The departure from ideality in the binary mixtures can be quantitatively described by applying the pseudophase approximation with quadratic and cubic terms in the excess free energy of mixing (GE) both at the surface and in the micelles. The same parameters that describe the binary interactions give a quantitative fit to the adsorbed fractions in the ternary mixture over a wide range of composition. A similar analysis is effective for the mixture containing sodium dodecyl sulfate instead of SLES. Of the set of six GE required to fit the ternary data, one is ideal (SLES-LAS) and three, LAS-C12E8 (micelle) and C12E8-SLES (micelle and surface), have minima occurring at a composition (mole fraction) of the anionic species of 1/3.

Understanding the electrostatics of lipid membranes at a molecular level has the potential to offer great insight into the mechanism of voltage-gated processes but has been challenging to study experimentally. In this study, we characterize the equilibrium electric field fluctuations at the interfacial region of lipid bilayers by using a combination of ultrafast time-resolved infrared spectroscopy, molecular dynamics (MD) simulations, and spectral modeling. By monitoring the dynamics of the ester carbonyl stretching vibration in hydrated phosphocholine lipid bilayers, we are able to measure a correlation function for the femtosecond and picosecond fluctuations in the local electric field of the membrane, estimating the standard deviation in these fluctuations as ∼10MV/cm. The addition of gramicidin D at a 1:20 mol ratio with DMPC results in the formation of protein-lipid hydrogen bonds, which alter the dynamics of the ester groups. Using MD simulations, we conclude that ultrafast local field fluctuations exist whether or not water interacts with the ester groups; however, water does accelerate the time-scale of these fluctuations.

It has previously been shown that irradiation with UV light increases the vitamin D content of certain mushroom species, but the effect on other nutrients is unknown, and is difficult to assess due to the complexity of the sample matrix. Here, an offline reversed phase × reversed phase two-dimensional liquid chromatography methodology was developed and applied to Agaricus bisporus mushrooms in order to demonstrate the potential of the technique and assess the effect of UV irradiation on the mushroom’s metabolic profile. The method allowed the detection of 158 peaks in a single analytical run. A total of 51 compounds including sugars, amino acids, organic and fatty acids and phenolic compounds were identified using certified reference standards. After irradiation of the mushrooms with UV for 30 s the number of peaks detected decreased from 158 to 150; 47 compounds increased in concentration while 72 substances decreased. This is the first time that two-dimensional liquid chromatography has been carried out for the metabolomic analysis of mushrooms. The data provide an overview of the gain/loss of nutritional value of the mushrooms following UV irradiation and demonstrate that the increased peak capacity and separation space of two-dimensional liquid chromatography has great potential in metabolomics.

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.

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.

Using a column packed with fully porous particles, four methods for controlling the flow rates at which gradient elution runs are conducted in very high pressure liquid chromatography (VHPLC) were tested to determine whether reproducible thermal conditions could be achieved, such that subsequent analyses would proceed at nearly the same initial temperature. In VHPLC high flow rates are achieved, producing fast analyses but requiring high inlet pressures. The combination of high flow rates and high inlet pressures generates local heat, leading to temperature changes in the column. Usually in this case a post-run time is input into the analytical method to allow the return of the column temperature to its initial state. An alternative strategy involves operating the column without a post-run equilibration period and maintaining constant temperature variations for subsequent analysis after conducting one or a few separations to bring the column to a reproducible starting temperature. A liquid chromatography instrument equipped with a pressure controller was used to perform constant pressure and constant flow rate VHPLC separations. Six replicate gradient separations of a nine component mixture consisting of acetophenone, propiophenone, butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone, and acetanilide dissolved in water/acetonitrile (65:35, v/v) were performed under various experimental conditions: constant flow rate, two sets of constant pressure, and constant pressure operation with a programmed flow rate. The relative standard deviations of the response factors for all the analytes are lower than 5% across the methods. Programming the flow rate to maintain a fairly constant pressure instead of using instrument controlled constant pressure improves the reproducibility of the retention times by a factor of 5, when plotting the chromatograms in time.

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.

Five methods were used to implement fast gradient separations: constant flow rate, constant column-wall temperature, constant inlet pressure at moderate and high pressures (controlled by a pressure controller), and programmed flow constant pressure. For programmed flow constant pressure, the flow rates and gradient compositions are controlled using input into the method instead of the pressure controller. Minor fluctuations in the inlet pressure do not affect the mobile phase flow rate in programmed flow. The reproducibilities of the retention times, the response factors, and the eluted band width of six successive separations of the same sample (9 components) were measured with different equilibration times between 0 and 15 min. The influence of the length of the equilibration time on these reproducibilities is discussed. The results show that the average column temperature may increase from one separation to the next and that this contributes to fluctuation of the results.

Two multidimensional HPLC separations of an Australian red wine are presented, >70% of the available separation space was used. A porous graphitic carbon (PGC) stationary phase was used as the first dimension in both separations with both RP core-shell and hydrophilic interaction chromatography fully porous columns used separately in the second dimension. To overcome peak analysis problems caused by signal noise and low detection limits, the data were pre-processed with penalised least-squares smoothing. The PGC × RP combination separated 85 peaks with a spreading angle of 71° and the PGC × hydrophilic interaction chromatography separated 207 peaks with a spreading angle of 80°. Both 2D-HPLC steps were completed in 76 min using a comprehensive stop-and-go approach. A smoothing step was added to peak-picking processes and was able to greatly reduce the number of false peaks present due to noise in the chromatograms. The required thresholds were not able to ignore the noise because of the small magnitude of the peaks; 1874 peaks were located in the non-smoothed PGC × RP separation that reduced to 227 peaks after smoothing was included.

An algorithm was developed to recognize peaks in a multidimensional separation and calculate their cumulative peak area. To find the retention times of peaks in a one dimensional chromatogram, the Savitzky–Golay smoothing filter was used to smooth and find the first through third derivatives of the experimental profiles. Close examination of the shape of these curves informs on the number of peaks that are present and provides starting values for fitting theoretical profiles. Due to the nature of comprehensive multidimensional HPLC, adjacent cut fractions may contain compounds common to more than one cut fraction. The algorithm determines which components were common in adjacent cuts and subsequently calculates the area of a two-dimensional peak profile by interpolating the surface of the 2D peaks between adjacent peaks. This algorithm was tested by calculating the cumulative peak area of a series of 2D-HPLC separations of alkylbenzenes, phenol and caffeine with varied concentrations. A good relationship was found between the concentration and the cumulative peak area.

A series of experiments was conducted to evaluate the degree of band broadening in very high pressure LC due to column connections. Different column manufacturers use slightly different designs for their column fittings. If the same column connections are repeatedly used to attach columns of different origins, different void volumes form between capillary tubes and column inlets. An Agilent Ultra Low Dispersion Kit (tubing id 75 μm) was installed on an Agilent Infinity 1290 ultra HPLC and used to connect successively an Agilent, a Phenomenex, and a Waters column. A series of uracil (unretained) samples were injected and eluted at a wide range of flow rates with a water/acetonitrile mixture as eluent. In order to determine the variance contribution from column connections as accurately as possible a nonretained probe compound was selected because the variance contribution from the column is the smallest for analytes, which have very low k values. Yet, this effect still has an impact on the resolution for moderately retained compounds (k > 2) for narrow-bore columns packed with fine particles, since variance contributions are additive for linear chromatographic systems. Each injection was replicated five times under the same experimental conditions. Then NanoViper column connections (tubing id 75 μm) were used and the same injections were made. This system was designed to minimize connection void volumes for any column. Band variances were calculated as the second central moment of elution peaks and used to assess the degree of band broadening due to the column connections. Band broadening may increase from 3.8 to 53.9% when conventional metal ferrules were used to join columns to connection sites. The results show that the variance contribution from improper connections can generate as much as 60.5% of the total variance observed. This demonstrates that column connections can play a larger role than the column packing with respect to band dispersion.