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.

Graphene nanodots-encaged porous gold electrode fabricated via ion beam sputtering deposition for electrochemical analysis of heavy metal ions

All rights reserved. A graphene nanodots-encaged porous gold electrode via ion beam sputtering deposition (IBSD) for electrochemical sensing is presented. The electrodes were fabricated using Au target, and a composite target of Al and graphene, which were simultaneously sputtered onto glass substrates by Ar ion beam, followed with hydrochloric acid corrosion. The as-prepared graphene nanodots-encaged porous gold electrodes were then used for the analysis of heavy metal ions, e.g. Cu2+ and Pb2+ by Osteryoung square wave voltammetry (OSWV). These porous electrodes exhibited enhanced detection range for the heavy metal ions due to the entrapped graphene nanodots in 3-D porous structure. In addition, it was also found that when the thickness of porous electrode reached 40 nm the detection sensitivity came into saturation. The linear detection range is 0.009-4 μM for Cu2+ and 0.006-2.5 μM for Pb2+. Good reusability and repeatability were also observed. The formation mechanism and 3-D structure of the porous electrode were also investigated using scanning electron microscope (SEM), transmission electron microscope (TEM) and X-ray photoelectron spectra (XPS). This graphene entrapped 3-D porous structure may envision promising applications in sensing devices.

Development of a resin based silica monolithic column encapsulation

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.

Chemiluminescence detection of MDMA in street drug samples using tris (2,2'-bipyridine) ruthenium (III)

Tris(2,2'-bipyridine)ruthenium(II) chemiluminescence was investigated for the detection of 3,4-methylenedioxymethamphetamine (MDMA) and several related compounds in street drug samples. Optimization using flow injection analysis showed that the selectivity of the reagent can be targeted towards the detection of secondary amines by altering the pH of the reaction environment. The greater selectivity of this mode of detection, compared to UV-absorbance, reduces the probability of false positive results from interfering compounds. The detection limit for MDMA under these conditions was 0.48 μM. A HPLC method incorporating post-column tris(2,2'-bipyridine)ruthenium(II) chemiluminescence detection was applied to the determination of MDMA in five street drug samples. The results obtained were in good agreement with quantification performed using traditional UV-absorbance detection, which demonstrates the viability of this method for confirmatory analysis of drug samples. This is the first report of tris(2,2'-bipyridine)ruthenium(II) chemiluminescence for the detection of MDMA and related amphetamine derivatives.

Electrogenerated chemiluminescence of tris(2,2' bipyridine)ruthenium(ii) using common biological buffers as co-reactant, pH buffer and supporting electrolyte

A series of aliphatic tertiary amines (HEPES, POPSO, EPPS and BIS-TRIS) commonly used to buffer the pH in biological experiments, were examined as alternative, non-toxic co-reactants for the electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(ii) ([Ru(bpy)3](2+)). These were found to be very attractive as "multi-tasking" reagents, serving not only as co-reactants, but also fulfiling the roles of pH buffer and supporting electrolyte within an aqueous environment; thus significantly simplifying the overall ECL analysis. Sub-nanomolar detection limits were obtained for [Ru(bpy)3](2+) in the presence of BIS-TRIS, making this species an valuable option for co-reactant ECL-based bioanalytical applications.

Mobile phone-based electrochemiluminescence sensing exploiting the 'USB On-The-Go' protocol

A low-cost system to generate, control and detect electrochemiluminescence using a mobile smartphone is described. A simple tone-detection integrated circuit is used to switch power sourced from the phone's Universal Serial Bus (USB) 'On-The-Go' (OTG) port, using audible tone pulses played over the device's audio jack. We have successfully applied this approach to smartphones from different manufacturers and with different operating system versions. ECL calibrations of a common luminophore, tris(2,2′-bipyridine)ruthenium(II) ([Ru(bpy)3]²⁺), with 2-(dibutylamino)ethanol (DBAE) as a co-reactant, showed no significant difference in light intensities when an electrochemical cell was controlled by a mobile phone in this manner, compared to the same calibration generated using a conventional potentiostat. Combining this novel approach to control the applied potential with the measurement of the emitted light through the smart phone camera (using an in-house built Android app), we explored the ECL properties of a water-soluble iridium(III) complex that emits in the blue region of the spectrum. The iridium(III) complex exhibited superior co-reactant ECL intensities and limits of detection to that of the conventional [Ru(bpy)3]²⁺ luminophore.

Simultaneous crystallization and decomposition of PVA/MMT composites during non-isothermal process

Decomposition of poly(vinyl alcohol)/montmorillonite clay (PVA/MMT) composites during melting-crystallization was experimentally confirmed by morphology and molecular structure changes. In particular, FTIR spectra show the shift of O-H stretching band as well as enhanced intensities of C-O stretching and CH2 rocking vibrational modes. Furthermore, Raman deconvolution indicates that C-H wagging, CH2-CH wagging, CH-CO bending and CH2 wagging modes in amorphous domains were all decreased greatly. Moreover, this decomposition leads to decreased melting enthalpy, melting point, crystallization enthalpy and crystallization temperature. Crystallization analysis shows that the MMT incorporated slows down the crystallization process in the PVA matrix regardless of the nucleation capability of MMT. Despite the severe decomposition, the crystallization kinetics still corroborated well with common classical models. As a result, molecular structure changes and crystallization retardation observed in this study clearly indicate the strong effects of the thermal degradation on the non-isothermal crystallization of PVA/MMT composites.

Corrosion protection afforded by praseodymium conversion film on Mg alloy AZNd in simulated biological fluid studied by scanning electrochemical microscopy

Surface passivation of AZNd Mg alloy with Pr(NO3)3 is studied using scanning electrochemical microscopy (SECM) in surface generation/tip collection (SG/TC) and AC modes. Corrosion protection afforded by the Pr treatment and the degradation mechanism in a simulated biological environment was examined on a local scale and compared with non-treated AZNd. SG/TC mode results revealed a drastic decrease in H2 evolution due to the Pr treatment. Mapping the local insulating characteristics using AC-SECM showed higher conductivity of the surface where H2 evolution was most favorable.

Analytically useful blue chemiluminescence from a water-soluble iridium(iii) complex containing a tetraethylene glycol functionalised triazolylpyridine ligand

We examine [Ir(df-ppy)2(pt-TEG)](+) as the first highly water soluble, blue-luminescent iridium(iii) complex for chemiluminescence detection. Marked differences in selectivity were observed between the new complex and the conventional [Ru(bpy)3](2+) reagent, which will enable this mode of detection to be extended to new areas of application.

A novel graphene nanodots inlaid porous gold electrode for electrochemically controlled drug release

A uniform graphene nanodots inlaid porous gold electrode was prepared via ion beam sputtering deposition (IBSD) and mild corrosion chemistry. HRTEM, SEM, AFM and XPS analyses revealed the successful fabrication of graphene nanodots inlaid porous gold electrode. The as-prepared porous electrode was used as π-orbital-rich drug loading platform to fabricate an electrochemically controlled drug release system with high performance. π-orbital-rich drugs with amino mioety, like doxorubicin (DOX) and tetracycline (TC), were loaded into the graphene nanodots inlaid porous gold electrode via non-covalent π-π stacking interaction. The amino groups in DOX and TC can be easily protonated at acidic medium to become positively-charged NH3(+), which allow these drug molecules to be desorbed from the porous electrode surface via electrostatic repulsion when positive potential is applied at the electrode. The drug loading and release experiment indicated that this graphene nanodots inlaid porous gold electrode can be used to conveniently and efficiently control the drug release electrochemically. Not only did our work provide a benign method to electrochemically controlled drug release via electrostatic repulsion process, it also enlighten the promising practical applications of micro electrode as a drug carrier for precisely and efficiently controlled drug release via embedding in the body.

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.

Overcoming solvent mismatch limitations in 2D-HPLC with temperature programming of isocratic mobile phases

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.

Investigations into the analytical applications and fundamental chemistry of the chemiluminescent reactions of Tris(2,2'-bipyridyl)ruthenium(III) with certain Papaver Somniferum alkaloids and other related compounds

The reaction of tris(2,2’-bipyridyl)ruthenium(III) (Ru(bipy) ₃³⁺) with various analytes to generate chemiluminescence has been well documented. This investigation sought to undertake a chemiluminometic study of the reactions of Ru(bipy) ₃³⁺ with selected Papaver Somniferum alkaloids and specifically synthesised phenethylamines. The investigation, based on a kinetic study, primarily addressed the effect of varying reaction conditions (pH) on Ru(bipy) ₃³⁺ chemiluminescence production. To monitor these reactions, a batch chemiluminometer was specifically designed, fabricated and automated to conduct an extensive study on the selected compounds of interest. The instrumentation incorporated a custom built reaction cell and comprised an ‘on-line’ sample preparation system with which calibration standards could be automatically prepared. The instrumentation provided both time-independent (peak area) and time-dependent (kinetic profile) information. A novel approach to the stabilisation of Ru(bipy) ₃³⁺ as a chemiluminescencent reagent was also investigated and a recirculating system was employed with the batch chemiluminometer to provide a stable supply of Ru(bipy) ₃³⁺. Codeine, thebaine and 6-methoxy-codeine were the Papaver Somniferum alkaloids selected for this study and several N-methylated and N,N-dimethylated phenethylamines and methoxy-substituted phenetheylamines were also synthesised to investigate the affect of pH on the chemiluminescence emission efficiency. The versatility of the batch chemiluminometer facilitated the kinetic study of numerous analytes over a broad pH range. The exemplary performance of the chemiluminometer as an analytical instrument, was demonstrated by the calibration functions, based on peak area data, which exhibited excellent linearity and sensitivity. The estimated detection limits (3s) for the selected alkaloids were in the range 2 x 10^-9 M to 7 x 10^-9 at pH 5.0 and above, which compared favourably to detection limits for the same compounds determined using FIA. Relative standard deviations (n=5) for peak areas ranged between 1% to 5% with a mean of 3.1% for all calibration standards above 2.5 x 10^-8 M. Correlation between concentration and peak area, irrespective of pH and analyte was excellent, with all but two calibration functions having r-squared values greater than 0.990. The analytical figures of merit exemplified the precision and robustness of the reagent delivery and ‘on-line’ sample preparation, as well as the sensitivity of the system. The employment of the chemiluminometer for the measurement of total chemiluminescence emission (peak area) was in itself a feasible analytical technique, which generated highly reproducible and consistent data. Excellent analytical figures of merit, based on peak area, were similarly achieved for the phenethylamines. The effects of analyte structure on chemiluminescence activity was also investigated for the alkaloids and the phenethylamines. Subtle structural variations between the three alkaloids resulted in either a moderately reduced or enhanced total emission that was two or three fold difference only. A significant difference in reaction kinetics was observed between thebaine and codeine/6-methoxy-codeine, which was dependent upon pH. The time-dependent data, namely the observed rate constants for the initial rise in intensity and for the subsequent decay rate, were obtained by fitting a mathematical function (based on the postulated reaction mechanism) to the raw data. The determination of these rate constants for chemiluminescence reactions highlighted the feasibility for utilising such measurements for quantitative analytical applications. The kinetic data were used to discriminate between analyte responses in order to determine the concentrations of individual analytes in a binary mixture. A preliminary, multi-component investigation performed on a binary mixture of codeine and 6-methoxy-codeine (1:1) successfully determined the concentrations of these individual components using such rate constant measurements. Consequently, variations in kinetics resulted in a significant difference between the relative chemiluminescence response based on peak area measurements and the relative response base on peak height measurements obtained using FIA. With regards to the observed reactivity of secondary amines and tertiary amines, chemiluminescence peak area determinations confirmed the vital role of pH on reaction efficiency, which was governed by structural features and kinetics. The tertiary amines investigated generally produced a greater emission under acidic conditions than the corresponding secondary amines. However, the measured chemiluminescence responses were highly dependent upon pH, with similar peak areas obtained for both amine groups under slightly alkaline conditions.

Chemistry, spectroscopy and analytical applications of certain chemiluminescent reactions

Chemiluminescence, the production of light from a chemical reaction, has found widespread use in analytical chemistry. Both tris (2, 2’-bipyridyl) ruthenium (II) and acidic potassium permanganate are chemiluminescence reagents that have been employed for the determination of a diverse range of analytes. This thesis encompasses some fundamental investigations into the chemistry and spectroscopy of these chemiluminescence reactions as well as extending the scope of their analytical applications. Specifically, a simple and robust capillary electrophoresis chemiluminescence detection system for the determination of codeine, O6-methylcodeine and thebaine is described, based upon the reaction of these analytes with chemically generated tris(2,2'-bipyridyl)ruthenium(III) prepared in sulfuric acid (0.05 M). The reagent solution was contained in a glass detection cell, which also held both the capillary and the cathode. The resultant chemiluminescence was monitored directly using a photomultiplier tube mounted flush against the base of the detection cell. The methodology, which incorporated a field amplification sample introduction procedure, realised detection limits (3a baseline noise) of 5 x 10~8 M for both codeine and O6-methylcodeine and 1 x 10~7 M for thebaine. The relative standard deviations of the migration times and the peak areas for the three analytes ranged from 2.2 % up to 2.5 % and 1.9 % up to 4.6 % respectively. Following minor instrumental modifications, morphine, oripavine and pseudomorphine were determined based upon their reaction with acidic potassium permanganate in the presence of sodium polyphosphate. To ensure no migration of the permanganate anion occurred, the anode was placed at the detector end whilst the electroosmotic flow was reversed by the addition of hexadimethrine bromide (0.001% m/v) to the electrolyte. The three analytes were separated counter to the electroosmotic flow via their interaction with a-cyclodextrin. The methodology realised detection limits (3 x S/N) of 2.5 x 10~7 M for both morphine and oripavine and 5 x 10~7 M for pseudomorphine. The relative standard deviations of the migration times and the peak heights for the three analytes ranged from 0.6 % up to 0.8 % and 1.5% up to 2.1 % respectively. Further improvements were made by incorporating a co-axial sheath flow detection cell. The methodology was validated by comparing the results realised using this technique with those obtained by high performance liquid chromatography (HPLC), for the determination of both morphine and oripavine in seven industrial process liquors. A complimentary capillary electrophoresis procedure with UV-absorption detection was also developed and applied to the determination of morphine, codeine, oripavine and thebaine in nine process liquors. The results were compared with those achieved using a standard HPLC method. Although over eighty papers have appeared in the literature on the analytical applications of acidic potassium permanganate chemiluminescence, little effort has been directed towards identifying the origin of the luminescence. It was found that chemiluminescence was generated during the manganese(III), manganese(IV) and manganese(VII) oxidations of sodium borohydride, sodium dithionite, sodium sulfite and hydrazine sulfate in acidic aqueous solution. From the corrected chemiluminescence spectra, the wavelengths of maximum emission were 689 ± 5 nm and 734 ± 5 nm when the reactions were performed in sodium hexametaphosphate and sodium dihydrogenorthophosphate or orthophosphoric acid environments respectively. The corrected phosphorescence spectrum of manganese(II) sulfate in a solution of sodium hexametaphosphate at 77 K, exhibited two peaks with maxima at 688 nm and 730 nm. The chemical and spectroscopic evidence presented strongly supported the postulation that the emission was an example of solution phase chemically induced phosphorescence of manganese(II). Thereby confirming earlier predictions that the chemiluminescence from acidic potassium permanganate reactions originated from an excited manganese(II) species. Additionally, these findings have had direct analytical application in that manganese(IV) was evaluated as a new reagent for chemiluminescence detection. The oxidations of twenty five organic and inorganic species, with solublised manganese(IV), were found to elicit analytically useful chemiluminescence with detection limits (3 x S/N) for Mn(II), Fe(II), morphine and codeine of 5 x 10-8 M, 2.5 x 10-7 M, 7.5 x 10-8 M and 5 x 10-8M, respectively. The corrected emission spectra from four different analytes gave wavelengths of maximum emission in the range from 733 nm up to 740 nm indicating that these chemiluminescence reactions also shared a common emitting species, excited manganese(II). Whilst several analytical problems were addressed in this thesis and answers to certain questions regarding the fundamentals of acidic potassium permanganate chemiluminescence were proposed, there are several areas that would benefit from further research. These are outlined in the final chapter of this thesis.

Contributions to analytical chemistry

This collection of seventy-five publications represents the authors contribution to spectroscopy, separation science and flow analysis. Of particular note are the fundamental investigations into chemiluminescence and the innovative strategies for its utilisation as a sensitive and selective means of detection for several important and challenging problems in analytical chemistry.