Light-emitting diodes and photodiodes in the deep ultra-violet range for absorption photometry in liquid chromatography, capillary electrophoresis and gas sensing

Absorbance measurement in the deep ultra-violet range (below 300 nm) has been one of the most widely used detection methods for analytical techniques as a large number of organic compounds have strong absorption bands in the deep UV region. The use of incandescent or discharge lamps coupled to a monochromator for the wavelength selection in a conventional UV detector makes it complex and costly. Light-emitting diodes (LEDs) for the deep UV range commercially available in recent years have become potential alternatives to thermal light sources. LEDs with their relatively narrow emission bandwidths (typically 20 nm) are well suited for absorption photometry in which a monochromator is not required. This dissertation, therefore, concerns the utilization LEDs and photodiodes (PDs) in the deep UV range as radiation sources and light detectors, respectively for absorption photometry in high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and gas sensing. LEDs were known to perform as light detectors. In measuring systems based on LEDs as light sources, PDs have been normally employed for detection devices. The practical reasons for the use of LEDs as alternatives to PDs, however, have not been demonstrated. Only an advantage of cost-saving was pointed out. In the first project, the performance of LEDs in the light intensity measurement was investigated and compared to that of standard silicon PDs in three different measuring configurations: current follower mode to measure to photocurrents, photovoltaic mode to determine the voltage developed across the diode on irradiation without load and discharge time mode to measure the rate to discharge the junction capacitance of diodes. LEDs as detectors were generally found to be adequate for the analytical work but PDs offered higher sensitivity and linearity as well as provided stable readings with faster settling times. Absorbance detectors for narrow-column HPLC (250 μm inner diameter) and CE (50 μm inner diameter) based on deep UV-LEDs and PDs selective for emission wavelengths were developed and evaluated in the quantification of model compounds at 255 and 280 nm. Absorbance measurements were directly obtained by the use of a beam splitter and PDs for reference signals and a logarithmic ratio amplifier-based circuitry to emulate the Lambert-Beer’s law. Narrow-column HPLC is useful for the applications in which the reduction in eluent consumption is desired or only limited amount of samples is available when utmost sensitivity is not required. In CE, the use of a capillary as the separation channel to minimize the peak broadening downscales the detection window to micrometer range which is even much narrower than that of a narrow-bore HPLC. This makes the design and construction of these LED-based detectors for narrow detection channels more challenging than for a standard HPLC as the higher efficiency for light coupling and stray light avoidance is essentially required. Additionally, high mechanical stability is needed to minimize the noise resulted from mechanical fluctuations. The performance of these optical devices at two measured wavelengths was excellent in terms of the baseline noise (low μAU range), linearity between absorbance values and concentrations (correlation coefficients > 0.999) and reproducibility of peak areas (about 1%). Not only was the potential of a deep UV-LED as a radiation source for absorption spectroscopy investigated for separation techniques but also for the detection of benzene, toluene, ethylbenzene and the xylenes compounds in the gas phase at 260 nm. In the first part of this work, its performance in the acoustic waves excitation was preliminarily investigated with some different measuring systems for the detection of the toluene vapor. It was found that the intensity of a deep UV-LED was insufficient to produce detectable acoustic signals. This was followed by the construction of an absorbance detector for the determination of these target compounds based on the combination of a deep UV-LED and PDs. This optical device was designed to use optical fibers for the light coupling from the LED to a measuring cell and a reference PD, that allows removing a beam splitter previously required for detectors of a narrow column HPLC and CE. Its performance with regard to linearity and reproducibility was satisfactory. Detection limits of about 1 ppm were determined. It could be concluded that viable absorbance detectors for narrow-column HPLC, CE and gas sensing based on deep UV-LEDs and PDs as light sources and light detectors, respectively can be constructed. The performance of these inexpensive LED-based optical devices with regard to linearity, reproducibility and baseline noise was satisfactory and found to be comparable to that of more complex and expensive commercial detectors. These detectors with features of low power consumption and small size are useful for portable battery-powered devices

ADVANCING THE SEPARATION SCIENCES THROUGH THE DELIVERY OF NEW MATERIALS, TECHNOLOGY AND METHODOLOGY.

A thesis and collection of works submitted to Plymouth University in partial fulfilment for the degree of DOCTOR OF SCIENC

Fluorescence determination of cochineal in strawberry jam in the presence of carmoisine as a quencher by means of four-way PARAFAC decomposition

The determination of cochineal (E-120) in strawberry jam was carried out in the presence of carmoisine (E-122) using the four-way PARAFAC decomposition and excitation-emission fluorescence matrices. In the measured conditions, there was no fluorescence signal for carmoisine due to a strong quenching effect and this colorant also led to a decrease of the fluorescence signal of cochineal. The European Union has fixed a maximum residue level, MRL, for cochineal in jam (100 mg kg−1). Therefore, the addition of other food colorant (carmoisine) in the jam could lead to false compliant decisions. The four-way PARAFAC decomposition avoided false compliant decisions caused by the quenching effect. Cochineal was unequivocally identified. Detection capability (CCβ) was 0.72 mg L−1 for probabilities of false positive and false negative fixed at 0.05. Cochineal was detected in the jam (104.63 mg kg−1) above the MRL. This amount was compared with the one obtained using a HPLC/DAD method.Spanish MINECO (AEI/FEDER, UE) through project CTQ2017‐88894-R and by Junta de Castilla y León through project BU012P17 (all co‐financed with European FEDER funds)

Determination of cochineal and erythrosine in cherries in syrup in the presence of quenching effect by means of excitation-emission fluorescence data and three-way PARAFAC decomposition

The simultaneous determination of two food colorants (cochineal (E-120) and erythrosine (E-127)) was achieved by means of excitation-emission fluorescence matrices and three-way PARAFAC decomposition together with the use of a calibration set that contained binary mixtures of both analytes. In the measured conditions, the amount of cochineal present in the sample affected the fluorescence signal of erythrosine since cochineal caused a quenching effect in the fluorescence of the other food additive. However, the signal of cochineal was not affected by the presence of erythrosine. A calibration line for erythrosine was built for each different concentration level of cochineal. The slopes of these regressions were different depending on the amount of quencher, whereas the intercepts were statistically equal to 0 at a 95% confidence level. The quantification of erythrosine was possible using the regression “amount of cochineal” versus “the slope of the calibration line for erythrosine”. Using this procedure, the mean of the absolute values of the relative errors in prediction for mixtures of both colorants were 5.86% (n = 10) for cochineal and 4.17% (n = 10) for erythrosine. Both analytes were unequivocally identified by the correlation between the pure spectra and the PARAFAC excitation and emission spectral loadings. Pitted cherries in syrup were analyzed. Cochineal and erythrosine were detected in those cherries at a concentration of 185.05 mg kg−1 and 10.76 mg kg−1, respectively. These concentration values were statistically equal to the ones obtained with a HPLC/DAD method.Spanish MINECO (AEI/FEDER, UE) through projects CTQ2014–53157-R and CTQ2017‐88894‐R and by Junta de Castilla y León through project BU012P1

Synthesis of Polyproline Spacers between NIR Dye Pairs for FRET to Enhance Photoacoustic Imaging

With the goal of enhancing the emission of ultra-sound (US) waves in photoacoustic imaging (PAI) for the detection of cancer we discovered an effective system for Förster Resonance Energy Transfer (FRET) composed of a near infrared fluorescent (NIRF) dye and a NIR quencher (NIRQ) dye separated by a polyproline peptide spacer. In PAI, a newly developed imaging technique, the signal is generated by excitation of cells using pulsed infrared lasers and measurement of resulting US waves using detectors. NIRF dyes can increase the sensitivity of PAI signal, particularly when used in targeted molecular imaging agents (TMIAs). However, most of the energy of absorption is likely lost during fluorescence. Our hypothesis was that if the fluorescence could be quenched by FRET, all of the energy from the laser could be converted to a substantially stronger US signal from the NIR dyes. In FRET, an optimized distance between the NIRF and NIRQ dyes is essential, but has not yet been determined for this dual dye system. To determine the appropriate distance, we designed and synthesized a polyproline spacer as a rigid “molecular ruler”. Multiple polyproline spacers (di, tetra, octa) were synthesized and coupled first to two NIRF dyes, Cy5.5 and IR770TI, using a modular synthetic approach developed by our group. When the emission resulting from FRET between NIRF dye pairs was evaluated, a strong FRET effect was observed from the octaproline dye pair, with twice the signal as the tetraproline. Furthermore, it was found that the FRET effect could be influenced by the solvents due to the dye aggregation. The polyproline approach was then applied to the synthesis and evaluation of NIRF-NIRQ dye pairs resulting in the discovery that tetra and octaproline NIRF-NIRQ systems yield nearly complete FRET quenching. The new FRET system is a valuable tool for future research in PAI to provide a higher, more sensitive signal. Samples have been submitted to collaborators for evaluation using PAI instrumentation at nearby cancer research centers

Multivariate analysis of organic acids in fermented food from reversed-phase high-performance liquid chromatography data

Multivariate calibration coupled to RP-HPLC with diode array detection (HPLC-DAD) was applied to the identification and the quantitative evaluation of the short chain organic acids (malic, oxalic, formic, lactic, acetic, citric, pyruvic, succinic, tartaric, propionic and α-cetoglutaric) in fermented food. The goal of the present study was to get the successful resolution of a system in the combined occurrence of strongly coeluting peaks, of distortions in the time sensors among chromatograms, and of the presence of unexpected compounds not included in the calibration step. Second-order HPLC-DAD data matrices were obtained in a short time (10 min) on a C18 column with a chromatographic system operating in isocratic mode (mobile phase was 20 mmol L−1 phosphate buffer at pH 2.20) and a flow-rate of 1.0 mL min−1 at room temperature. Parallel factor analysis (PARAFAC) and unfolded partial least-squares combined with residual bilinearization (U-PLS/RBL) were the second-order calibration algorithms select for data processing. The performance of the analytical parameters was good with an outstanding limit of detection (LODs) for acids ranging from 0.15 to 10.0 mmol L−1 in the validation samples. The improved method was applied to the analysis of many dairy products (yoghurt, cultured milk and cheese) and wine. The method was shown as an effective means for determining and following acid contents in fermented food and was characterized by reducibility with simple, high resolution and rapid procedure without derivatization of analytes.Fil: Mortera, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Zuljan, Federico Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bortolato, Santiago Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; ArgentinaFil: Alarcon, Sergio Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentin

Analytical Strategies for Fingerprinting of Antioxidants, Nutritional Substances, and Bioactive Compounds in Foodstuffs Based on High Performance Liquid Chromatography-Mass Spectrometry: An Overview

New technology development and globalisation have led to extreme changes in the agri-food sector in recent years that need an important food supply chain characterisation from plant materials to commercial productions. Many analytical strategies are commonly utilised in the agri-food industry, often using complementary technologies with different purposes. Chromatography on-line coupled to mass spectrometry (MS) is one of the most selective and sensitive analytical methodologies. The purpose of this overview is to present the most recent MS-based techniques applied to food analysis. An entire section is dedicated to the recent applications of high-resolution MS. Covered topics include liquid (LC)– and gas chromatography (GC)–MS analysis of natural bioactive substances, including carbohydrates, flavonoids and related compounds, lipids, phenolic compounds, vitamins, and other different molecules in foodstuffs from the perspectives of food composition, food authenticity and food adulteration. The results represent an important contribution to the utilisation of GC–MS and LC–MS in the field of natural bioactive compound identification and quantification

Selectivity enhancement in capillary electrophoresis – development of a two-dimensional separation and a dual detection system

For the analysis of complex samples such as biological or environmental ones, highly selective analytical strategies are required. The selectivity can be enhanced by means of hyphenation of orthogonal separation techniques or combination of complementary detectors as an alternative concept. In this work, an example for both concepts is presented, respectively. First, methodical and instrumental studies concerning the comprehensive hyphenation of the two most important instrumental techniques in ion analysis namely ion chromatography (IC) and capillary electrophoresis (CE) are described enabling the simultaneous determination of anions and cations. Further, the combination of capacitively coupled conductless conductivity detection (C4D) and mass spectrometry (MS) as dual detection concept for CE is introduced and applied using aqueous and non-aqueous background electrolyte systems. Another concept to enhance the selectivity is rendering the separation technique compatible to a highly selective detector such as MS. Thus, in a third part, an approach is presented enabling the coupling of surfactant-free microemulsion electrokinetic chromatography (SF-MEEKC) to MS. For the comprehensive hyphenation of capillary IC and CE, a modulator was developed ensuring well-controlled injections from the first to the second dimension. This was achieved by periodical injection of the IC effluent from a transfer capillary to the CE separation capillary by capillary batch injection. Important for the coupling was the compatibility of the two systems. Thus, the characteristics of the advanced capillary high performance IC with flow rates in the lower µL/min range facilitated the hyphenation being closer to CE conditions compared to IC with conventional columns. Due to the implemented capillary scale suppressor technology, the IC effluent consisted of analyte and pure water avoiding matrix concerned interferences and enabling exploitation of analyte stacking in CE. Further, fast CE measurements in the time range of seconds enabled the comprehensive coupling. Proof-of-concept measurements were performed using a model system containing nucleotides and their cyclic derivates. It was shown that the separation performance could be enhanced in the two-dimensional ICxCE-MS system compared to the single techniques. The work was further expanded to a methodical study concerning the simultaneous determination of positively and negatively charged analytes. A bypass system for IC was developed using a switching valve. This configuration enabled the cations bypassing the suppressor and thus they were not filtered out by the cation exchange membrane of the suppressor being not selective to eluent cations. Feasibility of the setup was demonstrated separating a model mixture of different arsenic species. The cations were eluted with water from the anion exchange column before driving a KOH gradient elution for the separation of anions. The injection parameters of the modulator introducing the IC effluent into the CE-MS system were studied taking into account the complex transport situation. It was found that the migration times were stable even for highly frequent injections with waiting times between the injections that were a factor four shorter than the migration time itself. Signal intensity of the MS was highest for positively charged species caused by the discriminating effect of the electrokinetic injection and the better ionization of the organic arsenic species by the electrospray ionization (ESI) source. Further, the coupling of two important detectors for CE is presented, namely capacitively coupled contactless conductivity detection (C4D) and electrospray ionization time-of-flight MS (ESI-TOF-MS). An experimental protocol was developed taking into account the requirements of the separation aspects and the compatibility with both detectors. ESI-TOF-MS requires background electrolytes consisting of volatile components such as ammonium acetate or formate. These, however, exhibit a rather high conductivity, which is disadvantageous for C4D. A 10 mM ammonium acetate/ammonia buffer was taken as compromise concerning the detection performance of both detectors. A sample containing various phenolic compounds serving as a model system was determined. The analytical characteristics showed the complementarity and suitability of this dual detection approach as C4D showed better response behavior towards m-cresol (limit of detection (LOD)=3.1 µM), while MS was more sensitive for determinations of m- and p-nitrophenol (LODs=0.8 µM) and 2,4-dinitrophenol (LOD=1.5 µM). The overall separation efficiency was excellent realizing the separation of counter-electroosmotic species with migration times of less than 60 s and illustrating that detector-induced band broadening could be neglected in the CE-C4D/MS system. In a second study, non-aqueous electrolytes were used for CE separations as they are highly compatible with both detectors due to their volatility and low background conductivity. A non-aqueous capillary electrophoresis (NACE)-C4D-MS method was developed using an acetonitrile based background electrolyte containing 2 M HAc and 4 mM NH4Ac enabling fast electrophoretic separations. Concentration changes of the background electrolyte and the choice of the inner diameter (ID) of the separation capillary were more critical for C4D than for MS. To choose a separation capillary with appropriate ID, the dependency of the sensitivity of the C4D on the ID was studied resulting in the use of a capillary with ID of 50 µm. The complementarity of the two detectors was demonstrated determining inorganic anions such as chloride, bromide, and nitrate (C4D) as well as organic biomolecules such as choline, thiamine, and acetylcholine (MS) simultaneously. A calibration was performed and the method applied on an extract of a food supplement quantifying the model analytes using the respective detector. MEEKC is a powerful tool for the separation of neutral species based on differences in their hydrophobic and hydrophilic properties. However, conventionally used SDS-based microemulsions are not compatible with ESI-MS. Enhancing the selectivity of the overall method, a surfactant-free microemulsion (SFME) based on water, ethanol, and 1-octanol was used as background electrolyte ensuring compatibility with ESI-TOF-MS. The small-angle X-ray scattering and dynamic light scattering measurements proved that the addition of ammonium acetate, which was necessary for the electrophoretic separation, was not altering aggregate formation in the SFME in the chosen concentration range. The separation performance of SF-MEEKC was demonstrated separating a model system consisting of hydrophobic and hydrophilic neutral vitamins, namely the vitamins B2 and D3, and the cationic vitamin B1 using UV/VIS detection. The influence of the ammonium acetate concentration on the separation performance was studied in detail. Characterization of the developed method was performed concerning reproducibility of migration times and peak areas and concerning the linearity of the calibration data. Further, the compatibility of SF-MEEKC with ESI-MS was shown determining the content of vitamin D3 in a commercial drug. Comparable sensitivity to aqueous CE-ESI-MS was achieved

Determination of Soybean Oil, Protein and Amino Acid Residues in Soybean Seeds by High Resolution Nuclear Magnetic Resonance (NMRS) and Near Infrared (NIRS)

A detailed account is presented of our high resolution nuclear magnetic resonance (HR-NMR) and near infrared (NIR) calibration models, methodologies and validation procedures, together with a large number of composition analyses for soybean seeds. NIR calibrations were developed based on both HR-NMR and analytical chemistry reference data for oil and twelve amino acid residues in mature soybeans and soybean embryos. This is our first report of HR-NMR determinations of amino acid profiles of proteins from whole soybean seeds, without protein extraction from the seed. It was found that the best results for both oil and protein calibrations were obtained with a Partial Least Squares Regression (PLS-1) analysis of our extensive NIR spectral data, acquired with either a DA7000 Dual Diode Array (Si and InGaAs detectors) instrument or with several Fourier Transform NIR (FT-NIR) spectrometers equipped with an integrating sphere/InGaAs detector accessory. In order to extend the bulk soybean samples calibration models to the analysis of single soybean seeds, we have analized in detail the component NIR spectra of all major soybean constituents through spectral deconvolutions for bulk, single and powdered soybean seeds. Baseline variations and light scattering effects in the NIR spectra were corrected, respectively, by calculating the first-order derivatives of the spectra and the Multiplicative Scattering Correction (MSC). The single soybean seed NIR spectra are broadly similar to those of bulk whole soybeans, with the exception of minor peaks in single soybean NIR spectra in the region from 950 to 1,000 nm. Based on previous experience with bulk soybean NIR calibrations, the PLS-1 calibration model was selected for protein, oil and moisture calibrations that we developed for single soybean seed analysis. In order to improve the reliability and robustness of our calibrations with the PLS-1 model we employed standard samples with a wide range of soybean constituent compositions: from 34% to 55% for protein, from 11% to 22% for oil and from 2% to 16% for moisture. Such calibrations are characterized by low standard errors and high degrees of correlation for all major soybean constituents. Morever, we obtained highly resolved NIR chemical images for selected regions of mature soybean embryos that allow for the quantitation of oil and protein components. Recent developments in high-resolution FT-NIR microspectroscopy extend the NIR sensitivity range to the picogram level, with submicron spatial resolution in the component distribution throughout intact soybean seeds and embryos. Such developments are potentially important for biotechnology applications that require rapid and ultra- sensitive analyses, such as those concerned with high-content microarrays in Genomics and Proteomics research. Other important applications of FT-NIR microspectroscopy are envisaged in biomedical research aimed at cancer prevention, the early detection of tumors by NIR-fluorescence, and identification of single cancer cells, or single virus particles in vivo by super-resolution microscopy/ microspectroscopy