A Mechanistic Investigation of the N-Hydroxyphthalimide Catalyzed Benzylic Oxidation Mediated by Sodium Chlorite.

A detailed investigation into the mechanistic course of N-hydroxyphthalimide catalyzed oxidation of benzylic centers using sodium chlorite as the stoichiometric oxidant is reported. Through a combination of experimental, spectroscopic, and computational techniques, the transformation is interrogated, providing improved reaction conditions and an enhanced understanding of the mechanism. Performing the transformation in the presence of acetic acid or a pH 4.5 buffer leads to extended reaction times but improves the catalyst lifetime, leading to the complete consumption of the starting material. Chlorine dioxide is identified as the active oxidant that is able to oxidize the N-hydroxyphthalimide anion to the phthalimide-N-oxyl radical, the proposed catalytically active species, which is able to abstract a hydrogen atom from the substrate. A second molecule of chlorine dioxide reacts with the resultant radical and, after loss of hypochlorous acid, leads to the observed product. Through a broad variety of techniques including UV/vis, EPR and Raman spectroscopy, isotopic labeling, and the use of radical traps, evidence for the mechanism is presented that is supported through electronic structural calculations.

Optical Screening and Classification of Drug Binding to Proteins in Human Blood Serum.

Protein-drug interactions in the human bloodstream are important factors in applications ranging from drug design, where protein binding influences efficacy and dose delivery, to biomedical diagnostics, where rapid, quantitative measurements could guide optimized treatment regimes. Current measurement approaches use multistep assays, which probe the protein-bound drug fraction indirectly and do not provide fundamental structural or dynamic information about the in vivo protein-drug interaction. We demonstrate that ultrafast 2D-IR spectroscopy can overcome these issues by providing a direct, label-free optical measurement of protein-drug binding in blood serum samples. Four commonly prescribed drugs, known to bind to human serum albumin (HSA), were added to pooled human serum at physiologically relevant concentrations. In each case, spectral changes to the amide I band of the serum sample were observed, consistent with binding to HSA, but were distinct for each of the four drugs. A machine-learning-based classification of the serum samples achieved a total cross-validation prediction accuracy of 92% when differentiating serum-only samples from those with a drug present. Identification on a per-drug basis achieved correct drug identification in 75% of cases. These unique spectroscopic signatures of the drug-protein interaction thus enable the detection and differentiation of drug containing samples and give structural insight into the binding process as well as quantitative information on protein-drug binding. Using currently available instrumentation, the 2D-IR data acquisition required just 1 min and 10 µL of serum per sample, and so these results pave the way to fast, specific, and quantitative measurements of protein-drug binding in vivo with potentially invaluable applications for the development of novel therapies and personalized medicine.

Non-invasive monitoring of the growth of metal-organic frameworks (MOFs) via Raman spectroscopy.

The applicability of Raman spectroscopy for phase discrimination of metal-organic frameworks (MOFs) has been demonstrated with F4_MIL-140A(Ce) and F4_UiO-66(Ce); analogues prepared from the same metal and ligand sources. Each analogue exhibits unique Raman peaks, with significant differences in the low frequency region, which is more sensitive to structural variations. Non-invasive Raman monitoring of F4_MIL-140A(Ce) synthesis indicated evolution of a unique MOF Raman peak with reaction progress; conversion of this Raman signal to extent of crystallisation was in good agreement with reported reaction kinetics determined via a synchrotron diffraction method. Additionally, Raman spectroscopy indicated initial rapid consumption of the nitric acid modulator present in the reaction coinciding with an expected high probability of nucleation. Raman spectroscopy is a promising technique for rapid screening of MOFs and can be used to study the mechanism of their formation in situ with kinetic insight into both the solution and solid phases of the reaction medium.

Measuring proteins in H2O using 2D-IR spectroscopy: pre-processing steps and applications toward a protein library.

The ability of two-dimensional infrared (2D-IR) spectroscopy to measure the amide I band of proteins in H2O rather than D2O-based solvents by evading the interfering water signals has enabled in vivo studies of proteins under physiological conditions and in biofluids. Future exploitation of 2D-IR in analytical settings, from diagnostics to protein screening, will, however, require comparisons between multiple datasets, necessitating control of data collection protocols to minimize measurement-to-measurement inconsistencies. Inspired by analytical spectroscopy applications in other disciplines, we describe a workflow for pre-processing 2D-IR data that aims to simplify spectral cross-comparisons. Our approach exploits the thermal water signal that is collected simultaneously with, but is temporally separated from the amide I response to guide custom baseline correction and spectral normalization strategies before combining them with Principal Component noise reduction tools. Case studies show that application of elements of the pre-processing workflow to previously published data enables improvements in quantification accuracy and detection limits. We subsequently apply the complete workflow in a new pilot study, testing the ability of a prototype library of 2D-IR spectra to quantify the four major protein constituents of blood serum in a single, label-free measurement. These advances show progress toward the robust data handling strategies that will be necessary for future applications of 2D-IR to pharmaceutical or biomedical problems.

Temperature Correction of Spectra to Improve Solute Concentration Monitoring by In Situ Ultraviolet and Mid-Infrared Spectrometries toward Isothermal Local Model Performance.

Changes in temperature can significantly affect spectroscopic-based methods for in situ monitoring of processes. As varying temperature is inherent to many processes, associated temperature effects on spectra are unavoidable, which can hinder solute concentration determination. Ultraviolet (UV) and mid-infrared (IR) data were acquired for l-ascorbic acid (LAA) in MeCN/H2O (80:20 w/w) at different concentrations and temperatures. For both techniques, global partial least squares (PLS) models for prediction of LAA concentration constructed without preprocessing of the spectra required a high number of latent variables to account for the effects of temperature on the spectra (root mean square error of cross validation (RMSECV) of 0.18 and 0.16 g/100 g solvent, for UV and IR datasets, respectively). The PLS models constructed on the first derivative spectra required fewer latent variables, yielding variable results in accuracy (RMSECV of 0.23 and 0.06 g/100 g solvent, respectively). Corresponding isothermal local models constructed indicated improved model performance that required fewer latent variables in the absence of temperature effects (RMSECV of 0.01 and 0.04 g/100 g solvent, respectively). Temperature correction of the spectral data via loading space standardization (LSS) enabled the construction of global models using the same number of latent variables as the corresponding local model, which exhibited comparable model performance (RMSECV of 0.06 and 0.04 g/100 g solvent, respectively). The additional chemometric effort required for LSS is justified if prediction of solute concentration is required for in situ monitoring and control of cooling crystallization with an accuracy and precision approaching that attainable using an isothermal local model. However, the model performance with minimal preprocessing may be sufficient, for example, in the early phase development of a cooling crystallization process, where high accuracy is not always required. UV and IR spectrometries were used to determine solubility diagrams for LAA in MeCN/H2O (80:20 w/w), which were found to be accurate compared to those obtained using the traditional techniques of transmittance and gravimetric measurement. For both UV and IR spectrometries, solubility values obtained from models with LSS temperature correction were in better agreement with those determined gravimetrically. In this first example of the application of LSS to UV spectra, significant improvement in the predicted solute concentration is achieved with the additional chemometric effort. There is no extra experimental burden associated with the use of LSS if a structured approach is employed to acquire calibration data that account for both temperature and concentration.

Detection of paracetamol binding to albumin in blood serum using 2D-IR spectroscopy.

Binding of drugs to blood serum proteins can influence both therapeutic efficacy and toxicity. The ability to measure the concentrations of protein-bound drug molecules quickly and with limited sample preparation could therefore have considerable benefits in biomedical and pharmaceutical applications. Vibrational spectroscopies provide data quickly but are hampered by complex, overlapping protein amide I band profiles and water absorption. Here, we show that two-dimensional infrared (2D-IR) spectroscopy can achieve rapid detection and quantification of paracetamol binding to serum albumin in blood serum at physiologically-relevant levels with no additional sample processing. By measuring changes to the amide I band of serum albumin caused by structural and dynamic impacts of paracetamol binding we show that drug concentrations as low as 7 µM can be detected and that the availability of albumin for paracetamol binding is less than 20% in serum samples, allowing identification of paracetamol levels consistent with a patient overdose.

Calibration model transfer in mid-infrared process analysis with in situ attenuated total reflectance immersion probes.

Process applications of mid-infrared (MIR) spectrometry may involve replacement of the spectrometer and/or measurement probe, which generally requires a calibration transfer method to maintain the accuracy of analysis. In this study, direct standardisation (DS), piecewise direct standardisation (PDS) and spectral space transformation (SST) were compared for analysis of ternary mixtures of acetone, ethanol and ethyl acetate. Three calibration transfer examples were considered: changing the spectrometer, multiplexing two probes to a spectrometer, and changing the diameter of the attenuated total reflectance (ATR) probe (as might be required when scaling up from lab to process analysis). In each case, DS, PDS and SST improved the accuracy of prediction for the test samples, analysed on a secondary spectrometer-probe combination, using a calibration model developed on the primary system. When the probe diameter was changed, a scaling step was incorporated into SST to compensate for the change in absorbance caused by the difference in ATR crystal size. SST had some advantages over DS and PDS: DS was sensitive to the choice of standardisation samples, and PDS required optimisation of the window size parameter (which also required an extra standardisation sample). SST only required a single parameter to be chosen: the number of principal components, which can be set equal to the number of standardisation samples when a low number of standards (n < 7) are used, which is preferred to minimise the time required to transfer the calibration model.

Improved prediction of tablet properties with near-infrared spectroscopy by a fusion of scatter correction techniques.

Near-infrared (NIR) spectra of pharmaceutical tablets get affected by light scattering phenomena, which mask the underlying peaks related to chemical components. Often the best performing scatter correction technique is selected from a pool of pre-selected techniques. However, the data corrected with different techniques may carry complementary information, hence, use of a single scatter correction technique is sub-optimal. In this study, the aim is to prove that NIR models related to pharmaceuticals can directly benefit from the fusion of complementary information extracted from multiple scatter correction techniques. To perform the fusion, sequential and parallel pre-processing fusion approaches were used. Two different open source NIR data sets were used for the demonstration where the assay uniformity and active ingredient (AI) content prediction was the aim. As a baseline, the fusion approach was compared to partial least-squares regression (PLSR) performed on standard normal variate (SNV) corrected data, which is a commonly used scatter correction technique. The results suggest that multiple scatter correction techniques extract complementary information and their complementary fusion is essential to obtain high-performance predictive models. In this study, the prediction error and bias were reduced by up to 15 % and 57 % respectively, compared to PLSR performed on SNV corrected data.

Determination of Bubble Size Distribution Using Ultrasound Array Imaging.

In this article, ultrasonic phased arrays are deployed as an imaging tool for industrial process analysis. Such arrays are typically used for sonar, medical diagnosis, and nondestructive testing; however, they have not yet been applied to industrial process analysis. The precise positioning of array elements and high frequencies possible with this technology mean that highly focused images can be generated, which cannot currently be achieved using ultrasound tomography. This article aims to highlight the potential of this technology for the measurement of bubble size distribution (BSD) and to demonstrate its application to both intrusive and noninvasive process measurements. Ultrasound images of bubble reflectors are generated using the total focusing method deployed using a 32-element, 5-MHz linear phased array, and an image processing algorithm for BSD determination is presented and evaluated under stationary and dynamic acquisition conditions. It is found that the sizing accuracy is within 10% for stationary reflectors larger than 4λ in diameter and that the algorithm is stable across the expected spatial variation of reflectors. The phased array is coupled to a six-axis robotic arm to scan a solid sample containing bubble reflectors at velocities up to 500 mms-1. The sizing accuracy is within 45% for bubbles larger than 4λ in diameter and at velocities up to 300 mms-1. However, above this velocity, the algorithm breaks down for reflectors smaller than 9λ in diameter. The ultrasound system is applied to a stream of air bubbles rising through water, which is verified via photographic analysis. Images were generated both intrusive and noninvasive, via a 10-mm Perspex barrier, to the process stream. The high bubble density in the process stream introduced scattering, limiting the measurement repeatability and the sample size in the measured distribution. Notwithstanding, this result demonstrates the potential of this technology to size bubbles for intrusive and noninvasive process analyses.

Reaction Monitoring Using SABRE-Hyperpolarized Benchtop (1 T) NMR Spectroscopy.

The conversion of [IrCl(COD)(IMes)] (COD = cis, cis-1,5-cyclooctadiene, IMes = 1,3-bis(2,4,6-trimethyl-phenyl)imidazole-2-ylidene) in the presence of an excess of para-hydrogen ( p-H2) and a substrate (4-aminopyridine (4-AP) or 4-methylpyridine (4-MP)) into [Ir(H)2(IMes)(substrate)3]Cl is monitored by 1H NMR spectroscopy using a benchtop (1 T) spectrometer in conjunction with the p-H2-based hyperpolarization technique signal amplification by reversible exchange (SABRE). A series of single-shot 1H NMR measurements are used to monitor the chemical changes that take place in solution through the lifetime of the hyperpolarized response. Non-hyperpolarized high-field 1H NMR control measurements were also undertaken to confirm that the observed time-dependent changes relate directly to the underlying chemical evolution. The formation of [Ir(H)2(IMes)(substrate)3]Cl is further linked to the hydrogen isotope exchange (HIE) reaction, which leads to the incorporation of deuterium into the ortho positions of 4-AP, where the source of deuterium is the solvent, methanol- d4. Comparable reaction monitoring results are achieved at both high-field (9.4 T) and low-field (1 T). It is notable that the low sensitivity of the benchtop (1 T) NMR enables the use of protio solvents, which when used here allows the effects of catalyst formation and substrate deuteration to be separated. Collectively, these methods illustrate how low-cost low-field NMR measurements provide unique insight into a complex catalytic process through a combination of hyperpolarization and relaxation data.

Quantitative In Situ Monitoring of Parahydrogen Fraction Using Raman Spectroscopy.

Raman spectroscopy has been used to provide a rapid, noninvasive, and nondestructive quantification method for determining the parahydrogen fraction of hydrogen gas. The basis of the method is the measurement of the ratio of the first two rotational bands of hydrogen at 355 cm-1 and 586 cm-1 corresponding to parahydrogen and orthohydrogen, respectively. The method has been used to determine the parahydrogen content during a production process and a reaction. In the first example, the performance of an in-house liquid nitrogen cooled parahydrogen generator was monitored both at-line and on-line. The Raman measurements showed that it took several hours for the generator to reach steady state and, hence, for maximum parahydrogen production (50%) to be reached. The results obtained using Raman spectroscopy were compared to those obtained by at-line low-field nuclear magnetic resonance (NMR) spectroscopy. While the results were in good agreement, Raman analysis has several advantages over NMR for this application. The Raman method does not require a reference sample, as both spin isomers (ortho and para) of hydrogen can be directly detected, which simplifies the procedure and eliminates some sources of error. In the second example, the method was used to monitor the fast conversion of parahydrogen to orthohydrogen in situ. Here the ability to acquire Raman spectra every 30 s enabled a conversion process with a rate constant of 27.4×10-4 s-1 to be monitored. The Raman method described here represents an improvement on previously reported work, in that it can be easily applied on-line and is approximately 500 times faster. This offers the potential of an industrially compatible method for determining parahydrogen content in applications that require the storage and usage of hydrogen.

Quantification of hyperpolarisation efficiency in SABRE and SABRE-Relay enhanced NMR spectroscopy.

para-Hydrogen (p-H2) induced polarisation (PHIP) is an increasingly popular method for sensitivity enhancement in NMR spectroscopy. Its growing popularity is due in part to the introduction of the signal amplification by reversible exchange (SABRE) method that generates renewable hyperpolarisation in target analytes in seconds. A key benefit of PHIP and SABRE is that p-H2 can be relatively easily and cheaply produced, with costs increasing with the desired level of p-H2 purity. In this work, the efficiency of the SABRE polarisation transfer is explored by measuring the level of analyte hyperpolarisation as a function of the level of p-H2 enrichment. A linear relationship was found between p-H2 enrichment and analyte 1H hyperpolarisation for a range of molecules, polarisation transfer catalysts, NMR detection fields and for both the SABRE and SABRE-Relay transfer mechanisms over the range 29-99% p-H2 purity. The gradient of these linear relationships were related to a simple theoretical model to define an overall efficiency parameter, E, that quantifies the net fraction of the available p-H2 polarisation that is transferred to the target analyte. We find that the efficiency of SABRE is independent of the NMR detection field and exceeds E = 20% for methyl-4,6-d2-nicotinate when using a previously optimised catalyst system. For the SABRE-Relay transfer mechanism, efficiencies of up to E = 1% were found for 1H polarisation of 1-propanol, when ammonia was used as the polarisation carrier.

SABRE hyperpolarization enables high-sensitivity 1H and 13C benchtop NMR spectroscopy.

Benchtop NMR spectrometers operating with low magnetic fields of 1-2 T at sub-ppm resolution show great promise as analytical platforms that can be used outside the traditional laboratory environment for industrial process monitoring. One current limitation that reduces the uptake of benchtop NMR is associated with the detection fields' reduced sensitivity. Here we demonstrate how para-hydrogen (p-H2) based signal amplification by reversible exchange (SABRE), a simple to achieve hyperpolarization technique, enhances agent detectability within the environment of a benchtop (1 T) NMR spectrometer so that informative 1H and 13C NMR spectra can be readily recorded for low-concentration analytes. SABRE-derived 1H NMR signal enhancements of up to 17 000-fold, corresponding to 1H polarization levels of P = 5.9%, were achieved for 26 mM pyridine in d4-methanol in a matter of seconds. Comparable enhancement levels can be achieved in both deuterated and protio solvents but now the SABRE-enhanced analyte signals dominate due to the comparatively weak thermally-polarized solvent response. The SABRE approach also enables the acquisition of 13C NMR spectra of analytes at natural isotopic abundance in a single scan as evidenced by hyperpolarized 13C NMR spectra of tens of millimolar concentrations of 4-methylpyridine. Now the associated signal enhancement factors are up to 45 500 fold (P = 4.0%) and achieved in just 15 s. Integration of an automated SABRE polarization system with the benchtop NMR spectrometer framework produces renewable and reproducible NMR signal enhancements that can be exploited for the collection of multi-dimensional NMR spectra, exemplified here by a SABRE-enhanced 2D COSY NMR spectrum.

A simple hand-held magnet array for efficient and reproducible SABRE hyperpolarisation using manual sample shaking.

Signal amplification by reversible exchange (SABRE) is a hyperpolarisation technique that catalytically transfers nuclear polarisation from parahydrogen, the singlet nuclear isomer of H2 , to a substrate in solution. The SABRE exchange reaction is carried out in a polarisation transfer field (PTF) of tens of gauss before transfer to a stronger magnetic field for nuclear magnetic resonance (NMR) detection. In the simplest implementation, polarisation transfer is achieved by shaking the sample in the stray field of a superconducting NMR magnet. Although convenient, this method suffers from limited reproducibility and cannot be used with NMR spectrometers that do not have appreciable stray fields, such as benchtop instruments. Here, we use a simple hand-held permanent magnet array to provide the necessary PTF during sample shaking. We find that the use of this array provides a 25% increase in SABRE enhancement over the stray field approach, while also providing improved reproducibility. Arrays with a range of PTFs were tested, and the PTF-dependent SABRE enhancements were found to be in excellent agreement with comparable experiments carried out using an automated flow system where an electromagnet is used to generate the PTF. We anticipate that this approach will improve the efficiency and reproducibility of SABRE experiments carried out using manual shaking and will be particularly useful for benchtop NMR, where a suitable stray field is not readily accessible. The ability to construct arrays with a range of PTFs will also enable the rapid optimisation of SABRE enhancement as function of PTF for new substrate and catalyst systems.

Spray Drying as a Reliable Route to Produce Metastable Carbamazepine Form IV.

Carbamazepine (CBZ) is an active pharmaceutical ingredient used in the treatment of epilepsy that can form at least 5 polymorphic forms. Metastable form IV was originally discovered from crystallization with polymer additives; however, it has not been observed from subsequent solvent-only crystallization efforts. This work reports the reproducible formation of phase pure crystalline form IV by spray drying of methanolic CBZ solution. Characterization of the material was carried out using diffraction, scanning electron microscopy, and differential scanning calorimetry. In situ Raman spectroscopy was used to monitor the spray-dried product during the spray drying process. This work demonstrates that spray drying provides a robust method for the production of form IV CBZ, and the combination of high supersaturation and rapid solid isolation from solution overcomes the apparent limitation of more traditional solution crystallization approaches to produce metastable crystalline forms.

Multivariate data analysis and metabolic profiling of artemisinin and related compounds in high yielding varieties of Artemisia annua field-grown in Madagascar.

An improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) protocol for rapid analysis of co-metabolites of A. annua in raw extracts was developed and extensively characterized. The new method was used to analyse metabolic profiles of 13 varieties of A. annua from an in-field growth programme in Madagascar. Several multivariate data analysis techniques consistently show the association of artemisinin with dihydroartemisinic acid. These data support the hypothesis of dihydroartemisinic acid being the late stage precursor to artemisinin in its biosynthetic pathway.

Monitoring of an esterification reaction by on-line direct liquid sampling mass spectrometry and in-line mid infrared spectrometry with an attenuated total reflectance probe.

A specially designed thermal vaporiser was used with a process mass spectrometer designed for gas analysis to monitor the esterification of butan-1-ol and acetic anhydride. The reaction was conducted at two scales: in a 150 mL flask and a 1L jacketed batch reactor, with liquid delivery flow rates to the vaporiser of 0.1 and 1.0 mLmin(-1), respectively. Mass spectrometry measurements were made at selected ion masses, and classical least squares multivariate linear regression was used to produce concentration profiles for the reactants, products and catalyst. The extent of reaction was obtained from the butyl acetate profile and found to be 83% and 76% at 40°C and 20°C, respectively, at the 1L scale. Reactions in the 1L reactor were also monitored by in-line mid-infrared (MIR) spectrometry; off-line gas chromatography (GC) was used as a reference technique when building partial least squares (PLS) multivariate calibration models for prediction of butyl acetate concentrations from the MIR spectra. In validation experiments, good agreement was achieved between the concentration of butyl acetate obtained from in-line MIR spectra and off-line GC. In the initial few minutes of the reaction the profiles for butyl acetate derived from on-line direct liquid sampling mass spectrometry (DLSMS) differed from those of in-line MIR spectrometry owing to the 2 min transfer time between the reactor and mass spectrometer. As the reaction proceeded, however, the difference between the concentration profiles became less noticeable. DLSMS had advantages over in-line MIR spectrometry as it was easier to generate concentration profiles for all the components in the reaction. Also, it was possible to detect the presence of a simulated impurity of ethanol (at levels of 2.6 and 9.1% mol/mol) in butan-1-ol, and the resulting production of ethyl acetate, by DLSMS, but not by in-line MIR spectrometry.

Variation in the transmission near-infrared signal with depth in turbid media.

Transmission near-infrared (NIR) measurements of a 1 mm thick aspirin disk were made at different positions as it was moved through a stack of eight 0.5 mm thick disks of microcrystalline cellulose (Avicel). The magnitude of the first derivative of absorbance for the aspirin interlayer at 8934 cm(-1) was lower when the disk was placed at the top or bottom of the stack of Avicel disks, with the largest signal observed when the aspirin was positioned at the central positions. The variation in signal with depth is consistent with that observed previously for transmission Raman spectrometry. In both cases, the trend observed can be attributed to lower photon density at the air-sample interface, relative to the center of the sample, owing to loss of photons to the air. This results in a reduction in the number of photons absorbed or Raman photons generated and subsequently detected when the interlayer occupies a near-surface position.

Towards better understanding of an industrial cell factory: investigating the feasibility of real-time metabolic flux analysis in Pichia pastoris.

BACKGROUND: Novel analytical tools, which shorten the long and costly development cycles of biopharmaceuticals are essential. Metabolic flux analysis (MFA) shows great promise in improving our understanding of the metabolism of cell factories in bioreactors, but currently only provides information post-process using conventional off-line methods. MFA combined with real time multianalyte process monitoring techniques provides a valuable platform technology allowing real time insights into metabolic responses of cell factories in bioreactors. This could have a major impact in the bioprocessing industry, ultimately improving product consistency, productivity and shortening development cycles. RESULTS: This is the first investigation using Near Infrared Spectroscopy (NIRS) in situ combined with metabolic flux modelling which is both a significant challenge and considerable extension of these techniques. We investigated the feasibility of our approach using the industrial workhorse Pichia pastoris in a simplified model system. A parental P. pastoris strain (i.e. which does not synthesize recombinant protein) was used to allow definition of distinct metabolic states focusing solely upon the prediction of intracellular fluxes in central carbon metabolism. Extracellular fluxes were determined using off-line conventional reference methods and on-line NIR predictions (calculated by multivariate analysis using the partial least squares algorithm, PLS). The results showed that the PLS-NIRS models for biomass and glycerol were accurate: correlation coefficients, R2, above 0.90 and the root mean square error of prediction, RMSEP, of 1.17 and 2.90 g/L, respectively. The analytical quality of the NIR models was demonstrated by direct comparison with the standard error of the laboratory (SEL), which showed that performance of the NIR models was suitable for quantifying biomass and glycerol for calculating extracellular metabolite rates and used as independent inputs for the MFA (RMSEP lower than 1.5 × SEL). Furthermore, the results for the MFA from both datasets passed consistency tests performed for each steady state, showing that the precision of on-line NIRS is equivalent to that obtained by the off-line measurements. CONCLUSIONS: The findings of this study show for the first time the potential of NIRS as an input generating for MFA models, contributing to the optimization of cell factory metabolism in real-time.

In situ monitoring of powder blending by non-invasive Raman spectrometry with wide area illumination.

A 785nm diode laser and probe with a 6mm spot size were used to obtain spectra of stationary powders and powders mixing at 50rpm in a high shear convective blender. Two methods of assessing the effect of particle characteristics on the Raman sampling depth for microcrystalline cellulose (Avicel), aspirin or sodium nitrate were compared: (i) the information depth, based on the diminishing Raman signal of TiO(2) in a reference plate as the depth of powder prior to the plate was increased, and (ii) the depth at which a sample became infinitely thick, based on the depth of powder at which the Raman signal of the compound became constant. The particle size, shape, density and/or light absorption capability of the compounds were shown to affect the "information" and "infinitely thick" depths of individual compounds. However, when different sized fractions of aspirin were added to Avicel as the main component, the depth values of aspirin were the same and matched that of the Avicel: 1.7mm for the "information" depth and 3.5mm for the "infinitely thick" depth. This latter value was considered to be the minimum Raman sampling depth when monitoring the addition of aspirin to Avicel in the blender. Mixing profiles for aspirin were obtained non-invasively through the glass wall of the vessel and could be used to assess how the aspirin blended into the main component, identify the end point of the mixing process (which varied with the particle size of the aspirin), and determine the concentration of aspirin in real time. The Raman procedure was compared to two other non-invasive monitoring techniques, near infrared (NIR) spectrometry and broadband acoustic emission spectrometry. The features of the mixing profiles generated by the three techniques were similar for addition of aspirin to Avicel. Although Raman was less sensitive than NIR spectrometry, Raman allowed compound specific mixing profiles to be generated by studying the mixing behaviour of an aspirin-aspartame-Avicel mixture.