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

A comparison of collision cross section values obtained via travelling wave ion mobility-mass spectrometry and ultra high performance liquid chromatography-ion mobility-mass spectrometry : application to the characterisation of metabolites in rat urine

A comprehensive Collision Cross Section (CCS) library was obtained via Travelling Wave Ion Guide mobility measurements through direct infusion (DI). The library consists of CCS and Mass Spectral (MS) data in negative and positive ElectroSpray Ionisation (ESI) mode for 463 and 479 endogenous metabolites, respectively. For both ionisation modes combined, TWCCSN2 data were obtained for 542 non-redundant metabolites. These data were acquired on two different ion mobility enabled orthogonal acceleration QToF MS systems in two different laboratories, with the majority of the resulting TWCCSN2 values (from detected compounds) found to be within 1% of one another. Validation of these results against two independent, external TWCCSN2 data sources and predicted TWCCSN2 values indicated to be within 1-2% of these other values. The same metabolites were then analysed using a rapid reversed-phase ultra (high) performance liquid chromatographic (U(H)PLC) separation combined with IM and MS (IM-MS) thus providing retention time (tr), m/z and TWCCSN2 values (with the latter compared with the DI-IM-MS data). Analytes for which TWCCSN2 values were obtained by U(H)PLC-IM-MS showed good agreement with the results obtained from DI-IM-MS. The repeatability of the TWCCSN2 values obtained for these metabolites on the different ion mobility QToF systems, using either DI or LC, encouraged the further evaluation of the U(H)PLC-IM-MS approach via the analysis of samples of rat urine, from control and methotrexate-treated animals, in order to assess the potential of the approach for metabolite identification and profiling in metabolic phenotyping studies. Based on the database derived from the standards 63 metabolites were identified in rat urine, using positive ESI, based on the combination of tr, TWCCSN2 and MS data.</p

Improved time-resolved measurements of inorganic ions in particulate matter by PILS-IC integrated with a sample pre-concentration system

A particle-into-liquid sampler coupled with ion chromatograph (PILS-IC) for the on-line measurement of inorganic ions has been modified by the insertion of two ion-exchange pre-concentration cartridges that enrich the sample during the period of the IC analysis. The limits of detection of the modified instrument were 10-15 times lower and the time coverage 24 times higher (from 2 to 48 min per hour) than those of the original PILS-IC setup. The instrumental performance in terms of recovery and break-through volume from the cartridges was satisfactory. The modified PILS-IC was operated in comparison with a diffusion denuder line and with a high-resolution time-of-flight aerosol mass spectrometer (HR-TOF-AMS) during a short intensive measurement period organized in the framework of the European Monitoring and Evaluation Programme (EMEP), a co-operative program for monitoring and evaluation of the long-range transmission of the air pollutants in Europe. The instrument showed a quantitative response in agreement with the results of the diffusion lines, and an ability to trace fine concentration variations not so different from the performance of the much more complex HR-TOF-AMS. From the time patterns of the ion concentrations measured by the modified PILS-IC, it was possible to obtain useful information about the variations in the air quality and in the strength of the particulate matter sourc

Nitrogen isotope analysis of ammonium and glycine

Stable isotope techniques can be used as a tool in nitrogen cycling studies of different ecosystems. The studies are based on measurement of the heavy (15N)- to- light (14N) isotopic ratios of nitrogen in different biospheric pools. Isotope ratio mass spectrometry (IRMS) is the most precise technique to use for analysis of nitrogen isotopic ratios. This thesis deals with the development of methods for compound-specific nitrogen isotope analysis of ammonium and glycine in aqueous solutions and soil extracts using Gas Chromatography - Combustion (GC-C) - IRMS. For ammonium, three different techniques were developed: equilibrium headspace analysis, solid phase microextraction (SPME) and the purge and trap (P & T) technique, which were all based on conversion of ammonium to ammonia with subsequent separation of ammonia for analysis. In the SPME and P & T approaches, custom-made absorbents were used for pre-concentration, followed by thermal desorption into the GC-C-IRMS system. For the equilibrium headspace technique, high precision measurements of the nitrogen isotopic ratio were obtained for concentrations above 420 mg N L-1. With further improvements and the use of suitable equipment, the method has the potential to be used for solutions containing ammonium in the low mg N L-1 range. The SPME technique increased the sensitivity by a factor of » 3 compared to the headspace technique, but was less precise. In addition, the NafionÒ material used for absorption showed a memory effect in the desorption step. With the P & T technique a large variation in the measured isotopic value was observed (using solutions containing 2 mg N L-1) which was due to a non-quantitative thermal desorption. However, with further improvements, the P & T technique has the potential to be used for samples containing below 1.0 µg N, which is a much lower amount than that possible with any method used today. A method for determination of the nitrogen isotopic ratio in free glycine in soil extracts was also developed. By a combination of sample pre-concentration and Isotope Dilution Mass Spectrometry (IDMS), it was possible to determine isotopic ratio in soil extracts with a glycine concentration of only 3 µM (0.042 mg N L-1). The precision obtained was sufficient for use with tracer studies and was higher by an order of magnitude than the precision obtained with conventional GC-MS

Synthesis of n-hexyl acetate in batch and chromatographic reactors

Petrochemical and fine chemical industries face a daunting problem in recovering acetic acid from its aqueous solutions. The recovery of acetic acid could be done through esterification reaction. However, esterification is an equilibrium limited reaction. Multi-functional reactors such as chromatographic reactor (CR) and reactive distillation column (RDC) are promising technologies mainly for equilibrium limited reactions wherein reaction and separation of products are carried out in a single equipment that tends to shift the equilibrium towards the desired direction which is not possible in a classical batch reactor. Physical and chemical characterisation of ion exchange resin catalysts such as scanning electron microscopy, Brunauer-Emmett-Teller (BET) surface area measurement, pore size distribution, elemental analysis, true density and particle size distribution were carried out to access the catalysts performance for n-hexyl acetate synthesis. Esterification of acetic acid with n-hexanol was studied with both dilute and concentrated acid in the presence of cation exchange resins (macroporous and gelular) in a jacketed stirred batch reactor to synthesise a value added ester, namely n-hexyl acetate and also to study the recovery of acetic acid from the waste aqueous streams. The effect of various parameters such as speed of agitation, catalyst particle size, feed mole ratio of n-hexanol to acetic acid, reaction temperature, catalyst loading and reusability of catalysts was studied for the optimisation of the reaction condition in a batch reactor. The non-ideality of each component in the reacting mixture was accounted for by using the activity coefficient via the use of the UNIFAC group contribution method. The kinetic data were correlated with both pseudo-homogeneous (PH) and adsorption based heterogeneous reaction rate models, e.g., Eley-Rideal (ER), Langmuir-Hinshelwood-Hougen-Watson (LHHW), and the modified LHHW (ML). Pseudo-homogeneous (PH) model gave the best representation of the kinetic data found experimentally. The feasibility of reactive distillation for the recovery of acetic acid using n-hexanol was evaluated through residue curve map (RCM) determination experiments. RCM provides information to a design engineer of the existence of separation boundaries imposed by the singular points corresponding to the reactive/non-reactive azeotropes, thus provides an insight into the feasibility of reactive distillation for this purpose. A laboratory scale batch chromatographic reactor was designed and constructed. Batch chromatographic reactor experiments were carried out using different parameters such as feed flow rate, feed mole ratio of n-hexanol to acetic acid, desorbent (n-hexanol) flow rate and reaction step to maximise the formation of n-hexyl acetate as well to achieve complete conversion of acetic acid. Continuous chromatographic reactor was designed, constructed and commissioned on the basis of the results obtained from the batch chromatographic reactor experiments. The experiments carried out in continuous chromatographic reactor correlated very well with the results from the batch chromatographic reactor for the optimised condition

Establishing the maximum carbon number for reliable quantitative gas chromatographic analysis of heavy ends hydrocarbons

This Thesis investigates the two main limitations of high temperature gas chromatography (HTGC) in the analysis of heavy n-alkanes: pyrolysis inside the GC column and incomplete elution. The former is studied by developing and reducing a radical pyrolysis model (7055 reactions) into a molecular pyrolysis model (127 reactions) capable of predicting low conversions of (nC14H30-nC80H162) at temperatures up to 430°C. Validation of predicted conversion with literature data for nC14H30, nC16H34 and nC25H52 yielded an error lower than 5.4%. The latter is addressed by developing an analytical model which solves recursively the diffusion and convection phenomena separately. The model is capable of predicting the position and molar distribution of components, using as main input the analytes’ distribution factors and yielded an error lower than 4.4% in the prediction of retention times. This thesis provides an extension of the data set of distribution factors of (nC12H26– nC98H198) in a SGE HT5 GC capillary column, based on isothermal GC measurements at both constant inlet pressure and flow rate. Finally, the above two models were coupled, yielding a maximum mass lost of 1.3 % in the case of nC80H162 due to pyrolysis and complete elution up to nC70H142, in a 12 m HT5 column

A practical investigation into the use of principal component analysis for the modelling and scale-up of high performance liquid chromatography

Liquid chromatography is becoming increasingly important for the final purification of biomolecules. Traditionally, chromatography has been modelled using mathematical techniques which require experimental determination of the physicochemical data for the separation of interest. These methods are both time-consuming and very complex and have only been truly successful in the prediction of binary separations and where the species in a mixture do not interact significantly. This thesis investigates the use of Principal Component Analysis (PCA), a multivariate statistical technique for the modelling and scale-up of chromatographic separations using a range of stationary phase chemistries and explores the utility of the approach as an engineering tool for rapid process development. The reversed-phase separation of a semi-purified erythromycin feed was used as the feedstock throughout the study. Separations were performed on columns with various geometries and stationary phases. An isocratic solvent comprising 45:55 (v/v) of acetonitrile/water was used to effect the separation into 4 major components and at least 10 minor components. In a first set of chromatograms, experimental design techniques were used to investigate the effects of four process variables (load volume, load concentration, temperature and pH of buffer) on the chromatogram shapes from each of 4 columns. The choice of appropriate data pre-processing prior to PCA was investigated in order to achieve maximal analytical performance from the statistical method. The results showed that when the retention times of elution peaks changed due to variations in temperature and pH, it was necessary to align the main product peak in order to gain most benefit from the PCA. Correlations were derived which enabled accurate chromatogram predictions (>95%) to be made using data from columns with fivefold change of scale and a fivefold change in sample size (25-fold scaling factor overall). A second set of chromatograms were generated in which only the amount of sample load was varied. Sample concentrations of 20mg/mL were separated by each column, the sample volume applied being in the range 1-10% of the bed volume of each column which included realistic non-linear, overload conditions. The principal components derived reflected similar properties of the chromatograms regardless of scale and stationary phase. These similarities were correlated, enabling predictions to be made from small (5cm length x 4.6mm diameter) to large-scale (up to 60mm diameter). The overall scaling factor in this set of chromatograms was in the region of 5000-fold. The use of data reduction techniques was investigated throughout the study so as to minimise the number of runs required at large-scale whilst maintaining highly accurate (>98% accuracy) predictions. Results showed that considerable reductions in the sizes of data sets could be made (>75% reduction) without significant loss in the quality of the data provided that attempts were not made to extrapolate too far outside the limits of the experimental conditions. PCA appears to be a very promising technique for the rapid and reliable modelling and scale-up of performance requiring minimal experimental work and achieving greater accuracy than with traditional mathematical approaches and makes recommendations for future work involving examining the potential relationships between PCA models and the underlying physico-chemical events controlling chromatographic separations

Diffusion and Adsorption Coefficients of Aromatic Hydrocarbons in Gas Chromatography Capillary Columns

This study focuses on a mathematical description of aromatic species elution peaks from a gas chromatographic BPX5 capillary column. Using the chromatographic peaks, statistical moments are calculated for toluene, naphthalene, phenol and 2-naphthol. This thesis reports two modelling approaches involving laminar gas flow, distribution coefficients (Ks) and diffusion coefficients in the stationary phase (Ds). Firstly, a model with equilibrium adsorption is considered to describe symmetric peaks for toluene and naphthalene. Moreover, a model with non-equilibrium adsorption is proposed to describe asymmetric peaks of phenol and 2-napthol. In addition to the Ks and Ds parameters, this model involves adsorption kinetic constants (kads). Validation of both mathematical models is developed by performing experiments at different carrier gas velocities and column temperatures (Tc). The model equilibrium adsorption, reports that the distribution coefficients, Ks, and the diffusion coefficients (Ds), solely depend on the solute and stationary phase properties. Furthermore, the model under non-equilibrium adsorption provides kads parameters for phenol and 2-naphthol. However, to fit the second moment, M2,exp, a revised model for the BPX5 column involving two classes of sites for solute adsorption is considered: one site with adsorption at equilibrium and the other site with adsorption at non-equilibrium. Thus, this PhD thesis establishes two chromatographic models for aromatic hydrocarbon species peaks eluted from a BPX5 capillary column. Both mathematical models represent an important contribution to the knowledge of solute interactions in capillary columns for GC. These models may potentially have a significant impact on the future of GC analysis of complex mixtures

The derivation of bioprocess understanding from mechanistic models of chromatography

This thesis, completed in collaboration with Purification Process Development of Pfizer Biotherapeutics, is concerned with how mechanistic models of chromatographic bioseparations can be applied in industry to accelerate development and increase robustness of industrial protein purification processes, whilst also realising the benefits of a systematic development approach based on fundamental process and product understanding. The first results chapter considers the application of mechanistic models to provide a link between high throughput screening (HTS) and scouting runs conducted during early process development. The chapter focuses on an anion exchange (AEX) weak partitioning chromatography (WPC) polishing step in a platform monoclonal antibody purification process. Adsorption isotherms are formulated from experimental multicomponent batch adsorption studies of monomer – aggregate. A new approach is taken where the adsorption equilibria is characterised as a function of the product partition coefficient, enabling the model to be applied to new candidate monoclonal antibodies without additional experimental effort. Stochastic simulations conducted at an early stage of process development identify promising operating parameter ranges for challenging separations, directs optimal performance, and reduces development times. A detailed analysis of model predictions increases fundamental knowledge and understanding of the complex WPC multidimensional design space, which enables better informed process development at Pfizer. Resin fouling over a chromatography columns lifetime can cause significant (undesired) changes in process performance. A lack of fundamental knowledge and mechanistic understanding of fouling in industrial bioseparations limits the application of mechanistic models in industry. An experimental investigation was conducted into fouling of the AEX WPC considered in the first results chapter. Analysis of fouled resin samples by batch uptake experiments, scanning electron microscopy, confocal laser scanning microscopy and scale down column studies, indicated significant blockage of the pores at the resin surface occurred that after successive batch cycles. Mass transport into resin particles was severely hindered, but saturation capacity was not affected. The increased understanding of resin fouling can direct future efforts to mitigate this detrimental phenomenon and maintain process performance, whilst providing a basis for the development of new fouling models. The third results chapter considers an industrial hydrophobic interaction chromatography (HIC) separation at a late stage of process development. Resin lot variability, combined with a variable feed stream, had resulted in serious performance issues during the purification of a therapeutic protein from crude feed material. The traditional approach to tackling this type of problem involves defining a design space based on an extensive experimental effort directed by factorial design of experiments conducted at great cost. The result is a fixed, inflexible manufacturing process, with a control strategy based on reproducibility rather than robustness, and little fundamental understanding of the source of the issue. In the third results chapter, the application of mechanistic models to identify robust operating conditions for the HIC is considered. A model is developed, validated experimentally, and used to generate probabilistic design spaces accounting the historical variability in the resin lots and load material. The stochastic simulation approach is extended to explore the impact of reducing variability in the load material on the design space. With historical process variability, no operating condition was found where the probability of meeting product quality specifications remained > 0.95 for all resin lots. Model simulations indicated that adopting an adaptive design space, where operating conditions are changed according to which resin lot is in use, is favorable for ensuring process robustness, which is a step change concept for bioprocessing. The conclusions and outcomes resulting from the application of mechanistic models to the two industrial systems in this thesis, provides a basis for the next generation purification process development platform

Advances in simulated moving bed : new operating modes : new design methodologies and product (FlexSMB-LSRE) development

Tese de doutoramento. Engenharia Química e Biológica. Faculdade de Engenharia. Universidade do Porto. 200