There have been significant advances in instrumentation concerned with the analysis of biopolymers in the last five years. The recent developments have provided physical methods for determining both purity and molecular weight of biopolymers with greater accuracy than by traditional methods. In the separation sciences, capillary electrophoresis has emerged as an invaluable tool for the biochemical analyst. In the spectroscopic world vast improvements in mass spectrometric instrumentation have provided an order of magnitude improvement in sensitivity. This factor, along with the revival of techniques such as laser irradiation and electrospray, (which along with several important discoveries concerning trivial chemistry during sample preparation in the former and realisation of the effect and importance of multiple charging in the latter) now means that the analysis of biopolymers by mass spectrometry is not only feasible but is becoming almost routine. In this thesis, in chapters 1, 2 and 3 the new techniques are described and applied to everyday analytical problems posed to the biochemical analyst in the pharmaceutical industry. In chapter 4 a comparison is made between an established liquid chromatography-mass spectrometry interface and electrospray, using a standard peptide mixture for reference. The importance of traditional methods of protein digestion by enzymes is highlighted in chapter 5, where novel use of an anomeric specific enzyme is made and the products analysed by two different mass spectrometric techniques. Chapter 6 describes the new role that mass spectrometry has claimed in biopolymer analysis by enabling a new protocol to be established in the search for novel pharmaceutical compounds which are both more efficient and more specific in their action.

This thesis describes the application of a microcomputer to the development of automatic analytical instrumentation. The basic structure of a typical microcomputer and the manner in which it is programmed are described, and the applications of microprocessors to analytical instrumentation are reviewed. The remainder of the thesis is devoted to detailed description of the stages involved in the development of an Automatic Titrator and an Automatic Viscometer; the microcomputer is used to both control the equipment and process the results. The computer-controlled titrator was developed for the sequential titration of a mixture of adipic acid and boric acid against sodium hydroxide for use in the manufacture of Nylon. The differential plot Apll/ OV is used for the determination of end-points. The effect on the results of varying several experimental parameters were examined. These included the orientation of the titrant delivery tube, stirring speed, and the time interval between the addition of titrant and recording the ADC reading. The accuracy and precision of the results obtained with the computer-controlled system compare favourably with the conventional titrator which titrates to present pH values. The instrument has been developed further to become one of general applicability. Attention was then turned to the development of an instrument for the determination of molecular weights of polymers, based on the measurement of viscosity. Two approaches were adopted: 1. Timing the rise of very small bubbles through the liquid medium of unknown viscosity; 2. Injecting a slug of liquid into a liquid flowing in a capillary tube and measuring the extent of dispersion and/or the viscous drag. In the first of these, considerable practical difficulties were encountered. The second approach was experimentally simple, and was adopted. Taylor proposed that the dispersion of the sample is a consequence of diffusion and the effect of the fluid flowing along the capillary. When the flow-rate is sufficiently slow, dispersion is predominantly by diffusion and formulae are derived which describe the sample concentration profile in terms of the diffusion coefficient of the sample into the carrier. Viscosity is related to the diffusion coefficient by the Stokes-Einstein equation. When dispersion is predominantly a consequence of fluid flow, then, according to Poiseuille's equation, the time between injection and detection is related to the viscosity of the sample. Both theories have been verified for several systems. Smooth calibration curves may be obtained for a given system. Sample volumes of 6:2 have been employed and measurements are typically made in less than one minute. The microcomputer is used to control the injection of samples and to measure the maximum concentration at the detector and the corresponding time from injection. The system has also been used for the determination of diffusion coefficients of a sample of orange oil into various solvents.

Chapter One involves a brief introduction to the different types of chromatography and mass spectrometry used in this thesis. A further explanation of the ionisation processes used and the interfaces for liquid chromatography and supercritical fluid chromatography coupling to mass spectrometry is provided. The basis of Chapter Two is the separation and analysis of various β-blockers using liquid chromatography-atmospheric pressure ionisation-mass spectrometry. Full analysis was conducted using an ion trap mass spectrometer. The advantages of using involatile phosphate buffers to effect separation was also investigated and novel liquid chromatography-mass spectrometry analysis using these buffers was performed on the patented self cleaning single quadrupole 'aQa' mass spectrometer. Chapter Three involves analysis by liquid chromatography-mass spectrometry of benzodiazepines. The effect of buffer concentration was studied and a comparison made of both atmospheric pressure chemical ionisation and electrospray ionisation, in terms of fragmentation data an detection limits. A comparison of liquid chromatography-mass spectrometry and supercritical fluid chromatography-mass spectrometry for a separation of non-steroidal anti-inflammatory drugs is discussed in Chapter Four. Good resolutions were obtained with both techniques; however, the latter had superior speeds of analysis. Online atmospheric pressure chemical ionisation and electrospray ionisation is discussed and compared. Chapter Five concentrates on packed column supercritical fluid chromatography-mass spectrometry of two differing classes of pesticides. Two analyses were necessary due to the different compound polarities, the first containing a mixture of ten phenylurea and triazine herbicides and the second a mixture of eight acidic herbicides. These analyses provided a useful comparison to the more frequently used liquid chromatography-mass spectrometry.

Recent research into the sterol content of marine invertebrates has shown that many sterol profiles are extremely complex. Prior to the application of far liquid chromatography in this field the separation of individual sterol molecules into pure components was rather difficult. Subsequently combined cas chromatography-mass spectrometry usinc C.C. stationary phases which are highly thermostable has been found to be extremely useful. The G.C.-M.S. evaluation of a now highly thermostable polar stationary phase PZ-176 for the analysis of complex sterol mixtures is described in Chapter two. Packed column gas chromatography is not capable of resolving all of the components of the most complex sterol mixtures. Therefore high resolution G.C. using glass S.C.O.T. columns has been investigated in detail and Chapter three presents data for a number of columns using cilia-based supports and a variety of polar and non-polar stationary phases. Since the techniques of open tubular G.C. receive scant attention in most published journals, the methods of preparation, installation and routine use of injection and S.C.O.T. columns suitable for sterol analysis are described in detail in Chapter three. Chapter four and five describe the use of mild gel chromatographic method using Sephadex UL-20 and Lipidex 5000, both as a means of obtaining a sterol fraction from an extracted total lipids sample and of fractionating complicated sterol mixtures, allowing minor sterols to be effectively separated from the bulk of the sterol sample prior to final analysis. The methods can be used instead of or in conjunction with conventional adsorption chromatography depending upon the type of sample encountered. Finally the high resolution G.C. and G.C.- M.S. analysis of the sterol content of the red spon Halidona iubens is described in Chapter Six. The sterol mixture is complex and contains several novel marine keto sterols.

This thesis describes studies made into flow injection analysis or unsegmented continuous flow analysis, a recently developed automatic method of analysis of solutions. The state of the art of flow injection analysis is presented in the form of a review. The aim of the experimental work was to develop apparatus and methods for trace multielement determinations particularly of metal ions. A novel and versatile miniature flow through phototransducer was developed with all solid state light components. The inherent stability of these components gives rise to very high sensitivity in colorimetric analysis. A particular orientation of the light components yields a transducer highly sensitive to refractive index allowing accurate refractometric determinations to be performed. The principle of operation of this refractometer has been determined and is described. The transducer was used as a colorimeter in two chemical systems to perform multielement determinations on a single sample plug. In the first, the reproducible concentration gradients of the sample-carrier interfaces are an integral part of the method. The pH-gradient flow injection analysis method uses a pH-gradient created by the injection of strong base into a system of weak acid(s) (or strong acid into a system of weak base(s)). pH changes occur across the sample such that selective conditions for the analysis of individual analytes are attained at different points along the pH gradient. The viability of the method is demonstrated with lead(II) and vanadium(V), cobalt(II) and manganese(II), copper(II) and nickel(II), and copper(II) and manganese(II). The second method utilises the reproducible timing between injection and analysis in a two point kinetic assay of cobalt(II) and nickel(II) using the differential reaction rates of the pseudo-first-order dissociation of the citrate complexes and a subsequent scavenging reaction. On stream masking of interfering ions with EDTA yields a highly selective analysis.

The aim of this research was to investigate the use of high spectral resolution sensors to recover more detailed information on the biochemical composition of forest canopies. The research was divided into two broad areas: laboratory and field studies. The laboratory studies were undertaken to determine the relationships between reflected radiation and the biochemical concentrations of foliage. A number of sample sets of slash pine, Sitka spruce, lodgepole pine and Japanese larch were analysed for their chlorophyll, water, nitrogen, lignin, cellulose, sugar and starch concentrations. Reflectance measurements were made for whole green, wet needles and dried whole needles. Stepwise regression analyses showed strong relationships between reflectance spectra and chlorophyll concentration. Wavelengths in the region of the red edge often had the strongest correlation with chlorophyll concentration. For the remaining biochemicals the relationships between concentration and reflectance were weaker. Also, for these biochemicals absorption features with the larget correlation with reflectance values could not be readily related to the wavelengths of absorption features determined from other work using near infrared spectroscopy techniques on dry ground samples. Field studies at two sites used similar statistical techniques to relate reflectance spectra (measured for canopies using the AVIRIS) to the biochemical concentration and content of the foliage. Except for chlorophyll and nitrogen the results of both the concentration and content data showed a weak comparison with laboratory results. Difference spectra between control plot canopy samples and fertilized plot canopy samples revealed a large number of apparent absorption features which could be related to the known absorption features of foliar biochemicals.

This thesis documents studies directed towards the development of robust carbon dioxide sensors based on two different transducing principles: optical and potentiometric measurement. Also described is the development and implementation of novel high throughput synthesis and measurement systems designed specifically for application in the development of polymeric sensing films and electrolytes. The experimental methods employed and the development and optimisation of high-throughput synthesis and measurement systems for experimentation with optically and electronically responsive, thin-film, gas sensing electrolytes are described in Chapters 2 and 3. The adaptation of existing machinery, the design of measurement apparatus and the visualisation of data is shown. Chapters 4 and 5 describe the investigation into the notionally ‘ideal’ components of a thin-film, optical carbon, dioxide sensor incorporating monomeric quaternary ammonium bases. Using criteria such as chemical and physical stability, an optimally stable sensor formulation is found and fully characterised for its response to carbon dioxide and compared to a sensor described in literature. In Chapter 6, the fabrication of optical CO₂ sensors using plasticized polymeric quaternary bases is described. Sixty-two sensors were synthesised, characterised and screened using the high-throughput system described in Chapters 2 and 3. Chapter 7 describes the fabrication and complete characterisation of a novel, planar, solid-state, Severinghaus-type CO₂ sensor incorporating a polymer electrolyte gel similar to the polymeric sensor described in Chapters 4 and 5. Chapter 8 describes the design and fabrication of an array of such Severinghaus-type electrodes for application in the high-throughput screening of electrolytes for acid/base gas sensing. Also described is a preliminary measurement of a single substrate of sixty-four sensors.

The Archaebacteria are thought to be a separate domain of ancient micro organisms which have evolved under the harsh conditions of temperature, salinity and absence of oxygen. The membrane of Archaebacteria is made of lipids of unusual structure and many functions are completed by their membrane lipids. In this thesis, studies of the structures of the lipids of two species of Archaebacteria, <I>Halobacterium salinarium</I> and <I>Natronobacterium magadii </I>have been undertaken to provide a better understanding of Archaebacteria. Early chapters introduce the Archaebacterium family, discuss the classification of lipids and the nature of the lipids present in Archaebacteria. This is followed by a discussion of the principles of chromatography and mass spectrometry with emphasis on the techniques used in these studies. Later TLC/scanning densitometry is demonstrated as a viable method for the isolation of the three major lipids of <I>H. salinarium</I> and supercritical fluid extraction (SFE) has been shown to be an alternative extraction method for the isolation of the lipids. The remaining chapters discuss the use of GC/EIMS, GC/CIMS, LC/APCIMS and LC/ESMS for the identification of TLC fractions. The GC/MS techniques used with permethylated, deuteriopermethylated and trimethylsilylated derivatives of the dialkylglycerol ethers obtained from the lipid extracts by removal of the phosphate groups enabled these ethers to be characterised. The LC-MS studies enabled the lipids to be studied and LC-APCIMS proved to be equally effective in both positive and negative ionisation modes, whereas LC-ESMS was most effective in the negative ionisation mode. In the case of <I>H. salinarium</I> studies of permethylated derivatives provided complementary structural information. <I>H. salinarium</I> was shown to contain PGP-ME, S-DGD, PC and PGS with C<SUB>20</SUB>-C<SUB>20</SUB> diethers and the presence of up to triunsaturated species was shown. PGP-Me', PGP-Me", PG' and PG" were found as the major compounds in <I>N. magadii </I>with C<SUB>20</SUB>-C<SUB>20 </SUB>and C<SUB>20</SUB>-C<SUB>25</SUB> diethers. Minor lipids, diglucosylglycerol diethers were also found in this extract. Use of direct injection of TLC samples with ES/MS and ES/MS/MS confirmed the structures of the major lipids present.

The relatively new separation technique of capillary electrophoresis, with its ability to provide rapid, high resolution separations based on electrophoretic mobility differences, has developed enormously over the last decade. Although the technique was first interfaced to mass spectrometry within a few years of its conception, when the studies described here were undertaken there were few reports of the use of such combined systems. The objectives of these studies were to conduct further investigations into the interfacing of capillary electrophoresis with mass spectrometry via atmospheric-pressure ionisation sources. The system was then to be used for the analysis of some polar and ionic compounds of interest to the pharmaceutical and agrochemical industries. The fundamentals of capillary electrophoresis, mass spectrometry and combined systems are reviewed. Interfaces developed in these studies for capillary electrophoresis-mass spectrometry for a range of capillary electrophoresis instruments and atmospheric-pressure ionisation sources are described and detailed practical instructions for their use are given. The results obtained for the separation and detection of analytes in three sets of compounds; some herbicide analogues, some <I>β</I>-blocker drugs and some small peptides are presented in Chapter 3. Transient capillary isotachophoresis methods have been developed and optimised for all three sets of analytes, improving concentration detection limits by at least an order of magnitude. The use of small-diameter capillaries has also been shown to improve sensitivity. Separation of two structurally similar herbicide analogues has been effected by the addition of an optimised concentration of a cyclodextrin to the electrophoresis buffer, and use of cyclodextrins in capillary electrophoresis-electrospray ionisation systems has been studied. Use of deuterated buffers in the combined system has been shown to improve certain separations and, through mass spectra showing deuterium exchange, provide further structural information for analysis. Practical considerations for successful completion of such studies are described.

Medical diagnosis and treatment of critically ill patients require frequent monitoring of carbon dioxide partial pressure in the human body. Along with the traditional methods (i.e. IR capnography and Severinghaus electrode), colorimetric and fluorimetric CO<SUB>2</SUB> sensors are playing an increasingly important role in detection of correct intubation, intensive care and blood CO<SUB>2</SUB> analysis, due to its advantages of cheapness in cost, miniature in size, and mechanically robust. In Chapter One an outline of the recent development of such sensor systems is given, and their applications background in the biomedical area is discussed. Chapter Two focuses on the experimental techniques used in these studies. In Chapters Three and Four the equilibrium responses of three new colorimetric and one fluorescence plastic film sensors for CO<SUB>2</SUB> as a function of % CO<SUB>2</SUB> and temperature are described, respectively. The results fit a model in which there is a 1:1 equilibrium reaction between the deprotonated form of the dye (present in the film as an ion pair) and CO<SUB>2</SUB>. The basic theory behind conventional colorimetric and fluorimetric optical sensors for CO<SUB>2</SUB> is proposed and examined in Chapter Five. Special attention is given to the effect on sensor response of the key parameters of initial base concentration and dye acid dissociation constant, K<SUB>D</SUB>. In Chapter Six the diffusion-controlled response and recovery behaviour of a naked optical film sensor with a hyperbolic-type reponse changes in analyte concentration in a system under test is approximated using a numerical model, followed by a second part in which a systematic, experimental investigation on the kinetics, responses and recovery behaviour of the colorimetric film sensors is described. Finally, in Chapter Seven, a plasticised and unplasticised polymer colorimetric film sensor for gaseous CO<SUB>2</SUB> is tested as a sensor for dissolved CO<SUB>2</SUB>. The plasticised form of the film sensor develops a measurable degree of opacity when exposed to aqueous solution, while an unplasticised polymer remains largely clear upon exposure to aqueous solution and it is shown that it also functions as a quantitative sensor for dissolved CO<SUB>2</SUB> over the range 0-4% CO<SUB>2</SUB>.