Procedimientos adecuados del ordeño

time-of-flight (TOF) mass spectrometer. This spectrometer is considerably smaller in size (approx. 1 m x 1 m x 0.3 m) than the ESI instrument and significantly easier to operate. Samples are prepared as shown in the experimental section.

As for ESI, MALDI is a very recently developed technique and much is still not understood about the desorption process. MALDI was developed by Karas and Hillenkamp58'59 and Tanaka60 in the latter part of the 1980’s although the method of laser desorption was developed over ten years previously by Hillenkamp61. Until this time laser desorption was not considered to be a viable ionisation method for polymers owing to the intense energy of the laser pulse and the fragility of the polymer chains. This generally restricted the mass range to approximately 1000 mus62. During laser desorption, the energy from the pulse of a laser focused on a solid sample is absorbed by the analyte molecules which are ionised and vaporised. An important advantage stems from the pulsed nature of the experiment which allows the use of TOF mass spectrometers that can generate a complete mass spectrum for every ionisation event. However it is the sample preparation that is vital to the MALDI process and it was found that the use of an effective matrix

Chapter 1: Introduction

significantly extended the mass limit of the desorption technique. Thus instead of directly irradiating a neat sample, the analyte is deposited from a solution containing a large excess of matrix which absorbs at the wavelength of the incident laser light. After removal of the solvent a residue remains which comprises well separated analyte molecules homogeneously dispersed in a ‘sea’ of matrix molecules63. The energy of the laser pulse is absorbed by the matrix and a small proportion of the matrix vaporises, carrying intact analyte molecules into the gas phase. This part of the process appears to be understood, but it is the next stage involving transfer of a charge to the polymer chains which is not entirely understood. Karas64 proposes that a shallow surface layer of the matrix is vaporised either by electronic (UV) or vibrational (LR) excitation and forms a gas plume which expands, gaining momentum, into the vacuum against the underlying solid bulk. Multiple collisions in the dense gas plume enhance this effect, thus further increasing the momentum resulting in a jet-like emission with supersonic velocities. Analyte molecules are entrained into the plume by multiple collisions and charged by collisions with reactive photoionised matrix ions, resulting in electron or proton transfer processes (photochemical ionisation). Karas65 notes that while this proton transfer reaction is likely to occur for proteins and peptides dissolved in aqueous media, it is less likely for polymers dissolved in organic solvents. The lack of understanding of this ionisation process and the fact that most of the matrices used so far are soluble in aqueous solvents and used for water soluble biopolymers, has led to a relatively slow growth in the MALDI analysis of most synthetic polymers over recent years. Clearly polar or more ionic polymers such as PEGs and PPGs may be examined using well-known matrices such as 2,5-dihydroxybenzoic acid and sinapinic acid used for biopolymer analysis, but these matrices do not mix well with non-polar polymers such as PS and polybutadiene. However the recent development of matrices more amenable to the analysis of polymers

insoluble in water has led to a renewed interest in the analysis of synthetic polymers using MALDI. Many of these ‘new’ matrices are liquids such as 2-nitrophenyl octyl ether58,62,66 (NPOE), 1,2,4-butanetriol, 2-cyano-5-phenyl-2,4-pentadienoic acid67 and quinizarin68 which offer the advantage of providing a homogeneous sample environment, improving sample and thus spectral reproducibility. The intimate mixing of the matrix and analyte (polymer) has been shown to be an essential requirement for the MALDI process and some researchers have integrated microscopes into their MALDI instruments to ensure that efficient mixing has occurred prior to analysis69. We have similarly examined mixtures of PNMMO with various matrices prior to MALDI analysis using an Olympus high power microscope and our results are shown in the results section of this thesis. Other new matrices also include solids such as indole acrylic acid70. These ‘new’ matrices have led to well-resolved MALDI spectra of poly(methyl methacrylate) (PMMA)66,71, PS70, polybutadiene68 and poly(vinyl acetate)70. The need for the introduction of salts to promote ionisation of many of the less polar synthetic polymers poses an additional problem but, as for the introduction of many ‘new’ matrices, so has there been considerable research leading to the use of many ‘new’ salts such as silver, copper and cobalt salts70.

Clearly the correct choice of matrix and salt to promote ionisation is vital to the MALDI process. Similarly, the relative concentration of matrix versus polymer used to obtain MALDI spectra is also variable and many researchers report many different relative amounts of matrix and polymer. Consequently the application, and thus the correct choice of experimental conditions, of MALDI to a polymer such as PNMMO for which no previous MALDI studies have been performed is unfortunately still somewhat of a ‘trial and error’ process. However the significant advantages offered by the MALDI technique strongly

Chapter 1: Introduction

suggest that MALDI spectra of PNMMO would facilitate the understanding of the degradation process.

1.9 LIQUID COLUMN CHROMATOGRAPHY

Various types of chromatographic technique are applied to the analysis of both undegraded and degraded polymers72'73. Chromatographic methods are among the most widely used methods for the analysis of polymer composition and for the determination of the molecular weight distribution74 7S. SEC is one of most well utilised of these techniques and it provides quick data pertaining to the molecular weight distribution of the polymer. However the limitations of this technique with respect to its accuracy and the difficulties associated with calibration have been discussed in section 1.8. In addition to these difficulties some researchers76 also propose that the mechanism of separation employed by SEC may involve solute-solvent-packing interactions that are not strictly dependent on molecular size. However, many researchers have successfully used SEC to analyse thermally degraded polymer samples such as PMMA and poly(alpha-methyl styrene)77 and photolytically degraded samples such as PMMA and poly(methyl vinyl ketone)78. As a result of the apparent limitations of the SEC technique, over the past 2 or 3 years researchers have coupled techniques such as SEC and ESI76 to separate and to analyse mass spectrometrically the various oligomeric components of polymers. In this way, the problems associated with the calibration of the SEC instrument are overcome. This technique is very much still in its infancy, but it has been successfully applied to the separation of a polydisperse mixture of octylphenoxypoly(ethoxy)ethanol76.

Although both ESI and SEC were locally available to us separately, it was not possible to couple these techniques as both instruments were in frequent demand. It was also

impossible to perform preparative SEC experiments with the SEC instrument available to us, i.e. the polymer sample could not be recovered for further characterisation during SEC analysis. Thus in an attempt to simulate the coupling of the SEC and ESI techniques, we used column liquid chromatography employing a 1 m column filled with a silica slurry to separate both undegraded and degraded PNMMO and the resulting fractions were analysed using ESI. Clearly this method of chromatographic separation also allows the resultant fractions to be analysed using IR, NMR and SEC. This appears to be the first time that this classical method of chromatographic separation has been coupled with ESI and the results of these experiments are summarised in the results and discussion section of this thesis.