Analysis of plant analytes using capillary electrophoresis and high performance liquid chromatography
Date of Award
Doctor of Philosophy
School of Natural Sciences
Faculty of Health, Engineering and Science
Associate Professor Mary C Boyce
Michael C Breadmore
Plants contain an enormous array of organic and inorganic components, the analysis for which may involve a wide range of methods. The focus of this study was to develop high performance liquid chromatography and capillary electrophoresis methods for the analysis of three classes of analytes: osmoregulants, minerals and amino acids.
Firstly, this study explored the potential of capillary electrophoresis for the analysis of three very common osmoregulants (proline, glycine betaine and mannitol). A diverse array of methods has been reported for determining each of these analytes, however, the literature on osmoregulants and their analysis is quite disjointed and traverses both biological and chemistry fields. Therefore, a comprehensive review of this literature has been completed (Chapter 2). Considerably fewer methods are available for the simultaneous determination of these osmoregulants, compared to individual analysis. In chapter 3, a method is described for the simultaneous analysis of proline and betaine by capillary electrophoresis at low pH and specifically various cationic probes for the indirect detection of proline and betaine were explored. Sulfanilamide was identified as a suitable probe and was employed to quantify proline and betaine in spinach and beetroot. However, this method could not detect mannitol as it is not charged at low pH.
In Chapter 4, a high performance liquid chromatography method for the simultaneous determination of all three osmoregulants is described. For separation, a NH2 column with formic acid and acetonitrile as the mobile phase were used. The high performance liquid chromatography evaporative light scattering detection method was applied to determine osmoregulants in Stylosanthes guianensis, Atriplex cinerea and Rhagodia baccata plant extracts. A complementary method, using a C18 column with heptafluorobutyric acid added to acetonitrile was used for verification of the analytes.
Secondly, the potential for using capillary electrophoresis was investigated to simplify and shorten the complex sample preparation procedure. Chapter 5 describes a capillary electrophoresis method that allows direct injection from plant tissues. The experiments highlighted that uncontrolled hydrodynamic injection of sample on piercing of food sample resulted in non-reproducibility. The addition of hydroxypropylmethlycellulose to the background reduced the uncontrolled hydrodynamic injection up to 95% for all of the analytes. The sample was injected electrokinetically and an imidazole buffer consisting of hydroxypropylmethlycellulose was used for separation. The issue of reducing the reliance on prior separation is also relevant to minerals, thus the developed capillary zone electrophoresis-UV method was applied for the direct injection of inorganic cations from apple, mushroom, zucchini, green bean and strawberries. The applicability of the method across fruit varieties was determined by analysing four apple varieties including red delicious, fuji, pink lady and royal gala.
Thirdly, the potential of the direct injection method was explored for the analysis of amino acids in zucchini. As amino acids are present at low concentrations and lack a chromophore, a more sensitive detector, capacitively coupled contactless conductivity, and pre-concentration of amino acids using isotachophoresis (leading electrolyte = HCl, terminating electrolyte = hydroxyproline) was performed. The separation of amino acids was carried using acetic acid. For minimising uncontrolled hydrodynamic injection poly(ethylene oxide) was used. Using this method sensitive detection of amino acids was possible (Chapter 6). In short, the developed methods allow for quick, inexpensive, sensitive and efficient analysis of plant components.
Kalsoom, U. (2015). Analysis of plant analytes using capillary electrophoresis and high performance liquid chromatography. https://ro.ecu.edu.au/theses/1690