Author Ma, Zhen
Title Analyses of phytohormones in tropical plants using liquid chromatogaphy
Institute Thesis (M.Sc.) National Institute of Education, Nanyang Technological University
Year 2009
Supervisor Tan, Swee Ngin ; Yong, Jean Wan Hong
Call no. QK898.H67 Ma
The overall aim of this thesis is to develop liquid chromatographic techniques for analyzing different classes of phytohormones in tropical plants. Today high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) are the key techniques in the field of biochemical analysis of complex mixtures due to the many advantages offered by these two methods. HPLC and LC-MS methods have been developed for the separation and identification of different classes of phytohormones in tropical plant materials, i.e. coconut water, sugar cane juice, and watermelon juice, etc. Such tropical fruit juices have been widely used in the tissue culture industry due to these media being rich sources of crude phytohormones.

Chapter 1 provides an introduction to HPLC and LC-MS techniques which are the most powerful techniques for the separation and analysis of chemical compounds from small molecules/ions to bio-macromolecules. Many articles and reviews published using HPLC and LC-MS over the last decade illustrating that these two techniques are now the most useful and widely applied tools in biochemical separation and analyses. Sample isolation and preparation, key to biochemical analyses, have been briefly introduced in this chapter.

Chapter 2 covers the theoretical aspects of HPLC and LC-MS. Several important parameters affecting separation, including capacity, efficiency, selectivity, and resolution, have been discussed. A variety of commercially available detectors for HPLC, several operation modes of HPLC, and the column used in HPLC, are also described. At the same time, some introductions of rapid resolution liquid chromatography (RRLC) are given in this chapter. Five distinct parts of MS, including the sample inlet, the ion source, the mass analyzer, the detector, and the data recorder are briefly introduced in this chapter. The introduction and application of a combination of liquid chromatography with mass spectrometry are also presented.

Chapter 3, the general characteristics of different classes of phytohormones are introduced, with a description about their chemical properties and their biological functions in plant growth and development. Brief introductions of different isolation and sample preparation methods, analytical techniques for phytohormones are presented. The discussion is not fully comprehensive but is intended to provide a brief description of those aspects of each technique.

Chapter 4, 5 and 6 are the results and discussion of this dissertation. These chapters describe the applications of HPLC and LC-MS for the analyses of different phytohormones and indole compounds in different tropical plant materials, such as coconut water, sugar cane juice and watermelon juice. The optimum HPLC separation and LC-MS identification conditions have been used for the analyses of different phytohormones and indole compounds in real samples. Some key findings of the phytohormones are as follows: indole-3-acetic acid (IAA, one of the most important auxins) and abscisic acid (ABA) have been detected in coconut water; IAA, 3-acetylindole, Indole-3-pyruvic acid (IPA) and indole-2-carboxylic acid have been found in sugar cane juice; and IAA and indole-3-butyric acid (IBA) have been found in the watermelon juice. Solid-phase extraction (SPE) with C18 columns has been used as an efficient method to pre-concentrate of different phytohormones and indole compounds. In addition, a new SPE method was developed for the isolation and pre-concentration of indole compounds in sugar cane juice successfully.

Chapter 7 presents a general conclusion for the successful application of HPLC and LC-MS for analyzing different classes of phtohormones and indole compounds in tropical plant materials. Further perspectives are also given in this chapter.