- Top of page
- Materials and Methods
- Results and Discussion
The elemental analysis of plants is an important tool for biologists in disciplines as diverse as ecology, physiology and agronomy. However, despite the routine application of digestion-based analytical techniques in many laboratories, the slow and often hazardous sample digestion process can create a bottleneck in the analysis of some elements, particularly where hundreds or even thousands of samples are to be analysed, as is the case for landscape-scale experiments in ecology or the rapid screening of new crop or biofuel varieties. Thus, the development of accurate and convenient high-throughput methods for assessing elemental concentrations in plants is of high importance. Here we describe a method for the rapid, safe and accurate elemental analysis of plant material using a portable X-ray fluorescence spectrometer (P-XRF). Although we concentrate on the measurement of phosphorus (P) and silicon (Si), both key elements for plant biologists and the latter notoriously difficult to analyse, P-XRF can potentially be applied to the simultaneous analysis of all elements from atomic number 12 (magnesium) up to atomic number 60 (neodymium).
Silicon typically constitutes between 0.1% and 5% of the dry weight of plants (Jones & Handreck, 1967). Despite being considered a nonessential element for the majority of higher plant species, Si can alter plant responses to a variety of environmental stresses, for instance by increasing drought and heavy metal tolerance (Neumann & zur Nieden, 2001; Hattori et al., 2005) or by acting as a defence against herbivores and fungal diseases (Fauteux et al., 2005; Massey & Hartley, 2006; Garbuzov et al., 2011). Soil Si application can boost crop health and yield, and its potential contribution to sustainable agriculture has recently been recognized (Datnoff et al., 2001). At the same time, an increasing global demand for biofuels requires the production of new plant varieties with low Si concentrations in their herbage, because Si particles that are dangerous to human health are emitted during the burning of the plant residuals (Blevins & Cauley, 2005), and Si forms sticky deposits on metal and refractory surfaces, thereby decreasing the burners’ performance (Miles et al., 1996). To date, advances in Si research are hindered by a lack of methods available for the economical, rapid, safe and accurate determination of Si in plant material.
In contrast to Si, the role of P in plant nutrition is, and has traditionally been, the focus of intense research. Phosphorus is an essential element for all life by being part of cell structural compounds such as nucleic acids and membranes, and by playing a key role in biochemical reactions such as photosynthesis and cell signalling. Soil P deficiencies frequently occur in both natural (Wardle et al., 2004) and agricultural (Cordell et al., 2009) systems, and investigations into plant P uptake mechanisms, for example by plant mutualistic mycorrhizal fungi, are of particular interest.
The most commonly applied methods to determine Si and P are based on alkaline fusion or acid digestion of the plant material (but see Masson et al., 2007), followed by spectrometric analyses of the obtained filtrate, using atomic absorption spectrometry (AAS; e.g. Hauptkorn et al., 2001), inductively coupled plasma spectrometry (ICP, e.g. Lopez Molinero et al., 1998), or colorimetric techniques (e.g. Fox et al., 1969; Allen, 1989). However, the accuracy of all these methods depends on the total destruction of the plant matrix, a process that can lead to element losses due to incomplete solubilization and, particularly in the case of Si, volatilization (Hoenig, 2001; Baffi et al., 2002). The accuracy of Si analysis by flame-AAS can be further decreased by matrix effects and oxide formation in the flame (Harris, 1991), whereas the performance of ICP can suffer from the dilution of the analyte with a large excess of the flux required for total dissolution of Si without volatilization (e.g. lithium metaborate) (Ramsey et al., 1995). Also, the digestion of the plant matrix usually requires the handling of dangerous chemicals, such as hydrofluoric, nitric, sulphuric and perchloric acid (e.g. Piper, 1942; Nayar et al., 1975; Haysom & Ostatek-Boczynski, 2006; but see Guntzer et al., 2010); digestion-based methods are not only hazardous but also very time-consuming considering the extensive weighing, heating, cooling and filtration steps involved. Furthermore, owing to the consumptive nature of all digestion-based techniques, the sample is inevitably lost during the analytical process, potentially a major problem in studies where only small amounts of test material are available and analyses of other aspects of plant chemical composition are required, or where researchers wish to re-analyse samples at a later date.
X-ray fluorescence spectrometry (XRF) provides a much faster, safer, nonconsumptive and potentially more accurate method to determine Si and P concentrations in plant material. XRF works on the principle of excitation of inner orbital electrons by an X-ray radiation source. As the excited electrons relax to the ground state, they fluoresce, thereby ejecting photons of energy and wavelength characteristic of the atoms present. Today, XRF instruments are widely used for the elemental analysis of building materials such as cement, glass or metals (Guerra, 1995; Lemberge et al., 2000), and their suitability for determining the elemental composition of plants, including Si, has been demonstrated in several studies (e.g. Evans, 1970; Gladney et al., 1989; Handson & Shelley, 1993; Guohui & Shouzhong, 1995; Richardson et al., 1995; Marguíet al., 2005; Queralt et al., 2005). However, despite several advantages of XRF over digestion-based techniques, such as its nonconsumptive nature and its often higher measurement accuracy, particularly in the case of Si (Ramsey et al., 1995), XRF has been largely confined to industrial applications and is not routinely used by biologists for the elemental analysis of plants. This might partly be due to the higher purchasing price of XRF instruments than that for equipment typically used in digestion-based elemental analysis techniques such as AAS or ICP. Furthermore, many XRF analysers require large quantities of plant material for analysis (typically between 1 and 10 g), limiting their use in studies where only small amounts of sample material are available.
Recently, the analytical power of portable X-ray fluorescence spectrometers (P-XRFs) has increased dramatically, and P-XRFs are now frequently applied in mining, soil exploration and in the analysis of consumer goods (Potts & West, 2008). The use of P-XRF instruments in plant analyses may provide important advantages over floor-standing or benchtop XRF instruments, including their much lower purchasing price, their very low running costs and their ability to analyse small amounts of plant material. Furthermore, these instruments are compact (the size of a small benchtop centrifuge) so can easily be moved and require very little laboratory or storage space. Also, P-XRFs constitute a valuable instrument for many laboratories by allowing in situ and in vitro measurements of, for instance, the distribution of nutrients or metals in soils (Argyraki et al., 1997). However, despite the ability of P-XRF to provide an economical and practical alternative to conventional XRF analysers, and to more time-consuming and potentially inaccurate digestion-based techniques, the suitability of P-XRF for the elemental analysis of plants has not yet been tested systematically, and a routine protocol for such measurements in plants has not been established.
Here, we describe a method for the rapid and accurate determination of two elements, Si and P, in plant material through the use of a P-XRF spectrometer. The method involves a quick, simple and inexpensive laboratory-based sample preparation procedure in which dried plant material is ground, pressed into pellets and analysed by exposing the pellets to X-rays for 30 s. The plant material does not need to be digested before analysis, making sample preparation fast, safe, convenient and cheap. Multiple elements can be determined simultaneously for the same sample, and the method is nondestructive so samples can be re-analysed at a later date.
We first established an empirical calibration for Si and P, then evaluated the analytical performance of the method through calculations of measurement bias, repeatability and intermediate precision (JCGM 200, 2012) using certified reference materials (CRMs) from different plant species, and one plant house reference material. We compared Si and P concentration data obtained by the analysis of plant material using P-XRF with those obtained by a digestion-based colorimetric technique. We tested empirically whether changes in sample mass are accompanied by changes in Si and P measurement intensity.