Rapid and accurate analyses of silicon and phosphorus in plants using a portable X-ray fluorescence spectrometer

Stefan Reidinger1*, Michael H. Ramsey2, Susan E. Hartley1

1Department of Biology, University of York, York, YO10 5DD, UK

2School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK

*Corresponding author:

Dr Stefan Reidinger

Telephone: +44 (0)1904 328590

Email:

Total word count for the main body of the text: 4270

Word count Introduction: 1351

Word count Materials and Methods: 985

Word count Results and Discussion: 1583

Number of Figures: 8

Number of Tables: 3

Summary

  • The elemental analysis of plant material is a frequently employed tool across biological disciplines, yet accurate, convenient and economical methods for the determination of some important elements are currently lacking. For instance, digestion-based techniques are often hazardous and time-consuming and, particularly in the case of silicon (Si), can suffer from low accuracy due to incomplete solubilisation and potential volatilization, whilst other methods may require large, expensive specialised equipment.
  • Here, we present a rapid, safe and accurate procedure for the simultaneous, non-consumptive analysis of Si and phosphorus (P) in as little as 0.1 g dried and ground plant material using a portable X-ray fluorescence spectrometer (P-XRF).
  • We used certified reference materials from different plant species to test the analytical performance of P-XRF and show that theanalysis suffers from very little bias and that the repeatability precision of the measurements is as good as or better than that of other methods.
  • Using this technique we were able to process and analyse 200 ground samples a day, so P-XRF could provide a particularly valuable tool for plant biologists requiring the simultaneous non-consumptive analysis of multiple elements, including those known to be difficult to measure such as Si, in large numbers of samples.

Keywords

Silicon, phosphorus, herbage, elemental analysis, portable X-ray fluorescence spectrometry (P-XRF)

Introduction

The elemental analysis of plants is an important tool for biologists in disciplines as diverse as ecology, physiology or 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 bottle-neck 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. Hence the development of new accurate and convenient high-throughput methods for assessing elemental concentrations in plants is of high importance. Here, we describe amethod for the rapid,safe and accurate elemental analysis of plant material using a portable X-ray fluorescence spectrometer (P-XRF). Although we concentrate here on the measurement of phosphorus (P) and silicon (Si), both key elements for plant biologists and 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).

Si typically constitutes between 0.1 and 5% of the dry weight of plants (Jones & Handreck, 1967). Despite being considered a non-essential 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 (NeumanzurNieden, 2001; Hattori etal., 2005) or by acting as a defence against herbivores and fungal diseases (Fauteuxetal., 2005; Massey & Hartley, 2006; Garbuzovet al., 2011). Soil Si application can boost crop health and yield, and its potential contribution to sustainable agriculture has recently been recognised (Datnoffet 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, since 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 etal., 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 etal., 2004) and agricultural (Cordell etal., 2009) systems, and investigations into plant P uptake mechanisms, e.g. 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, followed by spectrometric analyses of the obtained filtrate, using atomic absorption spectrometry (AAS; e.g. Hauptkornet al., 2001), inductively coupled plasma spectrometry (ICP, e.g. Lopez Molineroet 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 solubilisation and, particularly in the case of Si, volatilization (Hoenig, 2001; Baffietal., 2002). The accuracy of Si analysis by flame-AAS can be further decreased by matrix effects and oxide formation in the flame (Harris, 1998), whereas the performance of ICP can suffer from the dilution of the analytes 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 corrosive chemicals, such as hydrofluoric, nitric-, sulphuric- and perchloric acid, (e.g. Piper, 1942; Nayaretal., 1975; HaysomOstatek-Boczynski, 2006; but see Guntzeretal., 2010), and considering the extensive weighing, heating, cooling and filtration steps involved, digestion-based methods are not only hazardous but also very time consuming. Furthermore, due 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, non-consumptive 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 like cement, glass or metals (Guerra, 1995; Lembergeetal., 2000), and their suitability for determining the elemental composition of plants has been demonstrated in several studies(e.g. Evans, 1970; Gladneyet al., 1989; GuohuiShouzhong, 1995; Richardson etal., 1995; Marguíetal., 2003; Queraltetal., 2005). However, despite several advantages of XRF over digestion-based techniques, such as its non-consumptive nature and its often higher measurement accuracy, particular in the case of Si (Ramsey etal., 1995), XRFhas been largely confined to industrial applications and is not routinely used by biologists for the elemental analysis of plants.Thismightpartly be due tothe higher purchasing price ofXRF 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 1-10g), limiting their use in studies where only small amounts of sample material is 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 very compact (the size of a small benchtop centrifuge) so can easily be moved and require very little lab 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, nor has a routine protocol for such measurements in plants 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 seconds. The plant material does not need to be digested prior to analysis, making sample preparation fast, safe, convenient and cheap. Multiple elements can be determined simultaneously for the same sample, and the method is non-destructive 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, 2008) 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.

Materials and Methods

Empirical calibration

P-XRF instruments are usually equipped with a quantitative analysis software that uses the Fundamental Parameters Method for the analysis of the elemental composition of materials such as paint, soils or rocks (Potts & West, 2008). However, since no such software is commercially available for the quantitative measurement of elements in plant material, we established an empirical calibration function for Si and for P.

To test for the linearity of the Si calibration function we used synthetic methyl cellulose (Sigma-Aldrich, product number 274429) to simulate the plant matrix and precipitated silica powder (Fisher Scientific, product number S/0680/53) to spike the matrix with Si. We homogenized the spiked methyl cellulose powder by vigorous shaking and stirring to produce powders containing 0 (no silica added), 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% Si. In XRF analysis, samples composed of several elements, such as plants, yield multiple spectral lines that can interfere with each other (see below). However, initial tests showed that the fluorescence intensity emitted per unit Si did not differ between the simple matrix of the synthetic calibrators and the more complex matrices of the plant CRMs ‘Spinach’, ‘Tea’, ‘Bush Branches and Leaves’ and ‘Energy Grass’ (data not shown; Table 1a). Therefore, we established the empirical Si calibration by using synthetic calibrators only.

Synthetic P calibration material containing 0, 0.25, 0.5, 0.75 and 1% P was prepared by spiking methyl cellulose with sodium phosphate (Sigma-Aldrich, product number S5136). Whilst testing for the linearity of the P calibration function, it became apparent that these synthetic calibrators emitted a lower fluorescence intensity per unit P than the tested plant CRMs (Table 1b), and an inspection of the spectral lines showed interference between P and other elements present in the plant matrices, a common phenomenon in XRF analysis. We therefore used both synthetic and plant CRMs to establish a robust empirical P calibration, and accounted for elemental interference using standard procedures (see results).

Method validation

To determine the bias of the analytical method we used four different plant CRMs for Si and three plant CRMs for P (Table 1a). These CRMs were not previously used for establishing the empirical calibration and thus are independent test materials. We also included a house reference material (HRM) composed of a large homogenised sample of leaves of the grass Deschampsiacaespitosa (L.) Beauv.to quantify the repeatability of the method, its intermediate measurement precision, and the minimum amount of plant material required to obtain sufficiently accurate measurements.

Preparation of HRM material

We washed the D. caespitosa leaves under running tap water, then dried them in a fan assisted oven at 60° C for three days. Prior to grinding, the leaves were re-dried for 1 hour and roughly chopped using a conventional kitchen food processor. Grinding the leaf material for 90 seconds in a Pulverisette 23 ball mill (Fritsch GmbH, Germany) with a 5 ml stainless steel bowl and a 10mm stainless steel grinding ball at a rate of 50 beats sec-1 resulted in a fine and non-fibrous powder. Although we did not find any evidence in the present study thatSi and P measurement intensities changedwithincreasing grinding effort (data not shown), the emitted fluorescence intensity can be affected by the particle size of the powdered material, particularly in the case of Si which is mainly deposited close to the tissue surface. Increased grinding effort may reduce the size of particlesand hence theirsurface area,thereby reducing the emitted fluorescence (Evans, 1970). For reliable comparisons between contrasting plant samples, grinding time should be adjusted according to the toughness of the plant tissue to ensure particle sizes are similar.

Pellet preparation

Since X-ray fluorescence emitted from light elements such as Si and P is of low energy and has low penetrating power, the sample surface must be tight, flat and of equal density to obtain a repeatable photon flux from the sample to the XRF detector. We prepared the pellets without the addition of a binder since the powders showed good capacity to be compacted together. We pressed (if not otherwise stated) 0.7g of dried and ground material at 11 tonnes for 2 seconds using a manual hydraulic press (Specac, Orpington, UK) and a standard 13mm diameter die, resulting in a cylindrical pellet of around 5mm thickness. Pellets of any other size can be produced instead as long as their diameter exceeds 12mm. We used this procedure for both the synthetic calibration and plant materials.

P-XRF spectrometer system

We performed all analyses using a commercial P-XRF instrument (Niton XL3t900 GOLDD Analyzer, Thermo Scientific, UK). Instrument specifications and measurement conditions are shown in Table 2. Even though this analyser can be used as a hand-held instrument in the field, we used it in the laboratory in conjunction with a test stand (Thermo Scientific SmartStand),whichincreases the instrument’s performance when analysing light elements with low energy fluorescence such as Si and P. To avoid signal loss by air absorption, the instrument was connected to a (portable) gas cylinder containing low-grade helium, and all measurements were carried out in a helium atmosphere with a flow rate of 70 centilitres min-1. However, this is not essential and P-XRF analyses can also be conducted without helium, though this may increase the value of the detection limit of the method, particular for light elements such as silicon or phosphorus.

Si and P analysis using chemical digestion

To compare results obtained by P-XRF with those of a digestion-based technique, 5 plant samples (oneD. caespitosasample and two samples of Loliumperenne and Triticumaestivum) were analysed for silicon by fusing dried leaf samples (0.5g) in sodium hydroxide followed by analysis using the colorimetric silicomolybdate technique (Allen, 1989). Phosphorus analyses were carried out after triple digestion of 0.25g material from 3 plant CRMs (‘Coast Grass’, ‘Alpine Grass Mixture’ and ‘Rosa’; Table 1a), using the molybdenum blue method (Allen, 1989).

Results and Discussion

Empirical calibration

The linearity of the Si calibration function was confirmed by measuring the signal intensity in kilo counts per second (kcps) for two replicated methyl cellulose pellets containing 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% Si for 90 seconds each (Fig. 1a). We then established an empirical Si calibration using two types of calibration materials, one containing 0% Si and one spiked to contain 10% Si. This calibration strategy is optimal for analytical systems where it can be assumed that the calibration function is linear (Thompson, 2009). We measured the number of kcps for 10 spiked and 10 un-spiked calibration pellets and applied a linear regression to the data set

Whilst testing for the linearity of the P calibration function, it became apparent that the plant CRMs emitted higher fluorescence intensities per unit P than the synthetic calibrators (Fig. 1b), a phenomenon caused by spectral interference between the elements P, sulphur (S) and potassium (K) present in calibration CRMs, and silver (Ag) back-scatter from the instrument. To account for this interference, we established an empirical calibration for P by simultaneously measuring the fluorescence intensity in kcps for these 4 elements, for 5 replicated pellets of each CRM (Table 1b) and synthetic calibrator, and applied a linear regression model to the data using the LINEST function in MS excel to model the P fluorescence intensity In this equation, P, Ag, S and K in parenthesis stand for the kcps values of the according elements.

Next, we uploaded the Si and the P equation of the best fit line onto the P-XRF instrument, enabling the simultaneous analysis of both elements. Empirical calibrations for elements other than Si and P can be established and uploaded, allowing the user to measure a wide array of elements in a single plant sample within seconds.

Sensitivity and detection limit

The sensitivity of the instrument (i.e. net fluorescence intensity obtained per unit of analyte concentration), as calculated by the slope coefficient of the calibration graph was around 6 kcps per 1% Si, and around 5 kcps per 1% P. The detection limit was estimated as 0.014% for Si and 0.013% P, using three times the standard deviation of the percentage Si and P measured over a 10 minutes period for 15 different unspiked synthetic calibration pellets or 15 pellets of the CRM ‘Bush Branches and Leaves’ (Table 1b), respectively.