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
1
Department of Biology, University of York, York, YO10 5DD, UK
2
School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK
*Corresponding author:
Dr Stefan Reidinger
Telephone: +44 (0)1904 328590
Email: stefan.reidinger@york.ac.uk
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
1
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 the analysis 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.
2
Keywords
Silicon, phosphorus, herbage, elemental analysis, portable X-ray fluorescence
spectrometry (P-XRF)
3
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 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 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 (Neuman & 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
4
contribution to sustainable agriculture has recently been recognised (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, 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 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, 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.
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 solubilisation
and, particularly in the case of Si, volatilization (Hoenig, 2001; Baffi et al., 2002).
5
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; Nayar et al., 1975; Haysom & Ostatek-Boczynski, 2006; but see Guntzer
et al., 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, 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 like cement, glass or metals (Guerra, 1995;
Lemberge et al., 2000), and their suitability for determining the elemental
composition of plants has been demonstrated in several studies (e.g. Evans, 1970;
6
Gladney et al., 1989; Guohui & Shouzhong, 1995; Richardson et al., 1995; Marguí et
al., 2003; Queralt et al., 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 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 1-10g),
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 PXRF 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
7
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
8
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
9
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 Deschampsia caespitosa (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 redried 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 that Si and P measurement intensities changed
with increasing grinding effort (data not shown), the emitted fluorescence intensity
can be affected by the particle size of the powdered material, particularly in the case
10
of Si which is mainly deposited close to the tissue surface. Increased grinding effort
may reduce the size of particles and hence their surface 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
11
a test stand (Thermo Scientific SmartStand), which increases 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 (one D. caespitosa sample and two samples of Lolium
perenne and Triticum aestivum) 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
12
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 [% Si = (5.17 × kcps) + 0.91].
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 [% P = 8.25 ×
P
S
K
(Ag) − 0.53 × (Ag) + 0.14 × (Ag) − 0.14]. 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.
13
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.
Bias
To estimate the translational bias (i.e. constant over the whole analyte
concentration range) and rotational bias (i.e. proportional to analyte concentration) of
the method, we measured 10 pellets of each of the validation CRM materials (Table
1a) and fitted a linear functional relationship between the measured Si or P values and
the certified values. Linear regression requires the predictor variable to be measured
without error, but uncertainties on CRM values can be large, consequently violating
this assumption. Therefore, a functional relationship estimation by maximum
likelihood (FREML) analysis was applied to each data set, which provides estimates
of the intercept and slope of the line and plus their standard errors that do not suffer
from the biases introduced by the inappropriate use of regression (Ripley &
Thompson, 1987).
14
For Si, we found a good relationship with a non-significant rotational
(proportional) bias and a small significant translational (constant) bias of 0.082% m/m
(Fig. 2a), suggesting that readings for Si are slightly high, by 0.08% m/m above the
certified value of the CRMs. This translational bias may be caused by very low
concentrations of Si present as an impurity in the methyl cellulose. The value could be
subtracted from the Si concentrations measured in the samples to eliminate the bias
and improve the accuracy, if required. Poor agreement was obtained between Si
values from the chemical and those from the P-XRF analyses (Fig. 2b). Since we have
demonstrated above that the P-XRF technique suffers from very little bias, we are
confident that this lack of agreement arises from the low accuracy of the chemical
digestion technique, possibly due to an incomplete destruction of the plant matrix or
Si volatilization during the extraction process. This suggests that the use of P-XRF for
the determination of Si in plant material is not only faster and safer than conventional
digestion-based techniques, but also superior in terms of measurement accuracy.
We did not detect any rotational or translational bias for P (Fig. 3a), indicating
that P-XRF can provide highly accurate measurements for this element. Phosphorus
concentration data from the P-XRF analysis were closely correlated with those of the
digestion-based analysis (Fig. 3b), suggesting that both methods can provide high
accuracy. Nevertheless, P analysis using P-XRF is superior to conventional digestionbased methods in terms of safety and time expenditure, and does not lead to a loss of
the sample material during the measurement process, allowing samples to be reanalysed if required. Furthermore, P-XRF potentially allows the simultaneous analysis
of all elements between magnesium (atomic number 12) and neodymium (atomic
number 60) within seconds, a considerable time saving over using separate digestionbased methods for different elements.
15
Repeatability and precision
To evaluate the repeatability precision of the method, we prepared 10 pellets
of the HRM material (D. caespitosa leaves; mean Si = 1.03% m/m, mean P = 0.18%
m/m), measured each pellet once under identical experimental conditions and
calculated the relative standard deviation (RSD). The RSD was only 2.45% for Si and
3.69% for P, which is very low considering that these values include variation due to
sample homogenization as well as variation in measurement and instrument
performance. The repeatability caused by counting and instrument statistics alone was
0.63% for Si and 2.6% for P, as estimated by measuring one HRM pellet 10 times and
calculating the RSD.
The intermediate measurement precision of the method over time was
evaluated for Si by re-analysing one of the CRM materials (‘Bush Branches and
Leaves’; Table 1a) 10 times over a period of 3 months. Fresh pellets were made every
month because they start to deform some months after being pressed and stored in
sealed plastic bags, resulting in an uneven pellet surface that may influence the
measured Si concentration, though we did not find any evidence for this (data not
shown). The calculated RSD of CRM (which also includes the uncertainty due to
sample preparation) was only 2.04% for Si, which is in the range of the repeatability
of the method (see above), demonstrating that the measurements can be reliably
reproduced over time.
Sample mass
16
X-ray analyses are usually performed on samples that are ‘infinitely thick’, i.e.
the fluorescence emitted near the top of the sample pellet (the pellet surface not facing
the detector) is completely absorbed by the sample itself and does not influence the
measurements. However, since the amount of sample available for analysis is often
limited in biological investigations, we empirically evaluated the minimum amount
required to obtain sufficiently accurate Si and P measurements. From the HRM
material we prepared 10 different types of pellets, differing in mass and thickness: 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1g, resulting in a pellet thickness of
approximately 0.7mm per 0.1g plant powder. We did not press pellets weighing less
than 0.1g since these pellets broke easily during handling. We analysed Si and P
concentrations in four pellets of each type and plotted the concentration residuals
against pellet mass to confirm that the residuals show no sign of curvature (Fig. 4a, b).
Regression analyses showed that the measured Si and P intensity did not change with
pellet thickness (silicon: R2=0.06, p=0.134; phosphorus; R2=0.00, p=0.864),
demonstrating that the smallest amount of ground plant material required to be
pressed into a stable pellet (~0.1g) is sufficient to obtain reliable Si and P
measurements using P-XRF. Though the penetration power of secondary X-rays
typically increases with the atomic number of the element in question, other studies
have shown that even for very heavy elements such as lead, P-XRF fluorescence is
only emitted from a maximum sample depth of 0.15cm (Argyraki et al., 1997). This
lack of detectable effects of pellet thickness on the measurements demonstrates the
suitability of P-XRF to study variations in the elemental composition of plants on a
relatively small scale, such as analysing the effects of leaf age or position of leaves
within a plant on Si and P concentrations. Also, time consuming weighing of the
ground plant material prior to pressing it into pellets is unnecessary. In comparison,
17
analyses of P and Si with conventional, digestion-based techniques require
approximately 0.25g sample for P and 0.5g sample for Si (Allen, 1989), but due to the
destructive nature of these techniques the sample is inevitably lost and no further
analyses can be carried out, nor can samples be re-analysed at a later date.
Sample processing and analysis time
We recorded the time required to process and analyse samples starting with
ground plant material. Including the time spent cleaning the hydraulic press die
between samples and labelling the sample bags, we were able to press around 40
samples into pellets within 1 hour. The analyses of these pellets using P-XRF took
approximately 25 minutes, including sample labelling and changing samples between
measurements. This shows that it is feasible to analyse elemental concentrations in up
to 200 plant samples a day using this method, which is considerably faster than using
conventional methods based on the time consuming chemical digestion of the plant
material (as usually around 50 samples per day for one element alone).
Conclusions
We conclude that the use of P-XRF to analyse the elemental composition of
plants is superior to digestion-based techniques for several reasons. First, the plant
material does not need to be digested prior to analysis, thereby avoiding the time
consuming handling of expensive and hazardous chemicals. Second, XRF analyses
provide measurement accuracies (both in terms of method bias and precision) that are
rarely achieved by other digestion-based techniques such as AAS or ICP. Third, XRF
18
analyses are non-consumptive and the sample can be de-aggregated, re-pressed and
re-analysed at any time, and subsequently the same samples can be re-used for the
analysis of other aspects of plant chemical composition. The fact that the sample can
be reused enables the possibility of collecting smaller sample volumes, which has
obvious advantages in minimising the time and expense of collecting, storing and
processing plant material.
Further, the use of P-XRF instruments in plant analyses provides several
advantages over conventional XRF analysers. First, the purchasing price of P-XRF is
much lower than that for conventional analysers, and apart from very small amounts
of helium used during the measurement process the analyses are cost free. Second, PXRF instruments are able to analyse smaller amounts of plant material, a prerequisite
for many studies where the amount of sample material is limited. Third, P-XRF
instruments are very compact and easy to store, and they are a particularly valuable
and versatile instrument for many laboratories because they can be used for the
elemental analysis of soils, both in-situ and in-vitro, as well as plants.
Thus, P-XRF clearly has the potential to be more accurate and convenient
than digestion-based analytical techniques, particularly for difficult to analyse
elements such as Si, and may also provide a much more economical and practical
alternative to conventional XRF analysers, thereby providing a significant advance for
biologists requiring safe, rapid and accurate elemental analysis in plant ecology,
agronomy and other areas of plant biology.
19
Acknowledgments
This study was funded by a grant from the Natural Environment Research Council to
S.E.H (NE/F003137/1). We are grateful to Pietro Caria and John Hurley from Niton
for their support.
20
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Figure 1: Graph of (a) silicon concentrations in silicon-spiked methyl cellulose
calibrators and (b) phosphorus concentrations in plant (CRM) and spiked cellulose
calibrators, against the measured silicon and phosphorus fluorescence intensity in kilo
counts per second (kcps), respectively. For phosphorus (b), the slopes of the
regression lines differed significantly (analysis of covariance, P < 0.0001) between
CRM (6.69) and synthetic calibrators (5.07), indicating spectral interference between
P and other elements in the plant CRM calibrators.
Figure 2: (a) Estimation of the bias of the proposed analytical method for the
determination of silicon by comparison between measured (10 pellets per certified
reference material) and certified concentration values of reference materials using a
functional relationship estimation by maximum likelihood (FREML). Error bars show
95 % confidence interval of each mean. Percentage silicon measured = 0.082 +
0.932 × percentage silicon certified. (b) Relationship between silicon
concentrations of 5 grass samples measured by P-XRF and a traditional colorimetric
technique (1 measurement per sample/technique), showing poor agreement between
percentage silicon values obtained from P-XRF and colorimetric measurements
(Pearson correlation, r = 0.644, n = 5, p = 0.644).
Figure 3: (a) Estimation of the bias of the proposed analytical method for the
determination of phosphorus by comparison between measured (10 pellets per
certified reference material) and certified concentration values using a functional
relationship estimation by maximum likelihood (FREML). Error bars show 95 %
confidence interval of each mean.Percentage phosphorus measured = 0.020 +
26
1.136 × percentage phosphorus certified. (b) Relationship between phosphorus
concentrations of certified reference material ‘Rosa’, ‘Coast Grass’ and ‘Alpine Grass
Mixture’ measured by P-XRF and a traditional colorimetric technique (3
measurements per sample/technique). A good agreement was achieved between
percentage P measurements taken by P-XRF colorimetric analyses (Pearson
correlation, r = 0.979, n = 9, p < 0.001)
Figure 4: Plot of residuals for (a) silicon and (b) phosphorus versus sample mass.
27
Table 1a: Mean phosphorus or silicon concentration values (± 1 standard deviation) of
certified reference materials used for method validation, and their supplier.
Reference
material
Percentage phosphorus Percentage silicon
± 1 Std. Dev.
± 1 Std. Dev.
Supplier
NCS ZC73013
‘Spinach’
0.212 ± 0.024 %
China National
Analysis Center for
Iron & Steel
NCS ZC73014
‘Tea’
0.099 ± 0.008 %
China National
Analysis Center for
Iron & Steel
NCS DC73349
‘Bush Branches
and Leaves’
0.60 ± 0.07 %
China National
Analysis Center for
Iron & Steel
NJV 94-4
‘Energy Grass’
2.1 ± 0.24 %
Swedish University
of Agricultural
Sciences
IPE 101
‘Coastal Grass’
0.303 ± 0.0176 %
Wageningen
Evaluating Programs
for Analytical
Laboratories
IPE 106
‘Alpine Grass
Mixture’
0.396 ± 0.0201 %
Wageningen
Evaluating Programs
for Analytical
Laboratories
IPE 114
‘Rosa’
0.188 ± 0.0092 %
Wageningen
Evaluating Programs
for Analytical
Laboratories
28
Table 1b: Mean phosphorus concentration values (± 1 standard deviation) of certified
reference materials used for establishing the empirical P calibration, and their
supplier.
Reference material
Percentage phosphorus
± 1 Std. Dev.
Supplier
NCS ZC73013
‘Spinach’
0.32 ± 0.02%
China National Analysis Center
for Iron & Steel
NCS ZC73014
‘Tea’
0.43 ± 0.03%
China National Analysis Center
for Iron & Steel
NCS DC73349
‘Bush Branches and Leaves’
0.10 ± 0.004%
China National Analysis Center
for Iron & Steel
IPE 108
‘Parsley’
0.383 ± 0.0180%
Wageningen Evaluating Programs
for Analytical Laboratories
IPE 638
‘Maize’
0.200 ± 0.0117%
Wageningen Evaluating Programs
for Analytical Laboratories
IPE 682
‘Wheat’
0.0855 ± 0.0088%
Wageningen Evaluating Programs
for Analytical Laboratories
IPE 776
‘Lettuce’
0.611 ± 0.0451%
Wageningen Evaluating Programs
for Analytical Laboratories
IPE 977
‘Angelica’
0.829 ± 0.0544%
Wageningen Evaluating Programs
for Analytical Laboratories
29
Table 2: P-XRF instrument specifications and measurement conditions used.
P-XRF Unit
Niton XL3t900 GOLDD
Calibration Method
Empirical
Target X-Ray Tube
Ag
Detector
Silicon Drift Detector
X-Ray Tube Voltage
Si
6.2 kV
P
6.2 kV
Tube Current
100 µA
X-Ray Spot Diameter
8 mm
Primary Filter
Si
OFF
P
OFF
Si
Ka 1.740 keV; Kb 1.838 keV
P
Ka 2.015 keV; Kb 2.142 keV
Element Lines
30