Preconcentration of trace elements in biological materials by dry ashing for TTPIXE analysis A study of matrix effects

Nuclear Instruments

and Methods in Physics Research B 103 (1995) 466-472

Beam Interactions with Materials a Atoms

ELSEVIER

Preconcentration

of trace elements in biological materials by dry ashing for TTPIXE analysis A study of matrix effects

K-E. Saarela a, J-O. Lill b, F.J. Hernberg b, L. Harju a, A. Lindroos ‘, S-J. Heselius b,* ” Laboratory

ofAnalyticalChemistry.

h Accelerator

’

Department

Laboratory.

A’bo Akadcmi

A-bo Akademi

of Geology and Mineralogy.

Received 9 February

UniL,ersi?y, FIN-20500

University,

FIN-20500

A’bo Akadrmi Unicersip

Turku, Finland

Turku. Finlatld

FIN-20500

Turku, Finland

1995; revised form received 23 May 1995

Abstract Trace elemenrs in biological materials were preconcentrated by dry ashing at 550°C in order to improve the detection limits for thick-target PIXE analyses. The analytical procedure was calibrated with equally prepared ashes of certified biological standard reference materials (SRMS). The matrix effects were studied both theoretically and experimentally. A drawback of the ashing procedure is that volatile elements such as halogenides and sulfur can be partly lost depending on the composition of the material studied. Thick-target PIXE combined with dry ashing is a sensitive and reliable technique for thr determination of elements with atomic number > 20 in biological materials with a low ash content.

1. Introduction A method for direct quantitative analyses of biological materials by thick-target PIXE (TTPIXE) has been reported earlier by the present group [I]. The biological standard materials were pressed into pellets, consisting of pure graphite powder with the sample material on the surface [1,2]. The samples were irradiated in air. The integrated charge needed for quantification was determined by utilizing the light emission in air during irradiations. The precision of the method was good. However, the natural concentrations for some elements of interest in biological materials are close to the detection limits for direct analysis with TTPIXE. Therefore, dry ashing was evaluated in this work as a method for preconcentration of trace elements in biological materials. The chemistry of the dry ashing of organic matter is very complex. Good reviews on this topic have been given, for instance, by Bock [3] and Gorsuch [4,5]. The loss of mass during dry ashing can be mainly attributed to the removal of C but also N and H from the sample. The residue (ash) consists of inorganic matter which has bound oxygen during the ashing procedure. During ashing without additives, mainly halogenides, sulfur, selenium, mercury and arsenic have been reported to be lost [3,5].

* Corresponding author. Tel. + 358 2 I 2654 608, fax + 358 2 1 2654 912. e-mail [email protected].

Phosphorus, a main component especially in animal tissue. is expected to be retained in the ashes [3]. Low-temperature and wet ashing have been frequently used for sample preparation in thin-target PIXE analyses of biological materials [6,7]. Pallon and Malmqvist [8] have demonstrated low-temperature ashing as a useful technique for the preparation of biological samples to thick pellets for PIXE analyses. Dry ashing at high temperatures has been successfully used for preparations of tree rings to thin targets [9]. The use of this technique for the preparation of thick targets for PIXE analyses has not been found in the literature. Analyzing the ashes as thick targets has certain advantages compared with analytical methods involving dissolution of the ashes. A dissolution always means a dilution of the sample, including risks of contamination from the reagents used. It is also well documented that the use of acids does not guarantee a complete dissolution of the ash [3,5]. Incomplete dissolution giving two phases is problematic, especially in wet chemical analysis. In addition, the dissolution is also very time-consuming. Biological materials can be divided into groups according to their origin. The ashes of these materials have relatively similar compositions within the respective groups (Table la and Table lb). The matrix effects in TTPIXE analyses of ashes of samples within one group are thus expected to be small and predictable. This motivates the evaluation of dry ashing as: (a) a method for preconcentra-

0168-583X/95/509.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0168-583X(95)00643-5

K.-E. Saarela et al./Nucl.

Instr. and Meth. in Phys. Res. B IO3 (1995) 466-472

467

tion of biological samples, and (b) a way of preparing calibration standards from certified biological standard reference materials (SRMS) for the direct analyses of the ashed samples.

2. Experimental 2.1. Irradiation

conditions

The Abe Akademi 103 cm AVF cyclotron was used to produce a beam of 3 MeV protons incident on the target sample situated outside the cyclotron-vacuum system. A 7.5 pm Kapton foil was used as particle-beam exit window separating the cyclotron vacuum from the laboratory atmosphere. The beam was collimated to a diameter of 1 mm. The integrated charge on the target was measured indirectly utilizing measurement of the light emission induced in air by the particle beam [I]. The light is mainly due to molecular band transitions of N,. The light emission was measured with a PM tube. The integrated charge is needed in the normalization of the measured X-ray yields for the quantification of the results. An intrinsic germanium planar (IGP) detector with a 25 pm thick beryllium window was used in the measurements

[I]. The FWHM keV.

The

facing

samples

the particle

of the IGP were beam

detector

mounted and

was

with

the X-rays

Fig. 1. Schematic drawing of experimental setup for external beam TTPIXE analysis. The target sample faces the particle beam and the X-rays detected by the intrinsic germanium planar (IGP) detector at angles of 45”. The sample can be moved both horizontally and vertically with the help of a remotely controlled scanning device. The video camera monitors the target sample, and the PM tube is used for measuring the light emission in air during irradiation.

165 eV at 5.9

the planar detected

surface

with

by the

with a drilled hole of 0.5 mm diameter was used throughout the experiments.

detector at angles of 45” (Fig. 1). A scanning device uas developed enabling the remote control of the sample in vertical and horizontal directions without changing the irradiation/measuring geometry. The target sample was monitored with a video camera during the irradiations. The samples were irradiated for a total time of 10 min I(;P

Ash [%I

Vegetable Foods (mean) Leafy Vegetables Fruity Vegetables Roots Tomato Leaves (SRM) Pine Needles (SRM)

10.7 14.9 9.9 7.5 19.42 2.52

Matrix composition

Ash [%I

” Assumed

value.

10 5.6 8.3 4.32

every

data [12] and ashed biological

standard

Al

P

s

K

Ca

2.0 2.0 2.4 2.0 3.6

_

3.9 3.5 5.4 4.8 1.8

3.2 3.7 2.5 3.5 (2)

33 32 40 45 23

4.7

(1)

14.7

4.1 5.7 3.1 2.7 15.4 16.3

3.6 a

_ 2.2

Matrix composition

2.1 1.2

1.5 1.o

P 17 22 22 25

2 min.

An X-ray

reference

materials.

[%,I

Mg

Mg Cow Beef Liver Kidney Bovine Liver (SRM)

spot

The following NIST (National Institute of Science and Technology, Gaithersburg, MA, USA) biological standard

(b) Matrix compositions of ashed meat, edible bowels derived from documented The concentration of sulfur for Bovine Liver was estimated from PIXE spectra Material

of the beam

of 3 mm thick polycarbonate

2.2. Dry ushing of biological materials

Table 1 (a) Matrix compositions of ashed vegetable foods derived from documented Concentrations within parentheses are estimated from PIXE spectra Material

changing

filter

data [12] and ash of Bovine Liver SRM.

[%I s

K

17 14 16 (0)

31 20 20 23

Mn

_ _ 2.6

reference materials were ashcd: Pine Needles (SRM 1575). Bovine Liver (SRM 1577b) and Tomato Leaves (SRM 1573). The materials (about 400 mg) were exactly weighed into small porcelain crucibles which were then placed in an electric oven and the temperature was slowly raised. From 200 to 350°C the temperature was increased at a rate of about l”C/min to ensure complete charring of the material. Finally, the temperature was raised to 550°C and the samples were kept overnight at this temperature. The crucibles were removed from the oven and stored in a desiccator for a new weighing and later sample preparations. In order to verify the precision and reliability of the ashing procedure replicate ashings of two of the SRMs were performed. For Tomato Leaves the obtained ash content (as % per dry weight) was 19.42 + 0.09 (n = 5) and for Pine Needles 2.52 2 0.05 01 = 5).

where the integral represents the matrix effects and the integration is performed from the incident proton energy to zero. The cross-section for inner-shell ionization of the element Z is represented by cr,(E). Different theoretical approaches can be used for calculating the cross-section for K- or L-shell ionization (crk,z( E) or (rL,[( E)) caused by the impact of protons. The matrix stopping power S(E) is calculated using Bragg’s rule for linear combination of the major component stopping powers. The quantity r,( El describes the transmission of photons from successive depths in the matrix. The calibration factor K, for a standard reference sample S2 cannot be used directly for the quantification of the measured X-ray yield for a sample Sl with different matrix. The difference in composition between the samples has to be corrected for by a correction factor (CF):

2.3. Sunrple preparurion

CF=+

Target pellets for TTPIXE analyses of both the untreated SRMs and the ashes of these materials were prepared using the “sandwich” technique previously described [I .2]. The pellets consisted mainly of graphite with the sample material pressed onto the front surface. The prepared pellets were stored in a desiccator until analysis. The ashes were thoroughly mixed before application to the graphite surface in the pelletizing device. The amounts of ash pressed onto the surfaces of the graphite pellets resulted in ash layers which analytically represented thick targets. The pellet thickness and thereby also the measuring geometry during the irradiation were controlled by using a constant amount of graphite powder. This technique enables analysis of small amounts of sample material, such as the ash of biological materials having low ash content.

where I,,, and /zsz represent the integral in Eq. (2) the element Z in sample Sl and the reference sample respectively. The calibration factor K,,, for the sample is thus derived from the calibration factor Kzsz for standard reference sample S2 used for calibration:

for S2. Sl the

K /SI = CFK,,,.

(4)

3. Results and discussion 3.1. Calibration

factor

K,

ratio (K,), specific A signal/concentration element Z, has been introduced earlier [I] as K,=-

for the

’

where Y(Z) is the measured yield for one X-ray line of the element Z, Qpb, is the integrated current of the PM tube and C, the concentration of the element Z. The ratio K,, used for calibration, depends on the sample matrix. Introducing a constant k, for the element Z, the calibration factor can be written as K,=k,

0 %Z(Vz(E) I

El.

(3)

The concentration of an element can be calculated from the equation I$,,=

Z in the sample Sl

YlZl ”

(5)

CFK,,, Qm,

In this work the proton-induced ionization cross-sections have been calculated by a computer program using the parameter form of Johansson and Johansson [IO]. The parameters for each element were determined by fitting the fifth-order polynomial to cross-section data for six proton energies [I I]. Parameterizations for the stopping power of the matrix elements and the attenuation of X-rays in the sample matrix were implemented in the program code as suggested by Johansson and Campbell [I I]. Integration from the incident proton energy to zero was performed using the formula of Simpson. 3.2. Prediction

qf matrix

e8ect.y for ashed biological

mate-

riuls

Y(Z) C,Qlw

I

S(E)

dE

’

In order to obtain an overview of possible matrix effects in the analyses of ashed biological materials the average compositions of main elements in the different groups of terrestrial plant materials and animal products are given in Table la and Table 1b. respectively. The values given in the tables are calculated from concentrations and ash contents reported by Koivistoinen et al. [ 121, and are based on a very large amount of statistical material. Calculated concentrations of corresponding elements for some ashed SRMs are also included in the tables. The

K.-E. Saarela et al./Nucl.

Instr. and Meth. in Phys. Res. B 103 (1995) 466-472

ash contents of these SRMs used in the calculations were determined in this work (Section 2.2). Only elements with concentration above 1 wt.% are included in the tables. In Fig. 2a the matrix effects for the ashes of the three groups of vegetables and two SRMs are presented with the help of the correction factors (CF) given by Eq. (3). The ash of Vegetable Foods was used as reference material. The calculations were based on the data given in Table la. In the calculations of the matrix effects the remaining mass, after subtraction of the masses for the main elements given in Table la, is assumed to be due to oxygen. The matrix effects were calculated using the parameterization described in Section 3.1. The geometry described in Secnon 2.1 (Fig. 1) and 3 MeV protons were used as input in all calculations. At higher X-ray energies the matrix effects are of the same order as or smaller than the relative error of the analytical procedure used in this work (about 10%). These effects are due to variations in the proton stopping power. In the lower energy region the matrix effects are mainly due to attenuation of the X-rays produced. For the materials studied the effects of attenuation and stopping power cancel each other at one energy (- I I keV). No matrix effects are expected to appear at this energy for the materials studied. For vegetables (Fig. 2a) the largest error due to matrix effects was predicted for calcium as the attenuation of calcium K X-rays depends on the potassium concentration of the sample. Potassium concentrations of 33% (Vegetable Foods) and 45% (Roots) will be the worst case and are expected to cause a negative error of up to 20% for the calcium concentration in roots, assuming a calibration of the analyses with an ash of Vegetable Foods. The ashes of Tomato Leaves and Pine Needles SRMs differ from the other ashes in Table la by having relatively low concentrations of potassium. This is reflected in the figure as a peak in the energy region where the K X-rays for calcium are found in the PIXE spectra. The X-rays from calcium are less attenuated in the ashes of Pine Needles and Tomato Leaves. Analogous calculations of matrix effects were also made for animal tissues (Fig. 2b). The main components in ashes of animal material are theoretically potassium, phosphorus, sulfur and magnesium. In Fig. 2b the calculated matrix effects for the ashes of Liver, Kidney and the ash of the biological SRM Bovine Liver are presented using Cow Beef ash as reference material. Fig. 2c presents the correction factors for an ash of Vegetable Foods (Table la) with ash of animal tissue (Liver, Table 1b) as reference material. The graph shows severe matrix effects at energies representing K X-ray lines for potassium and calcium. These pronounced matrix effects can be related to a difference of 18% units in phosphorus concentration. The difference in phosphorus concentration between ashes of vegetables and ashes of animal tissue is typically in the order of 20% units.

15

’

I\1

11 ::

1.4

I1 1

1.3

1

469

1

’

1

1

Roots

Futy Vegetables ---Lady Vegetables .. .. Tomato Leaves Pine Needles - -

1.2 11 1 09 0.6 2

4

12

14

16

!-ray Argy$"]

Liver Kidney ---Bovine Lwer SRM

...

2

4

!-ray e:ergy

10 [keV]

12

14

16

’ 14

16

2r,,r,,, Vegetable Foods

1.2 -

l-

0.6

'1 i.

1-I’ 2

’ 4

’ ’ I ’ 6 8 10 12 X-ray energy[keV]

1

Fig. 2. (a) Predicted matrix effects for plant ashes with compositions given in Table la. The graphs represent the correction factors given by Eq. (3). Ash of Vegetable Foods (mean) was used as reference material. (b) Predicted matrix effects for ashed animal materials with compositions given in Table lb. The graphs represent the correction factors given by Eq. (3). Ash of Cow Beef was used as reference material. (c) Predicted matrix effects for ash of Vegetable Foods (mean) with composition given in Table la. The graph represents the correction factors given by Eq. (3). Ash of Liver (Table I b) was used as reference material.

The concentration of sulfur is normally much higher in animal tissues than in vegetable materials. This is not necessarily true for ashes of these materials. No sulfur was

K.-E. Saarelu et al./Nucl.

470

2

4

Instr. and Meth. in Phys. Res. B 103 (1995) 466-472

6

Fig. 3. X-ray spectra of non-ashed

Energy [kevl

P K

Ca Mn Fe CU Zn Br Rb

2.01 3.31 3.59 4.01 5.89 6.40 7.06 8.04 8.63 9.51 11.90 13.37

with respect to the potassium

ia K,,-peak

amplitude.

3.3. Quantitutice analysis of ashed biological samples

in the ash of Bovine Liver, although fully retained the concentration would have been 18%. On the other hand, sulfur is retained to a high degree in the ash of Tomato Leaves (Fig. 3). Therefore, the predicted matrix effects between the plant material and animal product as illustrated in Fig. 2c are not completely realistic for the determination of the potassium content. In these calculations sulfur is assumed to be preconcentrated in the ash without any loss. In real-life analyses calculations of matrix effects are preferably based on sulfur concentrations estimated from the X-ray spectra. This is done for the SRMs in Fig. 2a and Fig. 2b. However, Fig. 2 gives a good prediction of the amplitude of matrix effects for elements in ashes of biological materials. The matrix effects for elements heavier than iron are negligible. detected

Element

16

energly0[ke"j

and ashed Tomato Leaves (SRM 1573) normalized

Table 2 Calibration factors (K,) and relative errors calculated Non-certified values within parentheses

14

12 Lay

Fig. 3 presents the X-ray spectra of non-ashed and ashed Tomato Leaves normalized with respect to the potassium K,-peak amplitude. These normalized curves give a qualitative overview of eventual losses of elements and also information about the background in the X-ray spectra. Corresponding normalized curves for Bovine Liver (SRM 1577b) showed that sulfur is completely lost during ashing up to 550°C. Calibration factors (K,) calculated using Eq. (I) are given for both non-ashed and ashed Bovine Liver samples in Table 2. Certified values and the relative errors for elemental concentrations are also included in the table. The relative errors for the K, values were calculated using the

for non-ashed

and ashed Bovine Liver (SRM 1577b). Ash content

Non-ashed

4.32 wt.%.

Ashed

Certified value [%0 wt.]

Relative error [Yo]

Calibration factor K,

Il.0 9.94 9.94 0.116 0.0105 0.184 0.184 0.160 0.127 0.127 (0.0097) 0.0 137

2.7 0.2 0.2 3.4 16.2 8.2 8.2 5.0 12.6 12.6

0.0068 0.183 0.0300

8.0

Relative

Calibration

Relative

error [%I

factor K,

error [%I

0.005 1 0.095 0.0171 0.015 0.25 0.48 0.163 1.60 1.84 0.42

4.0 2.5 2.8 20.5 18.1 8.5 8.6 5.6 12.9 12.9

7.6 1.8

2.6

_ 0.53 0.86 0.270 2.27 2.48 0.52 (1.65) I .52

26.8 8.5 9.0 5.5 12.8 13.2 9.9

1.08

8.7

K.-E. Saarela et al./Nucl.

Insrr. and Meth. in Phys. Rex. B 103 11995) 466-472

error propagation formula [ 131. The individual errors taken into account were the statistical error of the integrated current of the PM tube, the peak-fitting error @AMP0 90, MicroSAMP Version 3.1 [ 141). the errors for certified concentrations and the error due to uncertainties in the determination of the preconcentration factor. The ratios of the calibration factors for non-ashed to ashed samples are in the range 1.4- 1.8 for most elements (Table 2). The lighter biological matrix is more favourable for the measurable yield of X-rays. However, the decrease in sensitivity for the ashes is small compared with the preconcentration factor, e.g. 23.1 for Bovine Liver calculated from the ash content 4.32%. Table 3 demonstrates dry ashing as a method for preconcentration of biological materials and for preparation of standards for thick-target PIXE analyses. Tomato Leaves (SRM 1573) is in this case used as the sample and calibrated against two other biological SRMs. The results obtained after corrections for differences in bulk composition of sample/standard ashes (Eq. (5)) are also included in the table. The obtained results are at least satisfactory; the relative errors are generally below 20% for most elements for the whole analytical procedure. As can already be predicted from Figs. 2a-2c, the matrix effects for most elements are small compared with the total analytical errors. For these elements no matrix corrections are necessary (Table 3). For calcium the obtained results after matrix corrections are somewhat controversial. The clearest improvements in the results can be

411

obtained for potassium, especially when the calibration is made against Bovine Liver. This is due to the greater difference in the bulk compositions between the ashes of these two biological materials. The main difference between the ashes of animal tissue and vegetables is due to the concentration of phosphorus.

4. Conclusion In order to evaluate the reliability of the thick-target PIXE analyses of biological samples preconcentrated by dry ashing, the quality of the whole analytical procedure was controlled. The analytical procedure included the use of ashes of certified biological standard reference materials for calibration of the method. As the ash content of the biological materials studied by this group varied between 0.2 and 20%, preconcentration factors up to 500 were achieved. The detection limits will thus be 0.01-I ppm for most elements in biological materials after preconcentration with the dry-ashing technique. Thick-target PIXE combined with dry ashing is thus a sensitive method for the analysis of biological samples. The precision of the ashing procedure, expressed as relative standard deviation, was about 1% when relatively small amounts (300-500 mg) of material were ashed. The ashing of an organic sample does not have to be absolutely complete. The main point is that the preconcentration factor is known exactly for the calculation of the concen-

Table 3 Concentrations obtained for elements in ash of Tomato Leaves SRM using ashes of the biological SRMs Pine Needles (PN) and Bovine Liver (BL) as reference materials. The indexation in the concentrations derived CC,,,, CPV2, C,,, , and C,,,) refers to the reference material used. Index 1 represents values obtained through direct calibration, whereas index 2 represents results obtained with corrections for differences in ash composition (Eq. (5)). All concentrations are given in %‘OO by weight. Non-certified value within parentheses Energy

Certified concentration

Obtained concentration

keV1

bl

c Ph’l

CPN2

17.5 + 1.9 230 f 21 230 f 21 154+ 14

61 274 218 159

154+ 14 1.23 & 0.12 3.55 + 0.34 3.55 * 0.34 0.057 + 0.007 0.32 f 0.04 0.32 f 0.04 0.032 f 0.003 0.032 i 0.003 (0.13)

159 1.28 3.00 2.94 0.072 A

Br

4.0 1 5.89 6.40 7.06 8.04 8.63 9.57 10.55 12.60 11.90

Rb Sr

13.37 14.14

0.085 + 0.008 0.23 f 0.02

Element

P K Ca

Mn Fe Cu Zn Pb

2.01 3.31 3.59 3.69

* No certified values given or not detected. b No matrix correction performed for L lines in this work.

[%o]

CBLI

c BL2

57 249 200 187

39 328 299

48 219 206

0.027 0.023 0.91

186 1.37 3.17 2.94 0.073 .I i b b 0.92

201 1.31 2.89 2.93 0.060 0.37 0.40 d d II

165 1.44 3.15 3.15 0.064 0.39 0.42 b b d

0.123 0.248

0.123 0.245

0.113 .I

0.115 il

il

11

312

K.-E.

Saarela

et al./~Vucl.

Instr. and

trations of elements in the original biological sample. Ashes are easy to homogenize and represent statistically large amounts of the original sample material. Ash samples down to a few mg can be analyzed by the millibeam technique utilizing the scanning device and the videocamera monitoring system developed for locating the sample. The ashed sample is free from further losses of volatile elements during the irradiation, which ensures a good reproducibility of the analyses. The calculations of matrix effects for ashes of different types of plant materials and animal tissue indicated that the errors due to matrix effects are relatively small in large parts of the PIXE spectra. In the low-energy region of the spectra, matrix corrections may be necessary. The results obtained for calcium and potassium were strongly affected by differences in bulk composition between the sample and the standard used for calibration. A correction for errors due to matrix effects is found to be necessary if matching SRMs are not available. This study shows that dry ashing combined with thicktarget PIXE is a sensitive and reliable analytical technique for the determination of trace elements in biological samples (e.g., food, clinical and environmental samples). Good quantitative results can be guaranteed by the proper choice of a biological standard reference material to be ashed and used for the calibration.

Acknowledgements The financial support provided by the University Council of ,&bo Akademi and The Foundation of Abe Akademi is gratefully acknowledged.

Meth.

in Phys. Res. B 103 (1995)

466-472

References

[II J-O. Lill. K-E. Saarela. F.J. Hemberg, S-J. Heselius and L. Harju, Nucl. Instr. and Meth. B 83 (1993) 387. Saarela, J-O. Lill, S-J. Heselius. I. Kresanov and V. Ninth, Proc. 6th Symp. on Medical Application of Cyclotrons, Turku. Finland. 1992, eds. L-M. Voipio-Pulkki and

121K-E.

U. Wegelius.

Ann. Univ. Turkuensis D 88 (1992) A131. of Decomposition Methods in Analytical Chemistry (International Textbook Company, Edinburgh, 1979). [41 T.T. Gorsuch, The Destruction of Organic Matter (Pergamon, Oxford. 1979). bl T.T. Gorsuch. in: Accuracy Sampling, Sample Handling and Analysis, vol. 1. NBS Spec. Pub]. 422 (NBS, Washington, 1976) p. 491. [61 N.F. Mangelson. M.W. Hill, K.K. Nielson, D.J. Eatough. J.J. Christensen, R.M. Izatr and D.O. Richards, Anal. Chem. 51 (1979) 1187. PI G.S. Hall, N. Roach, U. Simmons, H. Cong, M-I. Lee and E. Cummings, J. Radioanal. Nucl. Chem. 82 (1984) 329. [81J. Pallon and K.G. Malmqvist, Nucl. Instr. and Meth. 181 (1981) 71. [91 G.S. Hall, Nucl. Instr. and Meth. B 49 (1990) 60. 1101 S.A.E. Johansson and T.B. Johansson, Nucl. Instr. and Meth. 137 (1976) 473. [III S.A.E. Johansson and J.L. Campbell, PIXE: A Novel Technique for Elemental Analysis (Wiley, New York, 1988). Acta Agric. Stand., Suppl. 22 (1980) 75, [I21 P. Koivistoinen, 1 IO. [I31 G.F. Knoll, Radiation Detection and Measurement (Wiley, New York, 1989) p. 88. [141 P.A. Aamio, J.T. Routti and J.V. Sandberg, J. Radioanal. Nucl. Chem. 124 (1988) 457. [31 R. Bock, A Handbook