Microchim Acta (2007) DOI 10.

1007/s00604-007-0821-0 Printed in the Netherlands

Original Paper Estimation of uncertainty in electron probe microanalysis: iron determination in manuscripts, a case study
ino Sammelselg3 , Jelena Asari3 , Kristina Virro1 , Enn Mellikov2, Olga Volobujeva2 , Va 1 1 1 rgens , Ivo Leito Lilli Paama , Jaana Ju
1 2 3

Institute of Chemical Physics, University of Tartu, Tartu, Estonia Department of Materials Science, Tallinn Technical University, Tallinn, Estonia Institute of Physical Chemistry, University of Tartu, Tartu, Estonia

Received 16 November 2006; Accepted 31 May 2007; Published online 6 August 2007 # Springer-Verlag 2007

Abstract. A case study of ISO GUM-based estimation of measurement uncertainty of quantitative surface elemental analysis is presented. The analytical task was the measurement of iron content in the ink writing on the surface of an 18th century manuscript by electron probe microanalysis using a scanning electron microscope equipped with an energy-dispersive Xray spectrometer (SEM=EDS). The problems that arise in uncertainty estimation of quantitative surface analysis are outlined (dening the measurand, preparing calibration standards, non-uniformity of the surface, etc) and ways of overcoming them are suggested. The average iron content on the ink-covered surface of the sample manuscript was found to be 0.12 0.04 mg mm2 (at condence level 95.5%). Paper sheets with ink lines of known iron contents were used as calibration standards. The main source of uncertainty was the variability of the parallel measurement results from different locations of the sample surface (mostly due to the variations in the surface structure of paper),
Electronic Supplementary Material. The full uncertainty calculation le in MS Excel 2002 format is available as electronic supplementary material (ESM). This material is available online at http:==www.ut.ee=katsekoda=GUM_examples=EDS_ESM.xls. Correspondence: Ivo Leito, Institute of Chemical Physics, University of Tartu, Jakobi 2, 51014 Tartu, Estonia, e-mail: ivo. leito@ut.ee

which contributed 78% of the total uncertainty. It is concluded that EPMA using a SEM=EDS is suitable for at least semi-quantitative determination of iron in the writing of ink-written manuscripts. Several specic issues in uncertainty analysis are pointed out that need further investigation.
Keywords: Measurement uncertainty; scanning electron microscopy (SEM); electron probe microanalysis (EPMA); SEM=EDS; iron-gall ink; manuscript

Signicant progress in uncertainty estimation of chemical measurement results has been made in the recent years. In addition to conceptual works quite some practical case studies and examples are available. These are included as examples in guidance documents, such as the ISO GUM [1], the EURACHEM= CITAC uncertainty estimation guide [2] or the Nordtest handbook [3], in journal articles [49] and also on some measurement uncertainty related websites [10]. All these, however, relate only to the conventional macroscopic chemical analysis. Besides this, local and=or surface-analysis techniques are nowadays used more and more frequently. These measurements as any other have uncertainty. Due to the problems with dening the measurand, the (often high) non-uniformity of the surface and other problems, estimating

K. Virro et al.

uncertainty in surface analysis is not an easy task. Hence, not surprisingly, although there is no shortage of papers devoted to surface analysis of a great variety of objects, including quantitative analysis, the uncertainty of such measurements, especially for materials outside the alloy, mineral and semiconductor domain, has received only limited attention. The sources of uncertainty in such analysis have been studied, but far less is known about the evaluation of their magnitude [1115]. Works where the different uncertainty sources are quantied and combined according to a measurement model to yield the combined uncertainty of the result are to the best of our knowledge completely absent from the literature. Such approach would be valuable, as it would enable to analyze the contributions of different uncertainty sources and to point out ways to improve the measurement procedure. Among the objects that are analyzed using the surface analysis methods, old manuscripts have an important place. Manuscripts and drawings made with the iron-gall ink (active components are tannic acid and iron(II)sulfate) form a major part of the European cultural heritage. Iron-gall ink was the most common ink used before the 19th century [16]. Unfortunately, all such manuscripts are threatened by the so-called ink corrosion deterioration of paper by ink components like acids and ions of metals (e.g. Fe, Cu) [16, 17]. The paper looses its mechanical strength and the unique artifacts are damaged forever. This has become a serious problem in the eld of cultural heritage preservation. The problem has been recognized and large collaborative efforts have been undertaken to nd ways of stopping manuscript degradation. See the InkCor project homepage [17] and the Ink Corrosion Website [16] for a good overview of the state of the art in this eld. The mechanisms of the processes are still under investigation [16, 17]. Various parameters of the ink and the paper have their inuence on the corrosion and on the possible means of retarding and stopping it. An important factor among them is the iron content in the ink writing on the paper surface. Exact correlations have not been published, because there is not enough iron content data in manuscripts available. Therefore development of quantitative analysis methods are very important. The broader goal of our research in this direction is to evaluate the electron probe microanalysis approach based on SEM=EDS for elemental quantication in investigation of historic objects. The narrower aim of this paper is to tackle the problem of estimating un-

certainty of quantitative surface analysis on the example of measuring iron content on the paper surface (surface concentration) of historic manuscripts. In this context the term surface is to be understood in a broad sense: it means not only the monomolecular surface layer of the paper but a signicantly deeper layer, since a large part of the ink inltrates deeper into the paper at the time of writing. The exact denition of the term surface concentration is given below. Several rather difcult problems are associated with this task: quantication in the actual surface analysis, non-uniformity of the surface (e.g. in its porosity) and the elemental content on the surface area, preparation of calibration objects that are sufciently similar to the samples of unknown concentration. We present here an uncertainty estimation procedure according to the ISO GUM [1] and the EURACHEM= CITAC guide [2] applicable to SEM=EDS quantication of iron in paper surface of historic manuscripts. We explore and discuss the inuence of various experimental parameters on the uncertainty of the measurement result. In particular, our attention is devoted to the difcult issues of measurand denition and sample non-uniformity. Several specic problems in uncertainty analysis are outlined, that need further investigation. Different spectroscopic techniques are used to analyze old written manuscripts or drawings to identify inks [15, 1820], gain information about the deterioration of paper [2124], investigate the distribution and amounts of components in inks [1114, 22, 25 30] and the inuence of inhibitors to iron or evaluate conservation techniques or strategies [11, 2935]. The most important of them in the viewpoint of non-destructiveness are X-ray microprobe [29], microscopic X-ray uorescence analysis [15, 22, 27], Particle (proton) Induced X-ray Emission (PIXE) spectroscopy [1113, 18, 20, 31, 36], X-ray absorption near edge spectroscopy [27, 28] and SEM=EDS [14, 26, 33] (some of these methods, depending of the sample type, are more or less destructive). Although PIXE is generally considered the most powerful of them, fully meeting the requirements of analyzing objects of historical value, it has poorer lateral resolution and is less accessible than SEM=EDS. If quantitative results are desired, then if the investigated object is not a homogeneous material, the quantication with SEM=EDS has several difculties (see below) but the method has the important advantages of being sensitive, fast and it is possible to measure the analyte content on a highly

Uncertainty in electron probe microanalysis

localized area. All in all the SEM=EDS method is currently perhaps the most widely used method for localized elemental analysis of various surfaces and bulk materials. Experimental
Description of the analysis procedure The intensity of the X-rays corresponding to iron atoms (i.e. the EDS signal intensity of the Fe K line at 6.404 keV) generated in the paper surface by the electron probe was used as the analytical signal to determine the iron content of the sample surface. Quantication was carried out using the calibration graph method. The EDS signal intensity of standards (paper sheets with ink lines) of known surface concentration of iron was used for calibration.

method (using the standard conditions for iron determination at wavelength 248.3 nm).

Calculation of the surface concentrations of iron on the calibration samples With the term surface concentration we denote the mass (in mg) of iron per unit of surface area (mm2) irrespective of how deep iron has diffused into the paper. Under our experimental conditions the X-rays are emitted throughout the thickness of the paper (standards and sample). The surface concentrations of iron in the calibration standards were calculated using the following data: the lengths and widths of the lines, iron concentration and the density of the ink solution, and the mass difference of the fountain pen before and after drawing the lines.

The original manuscript Preparation of the calibration standards It is very important to carry out calibration with standards that resemble the actual analysis objects as closely as possible. For our analysis of manuscripts we prepared calibration standards in the form of paper sheets with ink lines of known iron surface concentration. The widths of the ink lines were adjusted to be as similar to the widths of the lines on the sample manuscript as possible. Pure Whatman cotton (without additives) paper (www.whatman. com) with gelatin sizing was used. Gelatin was a common surface sizing agent until 19th century. The other widespread method the alum-rosin sizing method was invented only in the early 19th century (and was in use until the 1970s) [37]. Test sheets for simulation of old writing were prepared by plotting 40 longitudinal lines (about 265 mm long and 0.54 mm wide) with three different model-ink solutions on the paper. The fountain pen used for plotting the lines was weighed before and after the plotting in order to nd out how much ink was consumed per area unit of a line. Three small samples from different places located diagonally over the investigated manuscript (18th century, from the Estonian National Archive) were analyzed. The spots were chosen with letters that visually looked similar to the model ink lines.

Analysis procedure ntec EDS Xash A SEM model Leo Supra 35 equipped with Ro 3001 detector was used. Accelerating voltage of 15.00 kV was chosen for the probe electrons. The beam cross-section was 0.5 mm, the beam current was 12 108 A. From each of the three standard sheets several cutouts were subjected to the analysis. Selected samples of approximately 0.5 cm2 were mounted on copper supports using double-sided adhesive tape. The standards and the manuscript samples were coated with a thin platinum layer of 2 nm thickness. The standards and samples were examined using SEM magnication around 300400 times (Fig. 1). For analysis both wide X-ray spectra and intensities of the Fe K lines on the manuscript sample (Fig. 2) and on standards (Fig. 3) were measured on the rectangular excitation areas on the ink lines (Fig. 1). More details are given in

Preparation of the ink solutions In order to achieve ink writing with known iron concentration (expressed in mg mm2 ), the iron concentration of the ink must be known. Iron gall inks were made from aqueous solutions of iron(II) sulfate, gallnuts (source of tannin) and gum arabic [32]. Preparation of iron-gall ink with known iron concentration is not straightforward. The main reason is that the ink is not completely homogenous and tends to form a precipitate. The generally used ink recipe, described by Neevel [32] was taken as the basis for standard solutions. Precise gravimetric preparation of the ink solution was complicated because of precipitation. Hence, we prepared three ink solutions: 12, 18 and 24 g of Mohrs salt (Reakhim, the former USSR, no website available) was dissolved in 200 mL of aqueous solution of gum arabic (Aldrich, www.sigmaaldrich.com) and then powdered tannin (Reakhim, the former USSR, no website available) was added. The tannin and gum arabic content was the same in all three models of inks: 0.05 and 0.03 g mL1 , respectively. The medium concentration of the three inks with the Fe to tannin molar ratio of 5.5 has been selected according to J. Neevel as a representative ratio in old inks [32]. These solutions were left standing and were ltered twice through a plug of cotton-wool. After such treatment the ink solutions were reasonably stable. The concentrations of iron in the ltered ink solutions were measured by the ame AAS

Fig. 1. Scanning electronmicrograph of the manuscript paper. The black rectangle on the ink line (the slightly lighter horizontal line) marks the excitation area

K. Virro et al.

Fig. 2. EDS spectrum of the manuscript paper

Fig. 3. EDS spectrum of a standard

the Electronic Supplementary Material (ESM). The averaged rate of Fe K-radiation counts measured with EDS over the rectangle areas was used as the analytical signal. The iron content of the clean original and standard paper was negligible so that no correction was necessary. The three most important matrix interferences (ZAF-corrections) in our case are the following: generation of characteristic X-ray radiation (involving Z-(atomic number) correction), absorption by the matrix (involving A-(absorption) correction) and secondary uorescence (involving F-correction). All three have low effect on the results of our analysis: differences in the X-ray generation conditions in standards and in the original paper are relatively small as all the matrixes are rather similar; absorption by the matrix is low because the matrix consists predominantly of light elements and the Fe K analytical line is relatively energetic; uorescence by the matrix

is also negligible because there are no heavier elements than iron in the matrix. Nevertheless it is reasonable to expect that there is at least some mismatch between the sample matrix and the matrices of the standards and this will be taken into account below.

Derivation of the uncertainty estimation procedure Specication of the measurand and dening the mathematical model The measurand is Csurf_orig the average total iron content (Fe2 and Fe3 ) in the surface layer (ex-

Uncertainty in electron probe microanalysis

pressed in mg per paper mm2 of paper surface) of the writing of the original manuscript: Csurf
orig

mFe orig ; A

where mFe_orig is the mass of iron in the written region of paper and A is the surface area of the written region. We have dened our measurement as a relative measurement: we used calibration with standards having similar matrix. The electron beam acceleration voltage was 15.00 kV in all our experiments and it is an intrinsic part of the measurand denition. The penetration depth (depth of diffusion into the paper) of ink is not evaluated in this work, as the detected Xrays are emitted throughout the paper thickness. This is evidenced by the appearance of the weak copper lines originating from the top layer of copper supports on which we rstly mounted the paper samples. Afterwards we used aluminum supports and as a result the copper lines are missing from the spectra. The emitted X-rays are affected by the porosity and inhomogeneity of the studied material. The surface concentration of the ink on the sample from the manuscripts sample Csurf_orig (mg mm2 ) was found using the calibration graph method. Calibration line was constructed with the following equation: Istdi I0 b Csurf
stdi ;

The slope and intercept of the calibration line are found according to the linear regression equations as follows: Pn Csurf stdi Istdi n C surf std Istd 4 b i1 Pn 2 2 C n C surf std i1 surf stdi I0 I std Pn
2 i1 Csurf stdi C surf std Pn 2 i1 Csurf stdi n

Pn

i1 Csurf stdi Istdi ; 2 C surf std

5 where Csurf_std(i) and Istd(i) are the same as above and n is the number of points on the calibration line. Surface concentrations of the model samples Csurf_std(i) were found as follows: mstdi ci AAS 1000; 6 Csurf stdi li wi stdi where mstd(i) (mg) is the mass of the i-th ink standard solution used for plotting the lines on the paper (found as the difference between the masses of fountain pen before and after plotting the 40 lines), c(i)AAS (mg mL1 ) is the concentration of the i-th standard ink solution determined by AAS, l(i) (mm) is the total length of the 40 lines of the i-th model sample, w(i) (mm) is the average width on the lines of the i-th model sample and std(i) (mg mL1 ) is the density of the i-th standard solution, and the multiplier 1000 (mg mg1 ) converts the units. Equations (3) to (6) taken together form our mathematical model. Identifying uncertainty sources Uncertainty of weighing includes the following sources: 1) repeatability uncertainty of weighing; 2) uncertainty caused by the drift of balance; 3) uncertainty caused by rounding of the reading of the balance. Air buoyancy is not taken into account here (densities of the sample and the standard are similar and the resulting uncertainty is small) and the weights read from the balance are considered to be the masses. Uncertainty of ink solution concentrations measurement by AAS. Detailed description of uncertainty estimation of the AAS results will be published elsewhere. For the purpose of this work we consider the uncertainty of the AAS results as a single uncertainty source. Uncertainty of the surface area of the lines. The uncertainty of length and width of the lines contribute to the uncertainty of the surface areas of the lines.

where Istd(i) denotes the average rate of signal counts observed with the i-th standard and Csurf_std(i) denotes the average surface concentration (mg mm2 ) of iron in the written region of the i-th standard. I0 and b are the intercept and the slope of the calibration line, respectively. The iron content on the sample surface was found as follows: Csurf
orig

Iorig I0 fmatrix b

eff ;

where Iorig is the averaged value of count rates from different locations of ink lines of the original manuscript and fmatrix_eff is the factor taking into account the slight mismatch between the matrices of the samples and the calibration standards. It is known that the EDS method is linear with respect to the surface concentration if the matrix composition of the samples is similar [38]. In the narrow range of concentrations that we used for the calibration graph we can assume that the matrix similarity is sufcient.

K. Virro et al.

The contribution of the length uncertainty is small and is determined by the accuracy of the length measurement. The uncertainty of the width of the lines is caused mainly by the uneven width of the lines themselves and to a lesser extent by the measurement uncertainty of measuring the line widths (using a measuring microscope). Uncertainty arising from the SEM=EDS measurements. The following uncertainty sources affect the signal intensity: 1) The variability of the count rates is caused by a) the non-uniformity of the paper surface; b) the nonuniform distribution of the ink particles on the surface; c) the non-uniform distribution of the Pt coating on the surface; d) the uctuations of the electron beam intensity, high voltage and time interval measurement; e) the fundamentally random nature of emission of Xray quanta. The joint contribution of these uncertainty sources can be estimated by making multiple measurements from different surface regions and averaging the results. It is important to mention that these uncertainty sources originate from the objects under study and are not caused by the SEM-EDS system. 2) Different wetting properties of the inks used in the originals and in the calibration samples. This effect causes different levels of ink diffusion into the depth of the paper material and thus different iron concentration depth proles. In our case this uncertainty source was minimized by a) the fact that the emission depth of the X-rays (determined by the penetration depth of the electrons) is deeper than the thickness of the papers (as evidenced by the presence of copper lines from the in the spectrum originating from the copper support) and b) careful selection of the paper and the width of the lines on the calibration sheets (both of these were matched to the original as closely as possible). 3) Matrix mismatch between the standards and the samples. Matrix mismatch is caused by different compositions and structure of the paper samples and the inks used for preparation of the standards and the actual samples. This effect causes different generation rate and attenuation of the X-rays emitted from the paper (see above). The paper for the preparation of the calibration standards was made of pure cotton bers, without any additives found in modern papers (see the Description of the Analysis Procedure). In addition, the paper was sized with gelatin to mimic as closely as possible the original paper. The same approach has

been used in other works dedicated to instrumental analysis of historic manuscripts [25, 3335]. 4) Overlap between the EDS peaks of iron and possible interfering elements. The only element that can signicantly interfere under our conditions is manganese. However, in our case the possibility of overlap between the peaks of iron (K 6.404 keV) and manganese (K 6.49 keV) in the EDS spectrum negligible, because even the strongest manganese line (K) at 5.899 keV was not detected in the investigated samples and standards (see the spectra in Figs. 2 and 3). Additionally to this, literature data indicate that manganese is not common to be found in paper or in ink, or if found, then its concentration is very low. Budnar et al. [12] and Remazeilles et al. [13] have detected manganese content in paper=ink to be by 1001000 times lower than that of iron. In our original paper or samples, no traces of manganese were detected at all under our experimental conditions. Therefore we can leave this uncertainty source out of consideration. Application example, quantifying the uncertainty components, calculating the combined uncertainty Uncertainty of ink mass measurement Sartorius ME235S balance was used for weighing the ink for preparation of the standards. The data on the repeatability of the balance was determined experimentally in the laboratory in the following manner: The fountain pen was weighed for 10 times before and 10 times after plotting with each standard ink solutions. From these data the uncertainty of the differences of the masses was calculated according to the EURACHEM=CITAC Guide [2]. The u(mstd(i), rep) was found 0.0138 mg. The drift of the balance was estimated from long-term experience from our labop ratory: umstdi ; drift 0:01= 3 0:0058 mg. The digital display of the balance has ve decimal places, hence the uncertainty caused by rounding of the pdig ital reading is: umstdi ; round 0:000005= 3 0:0029 mg. The equation of uncertainty of the mass of ink standards was found: v u u umstdi ; rep2 umstdi ; drift2 7 umstdi t umstdi ; round 2 The values of uncertainties of the ink masses used for preparation of the standards are given in Table 1.

Uncertainty in electron probe microanalysis Table 1. The uncertainty budgets of the surface concentrations of iron on the calibration standards Csurf_std(1) Values (mg mm2 ) uc(Csurf_std(i)) (mg mm2 ) Relative uc (%) Components of uc 0.121 0.005 3.7 Csurf_std(1) Value mstd(i) (mg) C(i)AAS (mg mL1 ) std(i) (mg mL1 ) l(i) (mm) w(i) (mm) 97.62 7.33 1033.1 10540.5 0.54 u 0.02 0.14 0.5 11.6 0.018 % 0 24.8 0 0.1 75.1 Csurf_std(2) 0.151 0.006 3.7 Csurf_std(2) Value 81.17 11.12 1045.0 10582.4 0.54 u 0.02 0.20 0.6 11.6 0.018 % 0 24.0 0 0.1 75.9 Csurf_std(3) 0.185 0.007 3.7 Csurf_std(3) Value 76.34 14.57 1060.4 10451.1 0.54 u 0.02 0.26 0.7 11.6 0.018 % 0 23.4 0 0.1 76.5

Uncertainty of the density and iron content of the Self-Prepared Ink The densities of the model ink solutions at temperature 22.6 0.2  C and their standard uncertainties expressed in milligrams per milliliter were the following: 1 1033.1, u(1) 0.5, 2 1045.0, u(2) 0.6 and 3 1060.4, u(3) 0.7. Pure water (density at the same temperature was 9976.6 mg mL1 ) was used as the reference. The iron contents of the three calibration ink solutions measured by AAS were as follows: 7.33, 11.12, 14.57 mg mL1 . The standard uncertainties of these concentrations are 0.14, 0.20, 0.26 mg mL1 , respectively . The AAS analysis method and the uncertainty estimates were validated by analyzing the 11.12 mg mL1 solution also with a different method (ICP-AES) at a different laboratory. The concentration was found as 11.00 0.44 mg mL1 , k 2, norm., which is well in line with our result. Uncertainty of the lengths and widths of the standard ink lines Uncertainty of the length of the lines was calculated through the following equation: q 8 ulline ulline ; cal2 ulline ; rep2 ; where the repeatability of uncertainty u(lline, rep) of the line length was estimated as the standard deviation of the mean of 20 readings (of the same length) and the calibration uncertainty was estimated as 0.5 p mm (rectangular distribution): ulline ; cal 0:5= 3 0:29 mm. The total lengths of standard (1), standard (2) and standard (3) were 10538.7, 10582.4 and 10451.1 respectively. The standard uncertainty of the three lengths

was the same: 11.6 mm. Each of the total length of the standard consisted of 40 short lines. The widths (w(i)) were determined making measurements in 22 different locations picked on the sheet diagonal (every location was on a different ink line) using a measuring microscope. The uncertainty arising from the measurement itself is negligible compared to the uncertainty due to the uneven width of the lines. The widths of the lines on the standards with their uncertainties are presented in Table 1. Uncertainty due to the SEM=EDS The electron beam (the probe current) of the used SEM=EDS is of high stability better than 0.2% per hour for analytical applications. Hence, the instrument instability contribution to the uncertainty of Csurf was considered of no importance. All the other direct uncertainty sources of the SEM=EDS measurement as well as uncertainty due to the uneven iron distribution
Table 2. The uncertainty budget of the surface concentration of iron in the original manuscript Results Value Set I Csurf_orig_I (mg mm ) Components of uc (xI in text) Csurf_std(1) (mg mm2 ) Csurf_std(2) (mg mm2 ) Csurf_std(3) (mg mm2 ) Sstd(1)_I Sstd(2)_I Sstd(3)_I Soriginal_I fmatrix_eff
2

uc Set II 0.121 Set I 0.010 u 0.005 0.006 0.007 1.3 1.8 2.0 2.2 0.032 Set II 0.020

0.124 Value 0.121 0.151 0.185 31.1 44.1 48.7 32.7 1.000

22.7 31.3 35.0 23.2 1.000

1.2 1.7 1.8 3.8 0.032

K. Virro et al. Table 3. The nal result Csurf_orig (mg mm2 ) 0.123 uc(Csurf_orig) (mg mm2 ) 0.016 Degrees of freedom 10 The uncertainty contributions to the result (%) u(Csurf_std(1)) 6.18 u(Csurf_std(2)) 0.45 u(Csurf_std(3)) 0.11 u(Sstd(1)) 10.51 u(Sstd(2)) 0.05 u(Sstd(3)) 0.02 u(Soriginal) 77.84 u(Matrix effect) 4.84 k(95.45%) 2.28 U(95.45%) (mg mm2 ) 0.036

and surface non-uniformity will be accounted for by the repeatability of the measurements. Parallel measurements on different locations of the lines were performed with each of the three standards and with the original manuscript (see the ESM for the calculations). The mean values and the standard uncertainties of the Sorig and Sstd(i) values are given in Table 2. The uncertainty sources 2 and 3 (see above) are taken into account by the matrix mismatch factor fmatrix_eff (Eq. (3)). This factor is dened in such a way that its value is equal to unity in order not to change the value of Csurf_orig. Its uncertainty is dened as the estimated relative standard uncertainty due to the matrix mismatch. To get a rough estimate of this contribution a model experiment was carried out. ZAF correction factors for pure paper (c(C) 98.35 m%, c(Fe) 0.88 m% and c(S) 0.77 m%) and a paper with additives (c(C) 81.65 m%, c(Fe) 2.99 m%, c(S) 0.95 m%, c(Si) 8.54 m%, c(Al) 0.36 m%, c(Na) 0.17 m%, c(K) 4.50 m%, c(Cl) 0.12 m% and c(Ca) 0.72 m%) were calculated using EPMA correction algorithm Stratagem [39] switched into bulk mode. This algorithm also permits to take the Pt coating into account. The difference between the calculated ZAF corrections was small: 3.2%. This value was used as the estimate of standard uncertainty due to the matrix mismatch (fmatrix_eff). Finding the combined uncertainty The combined standard uncertainty is calculated according to the ISO GUM approach [1], that is, the calculations were performed according to Eq. (9). v u 2 uX  @ Csurf orig t 9 uc Csurf orig uxi ; @ xi i where xi denotes the values of seven components of uncertainty in Table 2. The calculations were carried out with the MS Excel package using the Kragten Method [40] for approximating the partial derivatives.

The electron probe microanalysis with SEM=EDS was carried out twice on different days (Sets I and II) using the same standards and original manuscripts but different cutouts. The uncertainty budgets of iron concentrations on the surface of the standards are presented in Table 1. The results of the two separate analyses are presented in Table 2. The average surface concentration of iron in the original manuscript together with its combined uncertainty and the uncertainty budget containing the uncertainty contributions of the input quantities are presented in Table 3. Results and discussion Contributions of the uncertainty sources The obtained iron contents in the ink writing of the manuscript were 0.124 and 0.121 mg mm2 with standard uncertainties 0.010 and 0.020 mg mm2 , respectively. The large difference between the uncertainties of the two sets is caused by the different number of parallel measurements on the original (9 and 3, respectively, see the ESM). The average iron content on the ink-covered surface of the sample manuscript found as the weighed average from these measurement results was 0.123 mg mm2 with the standard uncertainty of 0.016 mg mm2 (see the ESM for the calculations). The uncertainty budget is heavily dominated by the variability of the count rates, which in turn is caused by inhomogeneity of the ink lines. Thus the number of effective degrees of freedom of this result can be estimated as completely determined by the low number of parallel measurements made from different locations of the sample leading to df 10 (see the ESM). The coverage factor at 95.45% condence level (corresponding to the k 2 level for the Normal Distribution) is therefore 2.28 leading to the expanded uncertainty 0.037 mg mm2 at 95.45% condence level (relative expanded uncertainty 30%). The main source of uncertainty with our sample type and equipment was the variability of the parallel

Uncertainty in electron probe microanalysis

measurement results from different locations of the manuscript surface contributing 54 and 84% of the overall uncertainty in the rst and second analysis set, respectively, and 78% to the uncertainty of the average result. This contribution is followed by the signal variability of the rst calibration standard (11%) and the iron content of the rst calibration standard (6%). The prevailing inuence of the sample heterogeneity fully validates the use of only 3 points for calibration in our case. Even in the impossible case that calibration graph would contribute no uncertainty to the result (meaning that there is innite number of points on the graph and there are no systematic effects affecting the points both conditions unrealizable), the standard uncertainty of the result would decrease only marginally: from 0.016 to 0.015 mg mm2 . The high contribution of the st calibration standard is not unexpected: the average rate of counts from the sample is very similar to that of the rst standard. The dominance of the signal variability (both in the case of samples and standards) in the uncertainty budget does not mean that the sample heterogeneity is the only uncertainty source worth considering. With a different sample the heterogeneity can be far lower and the calibration graph points can have large relative contribution to the uncertainty. The uncertainty contribution due to the variability of the signal can be reduced by increasing the number of measurements both from the calibration standards and from the sample. Both the purpose of the measurement and the economic effect have to be carefully considered. The uneven widths of the ink lines play the key role in determining standards preparation as the second most important source of uncertainty. The uncertainty contribution arising from the uneven width of the ink lines of the standards could in principle be reduced by drawing wider lines using the so-called reddish pen. The absolute uncertainty of the line widths would remain more or less the same but the relative uncertainty would drop several times. Because the uncertainty contribution of the width of the line is dependent on the relative uncertainty, the combined uncertainty of the result would seemingly decrease. However, this would destroy the similarity between the sample and standards (see the Preparation of the Calibration Standards Section): the diffusion of the ink in the broad lines would not be the same any more and a hard-to-quantify additional uncertainty source would be introduced.

Limit of detection All our samples, both standards and the original, had non-detectable content of iron on the unwritten area. Iron is a widespread element in the environment and the paper of any manuscript contains iron traces originating from contamination e.g. by paper verso sides. Based on the baseline noise level of the EDS spectra and the calibration line parameters a crude estimate of the detection limit of iron was obtained as 0.02 mg mm2 (signal to noise ratio 3) under our conditions. The signal=noise ratio for the lowest calibration standard was around 15. Thus, SEM=EDS is a suitable method to determine, at least semi-quantitatively, the surface concentration of iron in inks on the paper.

Comparison of our results with those of other authors This type of analysis is not done by laboratories on a regular basis. Thus it would be very desirable to compare both the element content levels and the uncertainties with those obtained by other authors in similar measurements. Studies on quantication of iron in old ink-written manuscripts have been published before [11, 13, 14, 25, 33]. Budnar et al. [11, 12, 36] and Remazeilles et al. [13] have done quantication of the elemental components of ink with PIXE. With destructive GFAAS method quantitative analysis of iron and copper in old manuscripts has been carried out by Wagner et al. [25]. The same group has quantied elements in iron-gall ink with energy dispersive X-ray uorescence analysis [14]. Semiquantitative analysis of inorganic components has been carried out by Odlyha et al. [33]. It is nevertheless difcult to compare our results to the literature results. Our results are presented as iron mass per area of written paper (unwritten area not included). The literature results are mostly presented in the form of mass of iron per mass of paper (unwritten area included). Analyzing our manuscript sample by AAS would have been a possible approach for comparing with an independent method, but that would destroy the sample and also it would be extremely difcult to nd the area of the ink lines as they were irregular and the whole manuscript was patchy. In some of the above-cited papers the issue of uncertainty has been addressed but a full ISO GUMbased uncertainty analysis has not been carried out. In Ref. [13] the authors have given the estimates of

K. Virro et al.

the variations between the results obtained from several spectra (measured with PIXE) from the same manuscript, but at different locations. Each spectrum was processed the same way and the authors looked at the variabilities of the results for the most typical elements. For iron the variability was found as 16% (presented as full spread of seven measurement results). In Ref. [11] the estimation of the variation has also been presented as the full spread of 10% (the number of measurements was not given). These uncertainties are lower than our uncertainty estimate. In Ref. [36] the accuracy of the analysis method (PIXE) was under discussion. The systematic error (520%) and precision (5%) were evaluated for the analysis of Fe and Cu. The relative standard uncertainty extracted from these data is in the range of 721%. This compares quite well with our relative standard uncertainty estimate of 13%. Conclusions SEM=EDS was used to determine the surface concentration of iron in the ink writing of an old manuscript. For quantication a calibration graph method utilizing AAS analyses of 3 standard ink solutions with different iron content was used. To estimate roughly the surface concentration of iron in the ink writing on the manuscript we recorded spectra on two days from altogether six different locations. The mean iron surface concentration on the written area was 0.123 mg mm2 with the expanded uncertainty 0.037 mg mm2 at 95.45% condence level, which we consider acceptable for such a complicated analysis task. Electron probe microanalysis with SEM=EDS is a suitable method for evaluating the iron content in old manuscripts. The method is universal (enables to quantify other elements in a similar manner), quite sensitive (down to few atomic percent detection limits) and fast (it takes around 5 min to record one spectrum and around 1 h to prepare a set of 1020 samples). Although non-destructive methods, like PIXE, m-XRF, etc exist and are in use for the analysis of old paper, the necessary equipment may not be available for everyday analysis, and getting high precision quantitative results from ink-on-paper samples is not a routine task also with those methods.
Acknowledgments. We are deeply indebted to Mr. Jaan Lehtaru, Dr. Viljar Pihl and Dr. Koit Herodes for valuable suggestions. This work was supported by the Estonian Science Foundation grants 5475 and 6651 and by the Basis nancing grant 06902 of University of Tartu.

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