J . Sci. Fd Agric. 1973, 24, 139-146

The Determination of Sulphur in Plant Material by X-ray Fluorescence Spectroscopy

Barbara Reed

Gouernment Chemical Laboratories, Perth, Australia

(Manuscript received 2.5 May 1972 and accepted 30 October 1972)

X-ray fluorescence spectroscopy is used for the determination of total sulphur in plant material.

One calibration curve can be used for pasture, clover, apple leaf, rape plant top and wheat plant samples.

The intensity of the sulphur Ka line radiation is measured on a pressed disc of sample backed with a phenol formaldehyde resin (“Excelite”) and this is related to a fused disc reference sample to give a ratio, x. A linear expression y =

1.0548~ - 0.0236 ( r = 0.9810) relating x, the ratio, and y , the chemically deter- mined sulphur content of the sample, is derived.

The technique is rapid (2 min/sample reading time). The reproducibility of readings is less than 1.5 % as a coefficient of variation, the standard error of pre- diction is 0.011 % sulphur and the detection limit is 13 pg/g.

1. Introduction

Chemical procedures1, for the determination of sulphur are usually slow and there is a need for a rapid, sensitive method that is universally applicable to plant material.

Brown and Kanaris-Sotiriou3 have shown that X-ray fluoresence spectroscopy is sufficiently sensitive to determine low levels of sulphur in soils. Kubota and Lazar4 determined sulphur in alfalfa, clover and grasses. Evans5 determined sulphur on a variety of plant samples diluted 1 + 1 with cellulose. McLachlan and Crawford6 have developed a technique for the determination of sulphur in subterranean clover Trifolium subterraneum L. and grass Phalaris tuberosa L.

Contrary to Kubota and Lazar4, who suggested that separate working curves may be necessary for each type of non-forage plant, McLachlan and Crawford6 have suggested that a single calibration curve may be sufficient for the determination of sulphur in different plant species.

Following this suggestion, the present study was undertaken to determine whether or not a single calibration curve was generally applicable to plant material because in these laboratories large numbers of sulphur analyses are often required on forage plants, cereals, fruit tree leaves, oil seed plants and particular plant parts.

Kubota and Lazar4 and McLachlan and Crawford6 used a sample packed loosely into a Mylar backed sample holder. Neither this technique nor dilution of the sample

139

140 B. Reed

with cellulose proved satisfactory. A pressed disc backed with Excelite was found preferable.

2. Experimental

2.1. Sample preparation The plant sample is dried at 80 "C and ground to pass a 1-mm screen in either a Wiley blade or Christy and Norris hammer mill.

10 g of this sample is ground in the 100 cm3 vial of a Tema Laboratory disc mill TlOO for 1 min or 1 g in the 10 cm3 vial for 2 min, to ensure a particle size to pass a BSI 100-mesh sieve, and dried under vacuum at 70 "C for 30 min.

1 g of the vacuum-dried sample backed with 4 g of Excelite (general purpose black moulding powder A168, available from Monsanto (Australia)) is compressed into a disc under vacuum and at 0.463 GN/m2 pressure (30 ton in-2).

All discs are stored under vacuum in a desiccator.

2.2. Standard control samples Discs of four pasture samples obtained from a collaborative study, clover grass- Kongaloon, winter fodder, white clover and clover grass-Glen Innes, were prepared and maintained as standard control samples.

2.3. Instrumental conditions All measurements are made with a Siemens X-ray Spectrometer SRSl using the accessories and settings shown in Table 1.

TABLE 1. Parameters for the determination of sulphur

X-ray tube Chromium Stabilised DC Generator Sample holder

Kristalloflex 4 operated at 45 kV, 20 mA. Liquid sample holder with 2 pm polycarbonate film

(Makrofol KG, manufactured by Farberfabriken Bayer AG, Leverkusen, Germany) and a nickel grid.

Path Vacuum. Analysing crystal 2d spacing Collimator Coarse 0.4" Detector

Penta-erythritol8.750 A.

Flow proportional counter fitted with 2 p m A1 flashed Makrofol with 90% argon, 10% methane counter gas. Dz voltage 1.670 kV.

Baseline 0.70 V, channel width 0.56 V, attenuation x 50. Pulse height analysis Line SKa 20 = 75.63" Background 73.63", 71.63". Count time Rotation Used.

2 x 40 s.

Set all parameters on the X-ray spectrometer. Before each series of samples it is necessary to optimise operating conditions on the

instrument with the flow counter voltage and the 28 angle of dispersion and using the fused disc7 reference sample (1 + 1 K,S04, SO,)-monitor- as the sulphur source.

Determination of sulphur in plant material 141

Traverse the peak across the window by altering the flow counter voltage in incre- ments of 0.005 kV to obtain the DzkV to give the maximum intensity. Set this DzkV. Select the 28 angle of dispersion to give the maximum intensity.

2.4. Procedure Read the monitor 10 x 40 s and each of the standard control samples 5 x 40 s. Average each set of results. Use previously determined background counts for these samples to calculate the ratio, x, of the net number of counts of the standard samples to the net number of counts of the monitor. If the ratio lies within the allowed limits of deviation (i 3 % of the mean) then proceed as follows.

Read the monitor 10 x 40 s, read ten samples 2 x 40 s and repeat this procedure with all remaining samples. After all line readings have been measured, read the monitor and every fifth sample at the two background positions. Average all background readings for each species of sample.

Calculate the net sample and the net monitor counts per 40 s and hence the ratio, x, for each sample and from this obtain the percentage sulphur dry basis from the line of best fit.

y = 1.0548~ - 0.0236.

2.5. Calibration curve On three separate occasions, 50 samples of pasture and clover were digested with nitric-perchloric acid and analysed for sulphur by a modified Johnson and Nishital method. The samples were chosen to cover the range 0.08 to 0.42 % sulphur dry basis. The sulphur content is normally distributed about the mean of 0.22% sulphur dry basis. This is a similar distribution to that found in pasture and clover samples analysed in these Laboratories.

The relationship between the averages of the chemical results and the ratios of the sulphur Kcr line intensity to the monitor is linear and is described by the equation,

y = 1.0548~ - 0.0236

where x is the ratio and y is the chemically determined sulphur content expressed as per cent dry basis.

Initially, separate calibration curves were established for the set of clover samples, the set of pasture samples and a combined curve of both the pasture and clover samples. A comparison between the results obtained from the combined calibration curve and the pasture and clover calibration curves, respectively, showed that both types of sample could be amalgamated into the one calibration curve, thus embracing a greater range of sulphur levels than would be possible if only one species was chosen. The correlation coefficient between the predicted results for pasture samples obtained from the combined calibration curve and the pasture calibration curve was 0.9999 and between the pre- dicted results for clover samples obtained from the combined calibration curve and the clover calibration curve was 0.995.

The sulphur content of 20 of the 50 pasture and clover samples used to establish the calibration curve, as analysed by X-ray fluorescence spectroscopy and a modified

142 B. Reed

Johnson and Nishital method, and including the highest and lowest value for each type of sample, are compared in Table 2. The fifty pairs of results are highly correlated (Y = 0.981).

TABLE 2. Sulphur content of pasture and clover samples by X-ray fluorescence and by chemical analysis

Sulphur (% dry basis)

Clover Pasture ,- . ,

Sample X.R.F. Chemical X.R.F. Chemical

1 2 3 4 5 6 7 8 9

10

0.077 0.155 0.153 0.206 0.225 0.227 0.229 0.245 0.290 0.385

0.067 0.144 0.158 0.209 0.225 0.232 0.236 0.245 0.291 0.394

0.094 0.115 0.158 0.180 0.206 0.243 0.252 0.294 0.326 0.420

0.094 0.111 0.158 0.179 0.213 0.239 0.261 0.291 0.322 0.407

Errors arise from two sources; the error in the determination of sulphur by the chemi- cal analysis and the error in the X-ray fluorescence analysis. Figure 1 illustrates the standard error of a prediction along the entire calibration curve. It is approximately 10.01 1 % sulphur dry basis.

Figure mined by

I I

0 4 -

0 3 -

(on 0 2 - -

-

I I I 0 1 0 2 0 3 0.4

*c

1 . Standard error of a prediction along calibration curve. S, = sulphur % dry X.R.F. analysis, S, = sulphur % dry basis determined by chemical analysis.

basis deter-

Determination of sulphur in plant material 143

3. Results

Pasture samples, clover samples, apple leaves, rape plant tops, wheat plant tops and parts of the wheat plant were analysed by both the described technique and a single chemical' determination.

Table 3 compares some of the results obtained and includes both the highest and lowest value for each type of sample.

TABLE 3. Sulphur content of samples by X-ray fluorescence and chemical analysis

Sulphur Sulphur (% dry basis) (% dry basis) -. ,-

Sample X.R.F. Chemical Sample X.R.F. Chemical

Pasture Wheat plant parts 1 0.124 0.112 Rachis 0.067 0.063 2 0.178 0.180 Sheath 0.092 0.082 3 0.371 0.378 Glumes 0.100 0.104

Clover Stem 0.101 0.116 1 0.202 0.214 Leaf 0.153 0.152 2 0.330 0.334 Flag 0.150 0.167 3 0.439 0.417 Grain 0.194 0.193

1 0.055 0.059 Rape plant tops 2 0.115 0.111 1 0.463 0.494 3 0.157 0.158 2 0.554 0.555

Wheat plant tops 3 0.651 0.640 1 0.178 0.183 2 0.212 0.210 3 0.234 0.233

Apple leaf

In all, 27 additional pasture and clover samples (r = 0.991), 20 apple leaf samples (r = 0.976), 20 samples of wheat plant tops (r = 0.955) and 12 samples of parts of the wheat plant (r = 0.957), were analysed by both X-ray fluorescence and chemical analysis. The agreement between the results is good as the correlation coefficients ( r ) are highly significant.

Although outside the range of the calibration curve, the results of twenty rape plant tops are not biased and the correlation coefficient (r = 0.874) is highly significant.

4. Discussion

4.1. Sample Preparation The evacuation time of the instrument was lengthy when loosely packed sample^,^,^ particularly pastures, were used. Even when separated from the remainder of the instrument by the polycarbonate film, some particles of the loose sample spread through- out the instrument and in at least one instance particles were found in the collimator. Dilution of the sample with cellulose5 reduced the count rate, increased preparation time

144 B. Reed

and some pressed discs, particularly flours, were not self-supporting. Extreme care is necessary in the handling of these discs.

The above were discarded in favour of a pressed disc backed with Excelite. The disc is durable and can be used repeatedly. Standard samples prepared in this manner have been in continuous use for ten months without any deterioration of the disc. The disc’s long life is valuable as it ensures that the disc can be used for determinations of other elements. Evacuation time of the sample chamber is rapid. Where evacuation time for a loose sample or a disc diluted with cellulose is three to four minutes, evacuation with the described disc occurs in 16 to 20 s.

4.2. Accuracy, precision and sensitivity

The proposed method was applied to a variety of standard samples which were analysed in these Laboratories and in two other Laboratories in Australia and New Zealand. The method is accurate, not only for forage samples but for cauliflower, citrus, banana, flour, wheat meal and maize grain samples. Table 4 shows the good agreement between the results.

TABLE 4. Collaborative analyses: sulphur content of samples by X-ray fluorescence and chemical analysis in three laboratories

Type of sample

Sulphur (% dry basis)

X.R.F. Chemical Average chemical

, Govt Chem. Lab. Aust. and N.Z.

Lucerne Clover grass-Kongaloon Winter fodder White clover Clover grass-Glen Innes Sudax Citrus Cauliflower Banana Flour Wheat meal Maize grain

0.34 0.25 0.32 0.25 0.18 0.11 0.29 1.02 0.23 0.12 0.1 3 0.12

0.35 0.25 0.33 0.24 0.20 0.11 0.28 1 .oo 0.20 0.1 I 0.12 0.11

0.34 0.24 0.31 0.24 0.20 0.12 0.29 1.03 0.23 0.11 0.12 0.12

TARLE 5. Reproducibility by X-ray fluorescence

Coefficient of Number of Mean sulphur Standard variation

Sample determinations (% dry basis) deviation ( %)

Clover grass 8 0.254 0.0036 1.42 Winter fodder 8 0.334 0.0040 1.20 White clover 8 0.249 0.0018 0.72 Clover grass 8 0.179 0.0022 1.23

The reproducibility with which a sample can be analysed by X-ray fluorescence spectroscopy is high as shown in Table 5. As previously shown the standard error of

Determination of sulphur in plant material 145

any prediction +0.011% depends upon the variance of both the chemical analyses and the X-ray fluorescence determinations used to establish the calibration curve.

Jenkins and De Vries6 describe the lower limit of detection as:

lower limit of detection = - Where R, is background count rate, TB is the time spent counting the background, m is the factor necessary to convert the deviation in counts into deviation in concentra- tion. Under the instrumental conditions used, the limit of detection of sulphur in plant material is 13 pg/g for a 40 s counting time.

At very low levels of sulphur in wheat plant samples, however, the agreement be- tween the sulphur content of samples as analysed chemically and by X-ray fluorescence spectroscopy is not good as shown in Table 6 . This disagreement could be attributed to three causes :

(i) the standard error of prediction being & O . O l l % sulphur; (ii) the results being well outside the lower limit of % sulphur used to establish the

(iii) the imprecise nature of chemical analyses at this level. calibration curve; and

TABLE 6. Very low levels of sulphur determined by X-ray fluorescence and chemical analysis

Sulphur (% dry basis)

Lab. no. X.R.F. Chemical

3548 3549 3551 3571 3572 3574 3575 3576 3595 3596 3620 3625

0.037 0.034 0.035 0.008 0.010 0.007 0.009 0.007 0.014 0.022 0.030 0.029

0.037 0.045 0.041 0.023 0.025 0.018 0.022 0.014 0.023 0.032 0.046 0.041

It is not clear why the X.R.F. results have a negative bias with respect to the chemical result.

5. Conclusion

Pasture, clover, apple leaf, wheat plant tops, rape plant tops, wheat plant parts, citrus, banana and cauliflower leaves, flour, wheat meal and maize grain samples can be satis- factorily analysed for sulphur by the rapid technique of X-ray fluorescence spectroscopy using one calibration curve.

146 B. Reed

Since the calibration curve established for pasture and clover samples can be applied to these other types of plant materials, the effect of the matrices of these materials on the mass absorption coefficient of the sulphur Kcc line fluorescent radiation is similar.

Acknowledgements

The author wishes to thank the Director of the Government Chemical Laboratories, 30 Plain Street, Perth, Western Australia, for permission to publish this paper and is also indebted to several colleagues of the Agriculture Division for their help. Gratitude is also expressed to Miss H. Nicol, Department of Agriculture, South Perth, W.A., for her assistance with the statistics and Mr M. F. Redies, Department of Agriculture, N.S.W., Biological and Chemical Research Institute, Rydalmere, N.S.W. for permission to publish the average chemical results of the standard samples shown in Table 4.

References 1. Johnson, C. M.; Nishita, H. Analyt. Chem. 1952, 24, 736. 2. Butters, B.; Chenery, E. M. Analyst, Lond. 1959,84,239. 3. Brown, G.; Kanaris-Sotiriou, R. Analyst, Lond. 1969,94,782. 4. Kubota, J. ; Lazar, V. A. Soil Testing and Plant Analysis Part 11, Soil Science Society of America,

Inc., Wisconsin, 1967. 5. Evans, C. C. Analyst, Lond. 1970, 95,919. 6. McLachlan, K. D. ; Crawford, M. J. Sci. Fd Agric. 1970,21,408. 7. Zussman, J. Physical Methods in Determinative Mineralogy Academic Press, London. 1967,

p. 206. 8. Jenkins, R.; De Vries, J. L. Analyst, Lond. 1969,94,447.