Element determination by total-reflection X-ray fluorescence spectrometry at the initial step of element speciation in biological matrices

ANALYTICA CHIMICA

ACW ELSEVIER

Analytica

Chimica Acta 309 (1995) 327-332

Element determination by total-reflection X-ray fluorescence spectrometry at the initial step of element speciation in biological matrices Klaus Giinther aj*, Alex von Bohlen ‘, Christoph Strompen ’ a Institut jiir Angewandte Physikalische Chemie, Forschungszentrum Jiilich GmbH, D-52425 Jiilich, Germany h Institut ftir Spektrochemie und Angewandte Spektroskopie, Bunsen-Kirchhoff-Str. I I, D-44139 Dortmund, Germany ’ Institut jiir Lebensmittelwissenschaft und Lebensmittelchemie der Uniwrsitiit Bonn, Endenicher Allee 1 I-13, D-531 I5 Bonn, Germany Received 22 November

1994; revised

25 January

1995; accepted 26 January

1995

Abstract Total-reflection X-ray fluorescence spectrometry (TXRF) is used for the simultaneous determination of Ca, Cu, Fe, K, Mn, Rb, Sr and Zn in 12 different vegetable foodstuffs and their cell fractions after mechanical cell breakdown. The vegetables are homogenized by liquid shearing (ultra-turrax treatment) in the presence of buffer. The homogenates are separated into cytosols (liquid fractions) and pellets (solid fractions) by centrifugation. The distribution of the elements between the fractions is determined. Before analyses all samples were digested with HNO,. The quick and simple method of cell breakdown as an initial step for speciation transfers Zn, Cu, Mn, Rb and K in significant percentages into the soluble form. Consequently, a large fraction of each of these elements is directly accessible to further analytical separation procedures. On average Sr, Ca and especially Fe are mainly bound to pellet components. TXRF proved to be an excellent analytical tool in these investigations. It requires only one internal standard and minute samples with simple preparation. Keywords:

X-ray fluorescence

spectrometry;

Element speciation;

Sample pretreatment;

1. Introduction Speciation necessary

of elements

for the precise

in different assessment

matrices

is

of their biologi-

since resorption and metabolism are often dependent on the given element species [l-3]. The initial step for element speciation in solid biological matrices is to transfer as many of the

cal effects.

* Corresponding 0003.2670/95/$09.50

author 0

SSDI 0003-2670(95)00087-9

1995

Elsevier Science B.V. All rights reserved

Vegetable

foodstuffs

elements as possible into a soluble form so that they will be easily accessible to the following analyses using traditional methods of analysis such as liquid chromatography or electrophoresis. As hardly any attention has been paid to this factor thus far, systematic studies of the solubility behaviour of elements in biological samples after cell breakdown were necessary. In the present investigation 12 different vegetable foodstuffs served as examples of biological matrices and total-reflection X-ray fluorescence spectrometry (TXRF) - a universal and economic method for

328

K. Giinther et al. /Analytica

Chimica Acta 309 (1995) 327-332

trace analysis - was used for the simultaneous multielement determination in the resulting samples. TXRF is a special analytical tool derived from energy-dispersive X-ray fluorescence spectrometry (ED-XRF). In TXRF the primary beam is directed onto an even surface of a sample carrier at a very small angle, so that the primary beam is totally reflected at the carrier. The sample has to be placed onto the carrier surface in the form of a thin but rough layer (Fig. 11. This geometry leads to low detection limits. Additionally, the thin-film type samples prevent matrix effects so that an easy calibration can be carried out by adding one single internal standard element [4-71.

cupitutu L. J rubu DC.); white cabbage leafs (Brussitu oleruceu L. var. cupitutu L. f: ulbu DC.). The plant parts were thoroughly washed with bidistilled water and chopped up with a clean glass shard. After adding Tris-HCl buffer (20 mmol, pH 8.0; weight ratio of fresh plant material to buffer = 1 to 2) the mixture was subjected to liquid shearing a treatment by the so-called ultra-turrax (apparatus T 45 Prof. P. Willems, Janke and Kunkel KG, IKA Werk, Staufen i. Breisgau) for 3 min at low temperature (5-10” 0. The homogenates were then centrifuged (centrifuge WKF G50K, 20,000 rpm) for 90 min at 5” C and 45,000 g. The pellet (solid fraction) obtained for subsequent washing was agitated two times with buffer and centrifuged for 15 min each at 45,000 g. The supernatants obtained are combined with the respective cytosols of the first centrifugation (liquid fraction). All plant fractions were frozen immediately after being prepared and stored in small portions for further use at -20” C. Three cytosols, pellets and homogenates of each experiment were digested with cont. HNO, for a period of 3 h at 160-170” C (Perkin-Elmer autoclave 3 und Berghof DAB II with PTFE inserts). The quantification of element concentrations was carried out with Ga as internal standard by means of total reflection X-ray fluorescence (EXTRA II, Rich. Seifert and Co, Ahrensburg, incorporating a MO tube and an energy-dispersive spectrometer with Si(Li) detector, system 860/500, Link Systems, London).

2. Experimental 2. I. Procedures The following ordinary plant materials were used: avocado fruits (Persea americana (Mill.)); banana fruits (Musu supientum L.); cabbage lettuce leafs (Luctucu sutivu L. var. cupitutu L.); chicory leafs (Chicorium intybus var. foliosum He@); Chinese cabbage leafs (Brussicu chinensis Juslen.); cucumber fruits (Cucumis sativus L.); dill stalks (Anethum gruveolens L.); paprika fruits (Capsicummis unnuum L.); parsley roots (Petroselinum crispum (Mill.) rudicosum (Alef.)); radish roots (Ruphunus sutivus L.); red cabbage leafs (Brussicu oleruceu L. var.

1 Si(Li)-

Detector

Sample

carrier

Fig. 1. Simplified instrumental

setup for TXRF.

K. Giinther et al. /Analytica

Each digestion was measured twice, and the measuring time was adjusted to 200 s. 2.2. Chemicals HNO, (suprapur), tris(hydroxymethyl)aminomethane (p.a.), NaN, (reinst) and Ga standard solution (Aldrich) were used. The buffer was 20 mmol Tris-HCl (pH 8.0)-l mmol NaN,.

3. Results and discussion To illustrate the multielement capability of the applied method, Fig. 2 shows the TXRF spectrum of a white cabbage homogenate. The Si peak at 1.74 keV originates from the quartz glass carrier but can be avoided if either glassy carbon or Perspex is used. The Ar signal at 2.96 keV results from the air in the gap between the carrier and the detector and can be suppressed by flooding the space with nitrogen. The metal contents determined in the twelve different vegetable foodstuffs are shown in Table 1. The total element contents of the plants were used in the representation of the cytosol/pellet distribution of metals normalised to 100% for the homogenates. The obtained distributions of the metals between cytosols and pellets were shown in Tables 2-5. The cytosol fractions of potassium (Table 2) and rubidium (Table 5) are more than 75% of the total, and

Chimica Acta 309 (1995) 327-332

329

well above this value for most of the foodstuffs. The concentration of rubidium in cabbage lettuce was too low to determine the distribution (see Tables 1, 5 and 6). Copper and zinc predominate in the examined cytosol too (Table 4) though to a lesser extent as potassium and rubidium. Only in the case of white cabbage, a high percentage of zinc is bound to pellet components. On average the cytosolic fractions of manganese, strontium and calcium amount to 46, 37 and 32%, respectively, for the vegetable foodstuffs in question. The solubility behaviour of these elements is rather heterogeneous. The cytosol fractions of these metals depends strongly on the plant concerned (e.g., different mobility of Mn in radish and banana or different mobility of Sr in white cabbage and banana, cf. Tables 3 and 5). Iron is not well transferred into a soluble form by ultra-turrax treatment. In all cases the main fraction of these metal is bound to pellet components (Table 3). The different concentrations of the elements determined in the obtained cytosols are demonstrated in Table 6. In summary, it can be stated that the quick and simple method of cell breakdown by ultra-turrax treatment is a useful method to transfer potassium, rubidium, copper, zinc and manganese in significant percentages into the soluble form. In some cases the results for calcium and strontium are satisfactory too.

8

Energy In keV

Fig. 2. TXRF spectrum of digested white cabbage

homogenate.

K. Giinther et al./Analytica

330

Chimica Acta 309 (1995) 327-332

Table 1 Total metal contents of plants referred to their fresh mass (dry mass components Plant [dry mass components

K ( pg/g)

Ca ( pg/g)

Mn ( cLg/g)

2850 (8.4%) 1110 (7.9%) 356 (0.9%) 425 (3.0%) 384 (3.7%) 1410 (9.3%) 790 (10.3%) 910 (8.1%) 1960 (2.2%) 870 (9.5%) 4150 (6.4%) 2810 (10.4%)

750 (9.9%) 297 (0.4%)

3.8 (10.4%)

Fe ( pg/g)

and standard deviations in parentheses,

n = 3)

Cu ( a/d

Zn ( a/d

Rb ( %/d

Sr ( a/P)

1.4 (7.9%) 0.34 (5.1%) 0.45 (15.3%) 0.14 (8.1%) 0.14 (4.8%) 1.2 (6.2%) 0.35 (2.5%) 0.74 (8.0%) 1.46 (8.1%) 0.36 (3.2%) 1.0 (8.7%) 2.1 (0.5%)

8.5 (2.3%)

0.6 (9.9%) 0.4 (16.7%) 0.07 (4.0%) 0.19 (2.2%) < d.1.

1.5 (6.5%) 1.0

(%)I Dill 19.91 Red cabbage 17.11 White cabbage

B.51 Cucumber L6.21 Cabbage lettuce r3.31 Paprika F6.91 Chinese cabbage

L4.61 Chicory L4.11 Parsley (roots) [20.7] Radish 15.01 Banana [25.0] Avocado L21.11

(470.3%) p27.0%) (637%) E9%0) 220 (9.6%) 104 (9.2%) 195 (2.2%) 287 (0.3%) 3a

:ls2.7%)

FZ5%59/,, 0.43 (3.9%) 0.14 (15.0%) 1.02 (1.0%) 0.84 (6,7%) 0.84 (7.4%) 0.4 (4.5%) 1.65 (1.2%) 0.3 (17.6%) 13.3 (1.0%) 1.6 (4.6%)

3.2 (5.9%) :;:2%, 1.8 (10.6%) 1.5 (6.5%) 4.8 (8.2%) 5.2 (16.4%) 4.2 (5.3%) 14.4 (4.6%) 6.0 (14.0%) 2.5 (11.9%) 4.9 (1.6%)

;;:6%, 1.2 (1.8%) 1.1 (12.7%) 1.2 (4.5%) 4.2 (8.3%) 3.6 (8.4%) 2.6 (3.9%) 6.4 (6.0%) 2.5 (1.3%) 2.0 (4.9%) 3.0 (2.7%)

0.5 (12.7%) 0.3 (15.0%) 0.26 (5.5%) 1.4 (8.1%) 0.24 a 0.74 (5.2%) 2.4 (4.5)

(8.7%) 0.1 (7.8%) 0.2 (0.9%) 0.23 (9.1%) 0.2 (15.5%) 0.37 (10.9%) 0.24 (3.6%) 1.12 (0.1%) 0.7 (22.8%) 0.4 (7.0%) 0.5 (8.1%)

d.1. = Detection limit. a Only one value above d.1.

Table 2 Percentage distribution of K and Ca between (standard deviations in parentheses, n = 3)

cytosol

and pellet after cell decomposition

by ultra-turrax

treatment

and centrifugation

Plant

K Cyt. (%)

K Pel. (o/o)

K Horn. (o/o) Cyt. + Pel. (%)

Ca Cyt. (%)

Ca Pel. (%)

Ca Horn (%)

Cyt. + Pel. (%)

Dill Red cabbage White cabbage Cucumber Cabbage lettuce Paprika Chinese cabbage Chicory Parsley (roots) Radish Banana Avocado

87.6 (4.6%) 77.4 (0%) 89.6 (4.2%) 87.6 (8.0%) 108.5 (9.4%) 83.3 (10.4%) 83.6 (5.0%) 95.0 (18.7%) 76.1 (12.2%) 100.9 (6.0%) 101.4 (6.4%) 104.6 (14.6%)

7.8 (8.7%) 17.6 (4.9%) 11.0 (8.8%) 9.8 (17.4%) 3.1 (13.8%) 5.3 (11.2%) 8.3 (12.1%) 3.6 (12.1%) 16.7 (9.1%) 4.7 (10.3%) 1.6 (8.8%) 8.4 (15.4%)

100 100 100 100 100 100 100 100 100 100 100 100

45.3 (5.4%) 41.9 (4.2%) 28.2 (2.6%) 56.6 (2.8%) 52.4 (12.4%) 22.5 (6.3%) 25.5 (4.5%) 29.1 (8.3%) 16.2 (5.6%) 40.1 (11.4%) < d.1. 31.5 (1.7%)

59.7 (17.6%) 66.8 (8.2%) 83.0 (1.3%) 41.6 (15.6%) 73.2 (5.5%) 80.9 (8.8%) 66.5 (1.2%) 72.0 (9.6%) 84.5 (6.9%) 66.0 (6.9%) 100.0 (8.8%) 63.8 (7.7%)

100 (9.6%) 100 (0.4%) 100 (10.3%) 100 (2.0%) 100 (3.7%) 100 (5.9%) 100 (9.6%) 100 (9.2%) 100 (2.2%) 100 (0.3%) 100 a 100 (12.7%)

105.0 108.7 111.2 98.2 125.6 103.4 92.0 101.1 100.7 106.1 100.0 95.3

d.1. = Detection limit. a Only one value above d.1.

(8.4%) (7.9%) (0.9%) (3.0%) (3.7%) (9.3%) (10.3%) (8.1%) (2.2%) (9.5%) (6.4%) (10.4%)

95.4 95.0 100.6 97.4 111.6 88.6 91.9 98.6 92.8 105.6 103.0 113.0

K. Giinther et al. /Analytica Table 3 Percentage distribution of Mn and Fe between (standard deviations in parentheses, n = 3)

cytosol

Chimica Acta 309 (1995) 327-332

and pellet after cell decomposition

by ultra-turrax

331

treatment

and centrifugation

Plant

Mn Cyt. (%)

Mn Pel. (%)

Mn Horn. (%)

Cyt. + Pel. (o/o) Fe Cyt. (%I

Fe Pel. (%I

Fe Hom (%)

Cyt. + Pet. (%)

Dill Red cabbage White cabbage Cucumber

38.3 49.0 43.8 52.1

(3.4%) (5.4%) (11.3%) (6.5%)

Cabbage lettuce Paprika Chinese cabbage Chicory Parsley (roots)

44.2 52.8 43.4 59.5 39.4

(6.2%) (15.8%) (7.7%) (4.2%) (3.5%)

Radish Banana

< d.1. 81.4 (3.8%) 51.5 (5.6%)

54.9 60.7 65.4 46.4 61.9 40.7 67.0 51.1 71.0 66.7 10.3 44.7

100 100 100 100 100 100 100 100 100 100 100 100

93.2 109.7 109.2 98.5 106.1 93.5 110.4 110.6 110.4 66.7 91.7 96.2

73.2 (14.3%) 73.7 (15.3%) 77.9 (14.8%) 78.3 (12.5%) 67.3 (13.3%) 75.5 (3.7%) 89.2 (3.4%) 74.3 (10.5%) 100.5 (8.0%) 82.4 (0%) 84.6 (3.2%) 83.4 (4.3%)

100 100 100 100 100 100 100 100 100 100 100 100

83.8 109.7 100.0 94.1 98.8 87.4 108.6 96.3 107.4 96.0 110.7 99.4

Avocado d.1. = Detection

(12.5%) (0.3%) (4.8%) (5.9%) (6.2%) (8.9%) (6.7%) (6.7%) (8.7%) (2.8%) (9.2%) (3.5%)

(10.4%) (8.5%) (3.9%) (15.0%) (1.0%) (6.7%) (7.5%) (4.5%) (1.2%) (17.6%) (1.0%) (4.6%)

10.6 (4.7%) 36.0 (1.3%) 22.1 (6.7%) 15.8 (4.6%) 31.5 (13.3%) 11.9 (2.7%) 19.4 (3.5%) 22.0 (8.4%) 6.9 (7.3%) 13.6 (14.4%) 26.1 (16.3%) 16.0 (10.5%)

(3.2%) (5.9%) (9.2%) (10.6%) (6.5%) (8.2%) (16.4%) (5.3%) (4.6%) (14.0%) (11.9%) (1.6%)

limit.

Table 4 Percentage distribution of Cu and Zn between (standard deviations in parentheses, n = 3)

cytosol

and pellet after cell decomposition

by ultra-turrax

treatment

and centrifugation

Plant

Cu Cyt. (%)

Cu Pet. (%o)

Cu Horn. (%)

Cyt. + Pel. (W) Zn Cyt. (%I

Zn Pet. (%)

Zn Horn (%)

Cyt. + Pet. (%b)

Dill Red cabbage White cabbage Cucumber Cabbage lettuce Paprika Chinese cabbage Chicory Parsley (roots) Radish Banana Avocado

73.5 68.5 75.0 55.7 88.0 73.2 71.6 79.3 81.9 62.0 76.7 64.9

26.7 48.2 40.1 42.9 18.3 14.9 37.9 19.1 24.6 33.9 40.8 24.7

100 100 100 100 100 100 100 100 100 100 100 100

100.2 116.7 115.1 98.6 106.3 88.1 109.5 98.4 106.5 95.9 117.5 89.6

45.4 48.0 73.2 24.2 20.3 14.8 17.1 14.9 28.9 26.6 20.0 27.4

100 100 100 100 100 100 100 100 100 100 100 100

102.1 104.9 105.3 94.6 95.7 96.9 94.7 92.3 99.9 90.2 94.2 110.6

Table 5 Percentage (standard

distribution deviations

(16.8%) (5.5%) (3.3%) (3.6%) (15.5%) (15.9%) (4.7%) (8.3%) (2.9%) (10.8%) (7.1%) (1.4%)

(0.1%) (8.9%) (4.2%) (14.7%) (15.5%) (7.1%) (4.7%) (6.6%) (2.6%) (3.7%) (1.7%) (7.6%)

of Rb and Sr between

in parentheses,

(7.9%) (5.5%) (15.3%) (8.1%) (4.8%) (6.2%) (2.0%) (8.0%) (8.1%) (3.2%) (8.7%) (0.5%)

cytosol

56.7 56.9 32.1 70.4 75.4 82.1 77.6 77.4 71.0 63.6 74.2 83.2

(6.7%) (4.4%) (8.1%) (4.6%) (9.6%) (15.0%) (2.0%) (5.1%) (3.4%) (6.4%) (16.1%) (1.8%)

and pellet after cell decomposition

(17.7%) (5.1%) (3.5%) (16.2%) (9.6%) (12.5%) (5.7%) (12.5%) (4.0%) (3.9%) (7.0%) (3.7%)

by ultra-turrax

(2.3%) (7.6%) (1.8%) (12.7%) (4.5%) (8.3%) (8.4%) (3.9%) (6.0%) (1.3%) (4.9%) (2.7%)

treatment

and centrifugation

n = 3)

Plant

Rb Cyt. (%)

Rb Pel. (%o)

Rb Horn. (%o) Cyt. + Pel. (%)

Sr Cyt. (%)

Sr Pel. (o/o)

Sr Horn (%,) Cyt. + Pel. (%Jo)

Dill Red cabbage White cabbage Cucumber Cabbage lettuce Paprika Chinese cabbage Chicory

82.1 (9.0%) 75.3 (7.3%) 90.1 (10.1%) 85.7 (6.5%) < d.1. 91.8 (5.6%) 90.7 (0%) 90.9 (7.3%)

Parsley (roots) Radish Banana

90.8 88.4 90.6 83.0

< d.1. 16.2 (7.9%) < d.1. 15.8 (10.1%) < d.1. 8.7 (8.1%) < d.1. 5.7 (9.8%) 14.1 (1.5%) < d.1. < d.1. 9.9 (16.8%)

100 (9.9%) 100 (16.7%) 100 (4.0%) 100 (2.2%) < d.1. 100 (12.7%) 100 (15.0%) 100 (5.5%) 100 (8.1%) 100 a 100 (5.2%) 100 (4.5%)

34.0 (7.9%) 18.6 (8.9%) < d.1. 38.0 (8.0%) 32.4 (13.1%) 33.8 (18.4%) 20.8 (19.9%) 51.3 (8.0%) 14.8 (12.5%) 22.7 (7.8%) 88.4 (19.5%) 82.9 (11.3%)

55.2 (11.7%) 85.9 (11.3%) 83.6 (7.8%) 54.0 (16.6%) 72.9 (7.1%) 67.6 (16.4%) 68.9 (16.4%) 52.5 (9.0%) 83.0 (5.3%) 77.7 (12.9%) 24.9 (7.0%) 9.4 (20.6%)

100(6.5%) 100 (8.7%) 100 (7.8%) 100 (0.9%) 100 (9.1%) 100 (15.5%) 100 (10.9%) 100 (3.6%) 100 (0.1%) 100 (3.9%) 100 (21.5%) 100 (8.1%)

Avocado

(4.7%) (7.5%) (21.3%) (1.8%)

d.1. = Detection limit. * Only one value above d.1.

82.1 91.5 90.1 101.5 100.5 90.7 96.6 104.9 88.4 90.6 92.9

89.2 104.5 83.6 92.0 105.3 101.4 89.7 103.8 97.8 100.4 113.3 Y2.3

332

K. Giinther et al. /Analytica

Table 6 Total metal contents in the isolated cytosols

(standard

deviations

Chimica Acta 309 (1995) 327-332

in parentheses,

n = 3)

Plant

K ( pg/g)

Ca ( pg/g)

Mn ( wg/g)

Fe ( pg/g)

Cu ( Kg/g)

Zn ( pg/g)

Rb (cLg/g)

Sr ( be/g)

Dill Red cabbage White cabbage Cucumber Cabbage lettuce Paprika Chinese cabbage Chicory Parsley (roots) Radish Banana Avocado

467 (4.6%)

64.1 (5.4%)

0.28 (3.4%)

0.82 (4.6%)

0.20 (16.8%)

0.90 (6.7%)

0.09 (9.0%)

210 200 260 270 240 180

30.3 (4.2%) 8.3 (2.6%) 28.0 (2.8%) 19.0 (12.4%) 2.4 (6.3%) 15.2 (4.5%) 6.6 (8.3%) 7.7 (5.6%) 31.0 (11.4%) < d.1. 4.0 (1.7%)

0.1 (5.4%) 0.13 (11.3%) 0.06 (6.5%) 0.3 (6.2%) 0.09 (15.8%) 0.1 (7.7%) 0.05 (4.2%) 0.16 (3.5%) < d.l 2.63 (3.8%) 0.27 (5.6%)

0.28 (1.3%) 0.34 (6.7%) 0.2 (4.6%) 0.31 (13.3%) 0.1 (2.7%) 0.27 (3.5%) 0.2 (8.4%) 0.23 (7.3%) 0.22 (14.4%) 0.16 (16.3%) 0.26 (10.5%)

0.06 (5.5%) 0.08 (3.3%) 0.08 (3.6%) 0.1 (15.5%) 0.2 (15.9%) 0.07 (4.7%) 0.13 (8.3%) 0.29 (2.9%) 0.06 (10.8%) 0.18 (7.1%) 0.44 (1.4%)

0.34 (4.4%) 0.27 (8.1%) 0.56 (4.6%) 0.61 (9.6%) 0.7 (15.0%) 0.76 (2.0%) 0.43 (5.1%) 1.20 (3.4%) 0.42 (6.4%) 0.40 (16.0%) 0.81 (1.8%)

0.078 (7.3%) 0.043 (10.1%) 0.115 (6.5%) < d.I. 0.09 (5.6%) 0.064 (0%) 0.052 (7.3%) 0.29 (4.7%) 0.043 (7.5%) 0.16 (21.3%) 0.66 (1.8%)

0.10 (7.9%) 0.045 (8.9%) < d.1. 0.054 (8.0%) 0.054 (13.1%) 0.01 (18.4%) 0.02 (19.9%) 0.027 (8.0%) 0.04 (12.5%) 0.05 (7.8%) 0.09 (19.5%) 0.14 (1.8%)

d.1. = Detection

(0%) (4.2%) (8.0%) (9.4%) (10.4%) (5.0%)

190 (18.7%) 340 (12.2%) 235 (6.0%) 1020 (6.4%) 980 (14.6%) limit.

During further investigation of the isolated cytosols by coupling of separation methods (e.g., gel permeation chromatography, GPC) and multielement analytical techniques (e.g., TXRF) a speciation of a significant amount of these elements could now be achieved. A GPC-TXRF off-line system for example was used for screening tasks in the speciation of 12 elements in vegetables. Exemplary cytosols of lamb’s lettuce and cauliflower were separated by a Sephadex G-50 column and the obtained fractions were analysed directly by TXRF with an internal cobalt standard [8]. For a first characterization of element species in solution this GPC-TXRF off-line coupling is a very suitable system. In the present study, total reflection X-ray fluorescence spectrometry is a very useful analytical technique for the simultaneous multielement determination in the resulting samples. TXRF analyses are rapid and require only a minute amount of sample (about 20 ~1). Especially the internal standardisation makes sample preparation quite simple, and a concentration range of about four orders of magnitude can be investigated reliably by this technique. Further, the non-destructive operation enables the same sample to be analysed repeatedly. In addition, other elements can be determined by TXRF in the resulting fractions after liquid shearing and ensuing centrifugation. Apart from the elements quoted in the tables, for example Se, Cr, Co, Tl, Pb, Pt belong into that group. If a W tube instead of a MO tube is used, for example the elements Cd, Ag, Sn. Sb can also be determined.

In further studies using different biological samples the solubility behaviour of these elements could also be elucidated by using TXRF as the analytical method in the initial step of element speciation.

Acknowledgements We are grateful to the Deutsche Forschungsgemeinschaft (Gu 248/1-l), the Ministerium fur Wissenschaft und Forschung des Landes NordrheinWestfalen and the Bundesministerium fur Forschung und Technologie for financial support.

References [l] J.A.C. Broekaert, S. Gicer and F. Adams, Metal speciation in the environment, NATO AS1 Series, Springer-Verlag, Berlin, 1990. [2] M. Bernhard, F.E. Brinckman and P.J. Sadler, The importance of chemical speciation in environmental processes, Dahlem Konferenzen, Springer-Verlag, Berlin, 1986. [3] P.H.E. Gardiner, in Topics in Current Chemistry, Vol. 141, Springer-Verlag, Berlin, 1987. [4] G. Tolg and R. Klockenkamper, Spectrochim. Acta, 48B (1993) 111. [5] R. Klockenkamper and A. von Bohlen, J. Anal. At. Spectrom., 7 (1992) 273. [6] A. Prange, Spectrochim. Acta, 44B (1989) 437. [7] U. Reus and A. Prange, Spectroscopy Europe, 5 (1993) 26. [8] K. Gunther and A. von Bohlen, Spectrochim. Acta, 46B (1991) 1413.