Ideal standards for quantitative x-ray microanalysis of biological specimens.

The use of ion exchange beads is proposed as standards for the estimation of various elements in biological tissues. Elements to be detected or quantitated in biological tissues occur in three forms: as free ions or salts, in a crystalline phase, and bound to a matrix. For the latter category a standard system using ChelexlOO -jon exchange beads is proposed. Cations can be attached in a controlled way to such an artificial polystyrene divinyl benzene matrix, furnished with negatively charged iminodiacetate functional groups by replacing the sodium ions present in the commercial product. In Fig. 1 beads measuring 35-75 ym in 0 are shown in the SEM-mode as standards for bulk analysis, attached to a graphite plate with graphite glue (Leit C). The analysis of Co-loaded beads is given in Fig. 2. Loading conditions from salt solutions are described elsewhere (1). The binding is controlled mainly by the pH of the reaction. After loading, the excess of the equilibrium solution can be washed away by rinsing with a buffer having the same pH as the equilibrium solution. When the beads are maximally loaded, the concentration of the various elements vary according to the differences in affinitybetween 2 and 15 w/w% (as far as we have measured them), but almost every concentration below the maximum value can be obtained by changing the equilibrium conditions. The concentration on the maximally loaded beads was determined at 40 or 60 kV on 35-75 ym 0 beads with either the pure metal or copper as a reference. On identical samples the concentration was determined by neutron activation (Interuniversitair Reactor Instituut, Delft; Table I). TABLE I. Mean maximum concentration on Chelex -beads (in X w/w). Zn Cu Ni Co Fe Mn Ca Mg Na H(Na) Neutron activation 12.2 12.7 9.8 12.1 11.5 9.4 6.5 4.1 6.3 0.01 40 kV 13.0 13.9 12.9 10.5 11.5 10.5 6.2 3.7 — 0 + S.D. 2.3 1.6 0.6 0.5 0.6 0.5 0.6 1.8 60 kV 12.6 13.7 11.5 10.5 11.4 8.2 5.5 4.9 — 0 + S.D. 0.8 0.5 0.4 0.4 0.7 0.5 0.5 0.7 In the spectra sometimes some residual CI concentration is present in addition to the requested peak, as in most cases chlorine salts are used for the exchange. The The use of nitrates will prevent that. Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842107 C2-470 JOURNAL DE PHYSIQUE F i g . 1. Chelex beads on a g r a p h i t e s u b s t r a t e i n SEN-mode. Bar: 100 um. I n s e t : funct i o n a l group e q u i l i b r a t e d w i t h Nat. F i g . 2. Spectrum o f coba l t l oaded Chelex beads 60 kV, 100 nm spo t s i ze . The convers i o n o f t h e Na i n t o Co beads i s complete. The c o b a l t c o n c e n t r a t i o n was e s t i mated t o be: 10.5 ( o r 12.1)% (Tab le I). F i g . 3. P.K.C.-program f o r measur ing beads and X-ray a n a l y s i s i n t h e centre.Bar: 10 um. F i g . 4. Ag-'1P 1-beads loaded w i t h phosphate, spectrum taken a t 60 kV,100 nm spo t p l . F i g . 5. Superimposed images o f a bead and i t s measuring by t h e P.R.C.'program, t o demonstrate t h e accuracy o f t h e measurement. Bar: 10 um. F i g . 6. Ag-MP 1-beads loaded w i t h bromine, which more comp le te l y removes the cohimerc i a l l y p resen t c h l o r i n e than t h e phosphate d i d . 60 kV, 100 nm s p o t s i z e .