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Methods for Environmental Trace Analysis
Figure 5.23 Details of Step 3 of the sequential extraction method (cf. Figure 5.20).
Clean all vessels in contact with samples or reagents with HNO3 (4 moll-1) overnight and then rinse with distilled water.
Determine the blank as follows. To one vessel from each batch, taken through the cleaning procedure, add 40 ml of acetic acid (Solution A, see below). Analyse this blank solution along with the sample solution from Step 1 (as described in Figure 5.21).
Use a mechanical shaker, preferably of the horizontal rotary or the end-over-end type, at a speed of 30 rpm. Carry out the centrifugation at 1500 x G.
The reagents to be used for the three steps are as follows:
Water. Glass-distilled water is suitable. Analyse a sample of distilled water with each batch of the Step 1 extracts.
Solution A (acetic acid; 0.11 mol l-1). Add in a fume cupboard, 25 ± 0.2 ml of glacial acetic acid (AnalaR grade or similar) to about 0.5 l of distilled water in a 1 l polyethylene bottle and make up to 1 l with distilled water. Then make up 250 ml of this solution (acetic acid; 0.43 moll-1) with distilled water to 1 l to obtain an acetic acid solution of 0.11 moll-1. Analyse a sample of each batch of Solution A.
Solution B (hydroxylamine hydrochloride or hydroxyammonium chloride; 0.1 moll-1). Dissolve 6.95 g of hydroxylamine hydrochloride in 900 ml of distilled water. Acidify with HNO3 to pH 2 and make up to 1 l with distilled water. Prepare this solution on the same day as the extraction is carried out. Analyse a sample of each batch of Solution B.
Solution C (hydrogenperoxide; 300 mgg-1, i.e. 8.8 moll-1). Use the H2O2 as supplied by the manufacturer, i.e. acid-stabilized to pH 2-3. Analyse a sample of Solution C.
Solution D (ammonium acetate; 1 moll-1). Dissolve 77.08 g of ammonium acetate in 900 ml of distilled water, adjust to pH 2 with HNO3 and make up to 1 l with distilled water. Analyse a sample of each batch of Solution D.
The sequential extraction procedure can be carried out for the following trace metals: Cd, Cr, Cu, Ni, Pb and Zn. A separate sub-sample of the sediment or soil should be dried in a layer of approximately 1 mm in depth in an oven at 105 ± 2° C for 2-3 h and then weighed. From this, a correction to dry mass can be obtained and applied to all of the analytical results obtained (quantity per g dry sediment/soil).
While the complete experimental details for the three steps in this sequential extraction method are shown in Figures 5.21 -5.23, it is important to note the following:
The calibrating solutions should be made up with the appropriate extracting solutions.
For every batch of extractions, a blank sample, i.e. a container with no sediment/soil, should be carried through the entire procedure.
The sediment/soil should be in complete suspension during the extraction. If this is not the case, adjust the shaking speed to ensure that the suspension is maintained.
The results obtained from the sequential extraction of a certified reference material (CRM 601) are shown in Table 5.11. Finally, it is recommended that for ICP analysis a final filtration (0.45 ^m) is carried out in order to prevent
Methods for Environmental Trace Analysis
Table 5.11 The results obtained from the sequential extraction of a certified reference material
(CRM 601) 
Metal Certified value (mg kg-1) Uncertainty (mg kg-1)
First step Cd 4.14 0.23
Cr 0.36 0.04
Cu 8.32 0.46
Ni 8.01 0.73
Pb 2.68 0.35
Zn 264.0 5.0
Second step Cd 3.08 0.17
Ni 6.05 1.09
Pb 33.1 10.0
Zn 182.0 11.0
Third step Cd 1.83 0.20
Ni 8.55 1.04
Pb 109.0 13.0
nebulizer blockages. If GFAAS is the method of analysis, it is recommended that the standard additions method of calibration is used (see Chapter 1).
5.7.5 Food Studies
In related studies, the simulated gastro-intestinal digestion of foodstuffs has been used as an indication of the bioavailable forms of metal species, and hence the potential for subsequent uptake from the intestine. A two-stage enzymolysis procedure has been developed and is shown in Figure 5.24. This procedure was developed to simulate in vitro enzyme treatments for investigating the speciation of metals from ingested foodstuffs. It consists of two stages, following by a post-enzymolysis acidification step. The first stage involves treatment of the foodstuff with pepsin at pH 2.5, followed in the second stage by pancreatin and amylase at pH 7. All enzymolysis extracts were analysed to assess the effectiveness of each stage to solubilize trace metals. Some selected results for the solubilization of trace metals from foodstuffs are shown in Table 5.12. The following conclusions were made: the solubility differs for different food items, thus reflecting the differences in the complex chemical nature of the original foodstuff; the enzy-molysis itself brings about speciation changes, e.g. the reduction in solubility of cadmium from wholemeal bread and copper from tomato and spinach, after treatment with pancreatin and amylase; the pH affects the analyte solubility, with