Acetic Acid Extraction Protocol

The extraction should be performed in 250 mL pre-cleaned borosilicate glass, polypropylene or PTFE bottles using an end-over-end shaker. All laboratory 

Extractants should be prepared according to the following procedure. 0.43 mol acetic acid should be prepared by adding in a fume cupboard 250 2 mL of redistilled glacial acetic acid to about 5 L of distilled water in a 10 L polyethylene container. The solution should be diluted with distilled water to 10 L volume, mixed well and stored in a stoppered polyethylene container. 

The sample for analysis should be taken as it is. Before a bottle is opened it should be manually shaken for 5 min to rehomogenize the content. The results should be corrected for dry mass this correction must be performed on a separate portion of 1 g taken at the same time from the same bottle by drying in an oven at 105 2°C for 2-3 h until constant mass is attained (successive weighings should not differ by more than 1 mg). 

NH4NO3, CaCl2 and NaN03 Extraction Protocols Used in the Certification (Indicative Values) 

In addition to EDTA and acetic acid, weak extraction procedures were applied by some laboratories in the course of the certification, e.g. 0.1 molL NH4NO3, 0.01 mol CaCl2 and 0.1 mol NaNOs. These procedures are outlined below. It is emphasized that they have not been validated by an interlaboratory trial and that the values given in this report are indicative only. 

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For the homogeneity studies, the extractants (0.05 mol L EDTA, 0.43 mol L" acetic acid and 0.005 mol L DTPA) were prepared as laid out in the certification reports [15, 17], The trace element contents (Cd, Cr, Cu, Ni, Pb and Zn) in the extracts were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) for the CRMs 483/484, flame atomic absorption spectrometry (FAAS) or electrothermal atomic absorption spectrometry with Zeeman background correction (ZETAAS) for the CRM 600. In the case of the CRM 483, little analytical difficulty was experienced as illustrated by the good agreement obtained between the within-bottle and between-bottle CVs for the CRM 484, lower extractable contents, closer to the detection limits and consequent poorer analytical precision was observed in particular for Cr (EDTA extractable contents), Cd and Pb (acetic acid extractable contents). No particular difficulties were experienced for the CRM 600. On the basis of these results, the materials were considered to be homogeneous at a level of 5 g for EDTA- and acetic acid-extractable contents and 10 g for DTPA-extractable contents (as specified in the extraction protocols). 

The protocols for staining with amido black, Coomassie blue, Ponceau S, and AuroDye follow the suppliers recommendations. It should be noted that when staining PVDF membranes with Coomassie blue before N-terminal sequencing, omitting acetic acid from both the stain and destain solution is recommended to minimize potential extraction of protein from the membrane (Speicher, 1989). 

Betalains Materials Solvent A 18/82(v/v) CH3OH/0.05 M KH2PO, adjust to pH 2.75 with H3P04 Solvent B. CH3OH Sephadex G-25 (Amersham Pharmacia Biotech) water slurry, pH 2.0 adjust pH with HC1 Sample of beet juice or beet tissue extract (see Support Protocol), pH 2.0 adjust pH with HC1 1% acetic acid Four 7.88-mm x 61-cm Bondapak C,8/Porasil B columns connected in series 17.8/81.2/1.0 (v/v/v) CH3OH/0.05 M KH2P04/acetic acid 0.1% HC1... 

In order to improve comparability between results of extraction protocols for soil plant-available PTMs obtained by different laboratories, the Measurement and Testing Programme (formerly BCR) of the European Commission has recently proposed two harmonised procedures for extractable Cd, Cu, Cr, Ni, Pb and Zn in soils on the basis of 0.05 M EDTA (pH 7.0) and 0.43 M acetic acid solutions (Rauret et al, 2001). Moreover, a suitable reference material (BCR-700 organic-rich soil) for these extractions was prepared, which enable the quality of the measurements to be controlled. 

The other popular sequential extraction procedure is the protocol proposed by the Community Bureau of Reference, Commission of the European Community (known as the BCR protocol). The method was proposed on the basis of interlaboratory smdies undertaken in order to harmonize conditions for soil and sediment sample analysis. Based on the research data, in 1992 it was stated that application of EDTA or acetic acid solution is appropriate and sufficient for elimination of the bioaccessible fi action of metals from soil samples [62]. In the case of other samples, best results were achieved after application of a three-stage procedure with the following extractants ... 

The project was started in 1987 by a consultation of Emopean experts examining the possibility of harmonizing single and/or sequential extraction schemes for soil and sediment analysis [60]. This inquiry was followed by the design of single extraction schemes (EDTA, acetic acid and ammonium acetate) and a sequential extraction protocol which were proposed to a group of ca. 30 laboratories for possible harmonization in 1989 [193]. Interlaboratory studies (two on soils and two on sediment) were carried out between 1989 and 1993 [194-196] and were followed by certification campaigns conducted between 1994 and 1996 [197-199]. 

Since both the temperature of extraction and the vigour of the mechanical shaking could be expected to affect the amount of metal extracted, limits for both of these parameters were prescribed in the detailed protocol for extraction and analysis issued to the participating laboratories along with the solid sample [193]. As a check on each laboratory s calibration, reference solutions containing the trace metals of concern (Cd, Cr, Cu, Ni, Pb and Zn) were prepared. Three types of single extractant were used EDTA (0.05 mol L" ), acetic acid (0.43 mol L ) and ammonium acetate (1 mol L ). 

A variety of protocols utilising combinations of liquid-liquid and solid-phase extractions (LLE and SPE) have been used to clean-up tissue extracts. Alkaline extracts are commonly made acidic, extracted into ethyl acetate and then back-extracted into aqueous buffer at alkaline pH. Acidic extracts have been extracted directly into ethyl acetate and then back-extracted into buffer. QCA and mQCA may act as acids or bases, and both of these properties have been utilized in the SPE clean-up of the buffered extracts from the initial liquid-liquid partitions. Extracts were acidified prior to clean-up on non-endcapped sex (strong cation exchange) SPE columns. The analytes of interest were eluted from the columns using a mixture of sodium hydroxide and methanol. Further clean-up and transfer to an appropriate solution for instmmental analysis was achieved by re-acidification... 

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