4.1 Method Modifications During the Purification Process

The original purification protocol is using 1 M HCl extracts but it is nowadays used for a diverse set of extracts (Tamburini et al. 2010, 2018; Pistocchi et al. 2017; Granger et al. 2018; Pfahler et al. 2020). Depending on the extract it is necessary to modify the purification protocol slightly. Also, each sample is different and despite thorough testing of the purification protocol, issues might occur.

4.1.1 Modification 1

The ideal pH for the precipitation of ammonium phosphomolybdate (APM; step A1 of purification protocol) is 1. When using acidic reagents that are weaker than 1 M HCl, for example, 0.2 M HNO3 for eluting resin P, it is therefore necessary to add concentrated sulphuric acid (H2SO4). Concentrated H2SO4 is a strong acid that could potentially hydrolyse any organic or condensed P present in the extract. Add concentrated H2SO4 slowly to the sample only after ammonium molybdate and ammonium nitrate are added to the extract in step A1 of the purification protocol. Usually, 1 ml concentrated H2SO4 is sufficient to facilitate the precipitation of APM.

4.1.2 Modification 2

If the initial extract, e.g., 0.5 M NaHCO3, has an alkaline pH, start with step A2 to avoid unnecessary pH adjustment. Indeed, step A2 of the purification protocol (magnesium ammonium phosphate (MAP) precipitation) requires an alkaline pH (>7). In these cases, the crystals obtained from the MAP step are never properly clean. So, after the dissolution of the MAP crystals, proceed with step A1, repeat step A2 and continue with the remaining steps.

4.1.3 Modification 3

Certain cations like iron (Fe), silica (Si) and calcium (Ca) can interfere with the purification protocol and therefore their concentrations need to be reduced prior to Step A1. High concentrations of Si can be present in volcanic soils and can interfere with the molybdate complexation in step A1 of the purification protocol. Ca can also interfere in step A1 due to the formation of crystals with molybdate. Fe could form colloids and co-precipitated with organic P. Brucite precipitation should be added before step A1 to reduce the concentrations of interfering cations. Alternatively, cation exchange resins could help in reducing the cation concentrations; however, this has, to the best of our knowledge, not yet been tested in case of the purification protocol.

4.2 Major Issues

4.2.1 Issue 1: No APM Precipitation

This is often the case when the initial extract is not acidic enough. In most cases modification 1 will help. In the presence of high carbonate concentrations, even the 1 M HCl extract might not be acidic enough (Pistocchi et al. 2017). Pistocchi et al. (2017) therefore adjusted the liquid-to-solid ratio for their samples during the extraction to 100:1 instead of 50:1. Too low P concentrations (<10 µmol) can also be a reason for no APM precipitation. In that case, the sample needs to be extracted again and a higher amount of material needs to be used. In this case, it is possible that multiple subsamples are extracted and then the extracts are combined. A brucite step is most likely needed in order to reduce the extract volume prior to APM precipitation.

4.2.2 Issue 2: High Organic Matter

In most cases, for example with low organic matter but high inorganic P concentrations, steps A1 and A2 are sufficient to remove organic matter from the initial extracts. Sometimes steps A1 and A2 are not sufficient and it is necessary to use the DAX-8 resin or brucite precipitation before proceeding with the purification protocol. Organic matter or colouration is also an issue in Olsen P extracts and charcoal is used to deal with this issue. However, charcoal is often contaminated with P and hence not ideal for the δ18OP method, unless acid cleaned and checked for P.

For conditioning the DAX-8 resin, proceed as follows: Take the equivalent of 10 ml of resin per sample and place it in a 500 ml plastic bottle. To condition the new resin, use 1.5 bed volumes (BV) of methanol, shake well, and let rest for 15 min. Discard methanol, rinse with 1.5 BV of ultrapure water, shake and let rest for 15 min. Carefully discard water. Add ultrapure water just to cover the surface of the resin. After use, the resin should be collected and washed with 1 M HCl + methanol. Store in methanol and at room temperature.

4.2.3 Issue 3: Crystals not Dissolving and Discolouration of Solution

Sometimes the ammonium phosphomolybdate (APM), which formed during step A1, does not dissolve immediately in the ammonium citrate solution (step A2). In this case, leave the solution for about 1 h and if it did not dissolve by then, filter it before continuing with the remaining parts of step A2. Likewise, magnesium ammonium phosphate (MAP), which formed during step A2, does not dissolve immediately when adding 0.5 M HNO3. Leave the solution for one hour; if not dissolved by then, filter the solution.

Another issue, which might occur, is the discolouration of the solution (Fig. 4.1). Dissolved APM and MAP should yield clear solutions. Filtering the solutions prior to continuing with the respective step (A2 or A3), usually reduces the discolouration.

Fig. 4.1
figure 1

Dissolved, but decoloured ammonium phosphomolybdate (APM)

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