1) Microwave Digestion Procedure
1. To a microwave digestion tube, add 0.1 g (+ 0.01 g) of ground, homogenised plant material. See Figure 1 for example of digestion vessels.
2. Add 9 mL of concentrated nitric acid and 1 mL of hydrogen peroxide to the plant material.
3. Allow the mixture to react in the vessel open in the fume hood for ~45 minutes. NOTE: Samples will effervesce and release gas. Some samples will effervesce strongly and may require that half the acid and hydrogen peroxide are added and allowed to react for 30 minutes before the other half of the acid and peroxide are added. This is to prevent samples spilling over the top of the digestion vessel.
4. Seal the microwave digestion vessels and digest for 15 minutes at 200°C.
5. Open vessels in the (ideally, scrubbed) fume hood once they have cooled below 60°C. Warning:Loosen the digestion tubes from their frames (if applicable) slowly as acid bubbles and aerosols may emerge from the seal between vessel and lid as pressure in the vessel is released. Once bubbling has stopped open the vessels. Toxic yellow/orange/brown fumes (NOx gases) will be released when lid is removed so vessels should be opened in a scrubbed fume cupboard. Approximately 9mL of solution will remain.
6. Fill the 50 mL conical tubes with 36 mL of MilliQ water so that acid digests are diluted to a lower concentration of P for analysis.
7. Pour the acid mixture of each digestion tube into a 50 mL conical tube. Store at 4°C until ready for analysis.
2) Preparation of Reagents for Segmented Flow Analysis
All reagents can be scaled down depending on how many samples need to be ran. Reagents and standards are prepared as outlined in Fishman & Friedman (1989), O’Dell (1993) and Skalar (2023).
1. Diluent: In a 5 L vessel add ~4.9 L of MilliQ water. Add 90 mL of concentrated nitric acid and 10 mL of hydrogen peroxide to mimic the background acid concentration of samples. Stability 1 month.
2. Sulphuric acid solution: Fill a 1 L volumetric flask with 800 mL of MilliQ water. Add 40 mL of sulphuric acid and dilute to the 1 L mark. Add 2 mL of FFD6.Stability 1 week.
3. Distilled water and FFD6: Fill a 1 L volumetric flask with MilliQ water then add 2 mL of FFD6 and mix. Stability 1 week.
4. Ammonium heptamolybdate solution: Add 800 mL Milli-Q water to a 1L volumetric flask and add 230 mg of potassium antimony oxide tartrate and swirl to dissolve. Add 69 mL of sulphuric acid (98%) and leave to cool. Add 6g of ammonium heptamolybdate using a plastic spoon. Swirl to mix. Dilute to the 1 L mark with Milli-Q water then add 2 mL of FFD6. Stability 1 week.
5. Ascorbic Acid Solution: Add 800 mL Milli-Q water to a 1 L volumetric flask, then add 11 g Ascorbic acid. Swirl to dissolve. Dilute to 1 L using Milli-Q water. Stability 1 week, keep refrigerated.
3) Preparation of Standards
Preparation of standards must be done on the day samples are being run. Make new standards each day.
1. Phosphorus Stock Solution (1000 mg P/L): Weight out 4.394 g of potassium dihydrogen phosphate. Add 800 mL Milli-Q water to a 1 L volumetric flask, then add the potassium dihydrogen phosphate. Dilute to the 1 L mark with Milli-Q water. Stable for 1 month.
2. Phosphorus Working Solution (10 mg P/L): Dilute 0.3 mL of stock solution to 30 mL using MilliQ water.
3. Using the autosampler (Skalar SA1074 Autosampler Random Access) program the preparation of standards 1-7 to be made up from the working solution. Figure 2 outlines the concentrations of standards that should be run for analysis.
4) Running Auto-Analyser
1. First run all lines with Milli-Q water to check for leaks.
2. Run reagents through the reaction manifold for approximately 10 minutes to prep the lines.
3. Add working standard and samples into the autosampler and run, as per the manufacturer’s instructions, using the 880 nm detector to detect for orthophosphate. Samples should be diluted 4-fold from the diluted digest samples.
4. At the end of a run, run Milli-Q water through all lines for approximately 10 mins. Turn off machine.
5. Assess peaks – if peaks are outside the calibration curve range they need to be further diluted and re-run.
6. Calculate phosphorus content as a percentage of plant material using the following equation:
Phosphorus (% of plant material) = (mg P/L * volume of acid digest (L) * dilution factor) / weight of plant material (mg) * 100