May 11, 2026
Hydrogen peroxide assisted soil sample digestion protocol for ICP-MS analysis of plant-available elements
- Sabine Ambrosius1,
- Maren Huppertz2,
- Sabine Metzger2,3
- 1Institute for Plant Sciences, Department of Biology, University of Colonge, Colonge, Germany;
- 2Biocenter MS-Platform, Department of Biology, University of Colonge, Colonge, Germany;
- 3Cluster of Excellence on Plant Sciences (CEPLAS), Colonge, Germany
Protocol Citation: Sabine Ambrosius, Maren Huppertz, Sabine Metzger 2026. Hydrogen peroxide assisted soil sample digestion protocol for ICP-MS analysis of plant-available elements. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp1j7pgzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 12, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 238437
Keywords: ICP-MS, soil material, digestion, nitric acid, hydrogen peroxide, plant available nutrients, soil sample digestion protocol for icp, soil sample digestion protocol, available elements from the soil, various elements from the soil, leachable soil material, hydrogen peroxide, available elements plant, ms analysis of plant, step instruction for the digestion, soil, trace element analysis, plasma mass spectrometry, nitric acid, available nutrient, roots as plant, hno₃, free solution suitable for icp, coupled plasma mass spectrometry, digestion
Funders Acknowledgements:
Deutsche Forschungsgemeinschaft (DFG), Collaborative Reasearch Centre TRR341 on 'Plant Ecological Genetics'
Grant ID: 456082119
Deutsche Forschungsgemeinschaft (DFG), Germany's Excellence Strategy, EXC-2048/1, CEPLAS
Grant ID: 39068611
Abstract
Plants absorb various elements from the soil through their roots as plant-available nutrients. There are several methods for identifying and quantifying these elements. One such method is trace element analysis using inductively coupled plasma mass spectrometry (ICP-MS). This protocol was developed as a guide for leaching the desired plant-available elements from the soil suitibale for ICP-MS. It provides a step-by-step instruction for the digestion of plant-available, leachable soil materials using 30% nitric acid (HNO₃) and 30% hydrogen peroxide (H₂O₂). Useful tips and notes are listed throughout the text in yellow note boxes. This protocol covers the sampling, reagent addition and heating parameters in preparation for the initial digest. Following digestion, samples are diluted and centrifuged to remove particles, resulting in a clear, particle- and precipitation-free solution suitable for ICP-MS analysis.
Guidelines
- Sampling: sample drying, weighing and filling the tubes.
- Digest of soil sample material with 30% HNO3 and 30% H2O2.
- Calculation of the dilution factor.
GOAL: The release of elements of interest that are plant-available in the soil into a final clear, particle-free sample solution suitable for measurement with an ICP-MS.
Materials
- Concentrated (67 - 69%) HNO3 (nitric acid), suitable for trace analysis in ppb range (e.g. Sigma Aldrich Nitric acid 69%, Suprapur - 115171000) - Diluted to 30% with de-ionized water.
- 30% H2O2 (hydrogen peroxide), suitable for trance analysis in ppb range (e.g. Sigma Aldrich Hydrogen peroxide solution ≥30%, for trace analysis - 95321).
- 15 mL conical centrifuge tubes, metal-free - One tube set for the digest and another set for the final samples, depending on the ICP-MS sample system, this can also vary. Check whether the tubes being used can withstand the centrifuge's rated force (e.g. VWR, Centrifuge Tubes, Metal-Free, with Screw Cap - 525 0629).
- OPTIONAL: 50 mL conical centrifuge tubes, metal-free - For storage of the dried soil material and larger samples sizes (e.g. VWR, Centrifuge Tubes, Metal-Free, with Screw Cap - 525 0631).
- highly de-ionized water - Type I ultrapure water (per ASTM D1193), with a resistivity of 18.2 MΩ·cm at 25 °C .
- Water bath - Should be able to reach a temparature of 100 °C .
- Conical centrifuge tube holder - To hold the centrifuge tubes.
- OPTIONAL: Multi-stepper pipette - Makes the life easier in the dilution step.
- Centrifuge with conical tube rotor (up to 12000 x g ) - If possible, adjustable to 4 °C , as we have found that this reduces the precipitation of samples further on.
Safety warnings
Danger
Strong acids - please take note of the safety data sheets.
Burns due to high temperatures and stronger chemical reactions between the acids while heating up.
Before start
IMPORTANT: Please remember and remind yourself about potential safety measures before starting this protocol. Depending on the sample type and the amount of material used, variations can occur in this protocol. The variations are indicated through the comments and notes and examples for these variations are given.
This protocol has been drawn up to demonstrate how soil samples are prepared for the analysis of plant-available trace elements using ICP-MS. In addition to soil material, other sample matrices, including plant and animal material, may also be analysed using ICP-MS. However, other protocols are more suitable for these sample materials.
IN GENERAL, THE FOLLOWING APPLIES TO THIS PROTOCOL: The goal is to create a clear, particle-free sample solution suitable for ICP-MS without introducing foreign elements of interest. This is NOT a total digestion process; rather, the aim is to leach the plant-available elements from the soil material. For da total digest, a different protocol must be followed using Aqua regia (a mixture of nitric acid and hydrochloric acid).
While this protocol provides general guidance for the procedure, modifications and adjustments may be required depending on the soil material, available equipment, and experimental conditions. This protocol focuses on leachable soil material and as such uses 30% HNO3 and 30% H2O2 for the digestion.
Sampling of soil material
1d
Collect and prepare the soil samples. These samples should be representative of the experiment site and the experiment question. It is therefore advisable to take samples from several locations within the experiment area and mix them. These samples may also be non-homogeneous.
Note
- Depending on the properties of the soil samples, the drying procedure may vary. In agricultural science, several soil preparation protocols for trace element analysis have already been developed and established. As such they can be used as guidelines for different soil types if problems occur during this protocol.
- This protocol focuses on leaching elements from the soil that are available to plants, as such the depth from which the soil sample is taken should also be considered. Different plants have different types of roots and reache different depth in the soil.
Mix samples thoroughly and remove large samples objects such as stones, wood, or plants if necessary. During this step, care should be taken to ensure that the samples remain representative. Larger particles should be verified for their relevance to the experiment and research question before removal. Afterwards the samples are dried in an oven at 40 °C to 60 °C until no more weight fluctuation is observed.
Note
- The drying step can be done in 50 mL conical centrifuge tubes or any other suitable tubes, as well as trays.
- It should be noted that the drying time increases when done in tubes.
If big soil pieces are present, dried soil samples should be hand-milled with a metal-free mortar and pestle, or crushed by hand to aid later leaching by digestion.
Note
- The soil material can also be sieved to remove big pieces that cannot be reduced in size, but it is important to avoid contamination with elements of interests, e.g. metals from a metal sieve.
- It should be noted, however, that sieving the soil sample may remove more than just the unwanted foreign matter from the sample. It should therefore first be assessed whether sieving is the correct approach or whether it is better to remove foreign matter individually.
Label two sets conical centrifuge tubes for digest.
Note
- Two sets of metal free 15 mL conical centrifuge tubes are needed in this protocol. The digest is done in the first set, and the final soil samples are stored in the second set.
- Keep in mind that, depending on the composition of the soil sample, strong reactions may occur during digestion with HNO3 and H2O2. Therefore, a preliminary test should be conducted to determine how the samples may react.
- Bigger 50 mL metal free conical centrifuge tubes can also be used to give the digestion reaction more space.
- Soil samples that are very high in calcium or iron tend to foam strongly when digested with H₂O₂. In addition, we have found that conventional German potting soil produces little to no foam when digested with H₂O₂. In contrast, some soil samples taken from the wild occasionally react very strongly to the addition of H₂O₂.
Weigh around 100 mg of the dried soil material into the first set of metal free 15 mL conical centrifuge tubes, then proceed with the digestion.
Note
- Note down the exact weight of the samples, since they are needed of the calculation for the dilution factor later on.
- If it is unclear how the soil samples will react to the digestion process, it makes sense to start by using smaller amounts of sample material and larger tubes.
- If more than 100 mg of soil samples have to be digested, 50 mL metal free conical centrifuge tubes should be used instead of the metal free 15 mL conical centrifuge tubes.
Digest of soil material with HNO3 and H2O2
19h
Add 500 µL 30% HNO3 to each sample and incubate at Room temperature for at least 02:00:00 .
Note
- Depending on the soil material used, the HNO3 can be completely absorbed by the soil material and form a slurrry. This is normal. Continue as usual.
2h
Add another 500 µL 30% HNO3 and incubate the samples Overnight at 65 °C in a water bath.
Note
- To prevent pressure generation and sample dilution with unwanted water from the condensation in the water bath, place the screw caps loosely tighten on the conical centrifuge tubes.
- At the end of steps 6 and 7, the total amoungt of 30% HNO3 that has to be added to the sample is 1 mL .
14h
The next day, check the samples before continuing. The samples, which became slurries when acid was added, should have been separated back into two phases by now. This is important because a liquid phase must be present for the rest of the protocol.
Note
- If this is not the case, the amount of 30% HNO3 needs to be increased and the Overnight step repeated.
- If the volume of 30% HNO3 needs to be increased and the previous steps repeated, the amount of all other reaction components (30% H2O2 and Milli-Q H2O introduced in steps 10 and 11) have to be increased in relation to the 30% HNO3 volume.
- If the slurry has not been separated into two phases and the amount of acid to be added exceeds the capacity of the conical centrifuge tube being used or is disproportionately large, it may be advisable to start the experiment again using smaller sample sizes or larger conical centrifuge tubes.
After checking the samples, increase the temperature of the water bath to 95 °C and boil the samples for at least 00:30:00 .
Note
- Be careful not to let it boil over and watch out for acid fumes when opening the tubes.
30m
Take the tubes out of the water bath and let the samples cool down completely before moving to the new step.
Note
- This step is crucial to reduce the reaction potential of the 30% H2O2, which is introduced in the next step, as well as reduce potential reactions between the heated up elements in the sample and the 30% H2O2.
- Please make sure that both the HNO₃ and H₂O₂ are used at the specified concentrations (30% each), as mixing these two concentrated acids will cause a violent reaction.
Add 200 µL 30% H2O2 and place tubes back into the water bath at 95 °C for 00:30:00 .
Note
- Be careful when adding the 30% H2O2, since hydrogen peroxide is highly reactive, has a tendency to foam due to its rapid oxidation into water and oxygen.
- If the amount of 30% HNO3 was increased in earlier steps, more 30% H2O2 needs to be added in relation to the increased amount of HNO3, e.g. if the total amount of 30% HNO3 was 1.5 mL instead of the 1 mL , 300 µL of 30% H2O2 needs to be added instead of the 200 µL .
- H₂O₂ is used in this protocol in addition to HNO₃ because it is a strong oxidizing agent that breaks down organic components, thereby allowing the release of the elements of interest bound within the samples.
30m
Let the samples cool down completely. Slowly add 8.8 mL highly de-ionized H2O. Invert the tubes to mix the samples.
Note
- It is important to use highly de-ionized H2O instead of any other water, since ICP-MS measures in the ppb range and even minor contaminants can be detected immediately.
- For the ICP-MS, Type I ultrapure water (per ASTM D1193) is required, with a resistivity of 18.2 MΩ·cm at 25 °C .
- Be careful when adding water to avoid splashing.
- If the amount of 30% HNO3 and 30% H2O2 was increased in the previous steps, the amount of water needs to be increased in the same ratio.
- If the the volume of all reaction components is increased in large amounts, a bigger tube should be used for the digestion reaction, such as 50 mL conical centrifuge tubes.
Store samples Overnight at 4 °C .
Samples can be stored longer at 4 °C if needed.
Note
- We found out, that storing the samples overnight at 4 °C before centrifugation can prevent further precipitation later on.
Centrifuge the samples with 12000 x g, 4°C, 02:00:00 and ensure that the remaining soil particles are all settled to the bottom of the tube.
Note
- Check before starting, if the conical centrifuge tubes are able to presist the 12000 x g force, otherwise the tube can break and the sample is lost.
- If necessary, reduce the relative centrifuge force [g] and increase the duration.
- If it is not possible to use a centrifuge with a cooling function, it is sufficient to keep the samples at 4 °C beforehand. However, we have noticed that this may lead to an additional centrifugation step later on to remove possible precipitates that occur more frequently.
- If needed, centrifuge the samples longer.
- IN GENERAL: the longer the samples are centrifuged, the better the clean-up.
- In some cases the remaining soil material will not settle to the bottom of the tube. If this happens, the samples need to be filtered.
2h
Calculation of the preliminary dilution factor [pre-DF] with the sample weight: pre-DF = (final volume of the whole reaction [mL] *1000) /sample weight [mg].
Note
- The difference in units between the 0 mg of soil material and the 0 mL of the final volume must be taken into account in the calculation, using the multiplication factor 1000. If all values were measured in the same unit, this adjustment can be omitted from the calculation.
Fill each tube of the second conical centrifuge tube set with 3.2 mL 2% HNO3. Add 800 µL of sample supernatant to the second set of conical centrifuge tubes.
Note
- This is a 1:5 dilution with 2% HNO3.
CRITICAL: !!! the final acid concentration should not be above 5% to prevent damages of the ICP-MS machine !!!
Store the samples at 4 °C until the ICP-MS measurement can be performed.
Note
- Please keep in mind that the samples cannot be stored forever, since some of the elements of interest are not stable over a long period of time.
Final check before starting ICP-MS measurements.
Note
- Check your samples directly before measuring by ICP-MS, since the samples can precipitate even when stored at 4 °C . If this happens, centrifuge your samples again beforehand at 4 °C for at least 00:30:00 and transfer the supernatant to a new conical centrifuge tube.
Dilution factor calculation summary
The dilution factor has already been mentioned in the steps 14 and 15. The various steps of the calculation are outlined again here. The dilution factor calculated here is always applied to the measurement data. Any further dilutions that may need to be done, are applied to the results as additional dilution factors.
First part of the calculation:
Preliminary dilution factor [pre-DF]: pre-DF = (final volume of the whole reaction [mL] *1000) /sample weight [mg].
Note
- The sample weight used is recorded at the beginning of the sampeling in step 5.
- The difference in units must be taken into account in the calculation, using the multiplication factor 1000 for the calculation. If all values were measured in the same unit, this adjustment can be omitted from the calculation.
- For simplicity’s sake, 1 ml is roughly equivalent to 1000 mg, so the unit can be dropped.
- The total volume of the reaction is measured after the step 11, including both the supernatant and the precipitate. This volume can usually be read directly from the scale on the tubes. If this is not possible, the conical centrifuge tubes should be weighed empty beforehand and again before the centrifugation step.
- If the weight of the tubes is used instead of the volume of the sample, the calculation looks as follows: pre-DF = (final weight of the tube [mg] - empty weight of the tube [mg]) /sample weight [mg].
Second part of the calcuation:
Dilution factor [DF] = pre-DF * 5
Note
- In the final dilution described in this protocol (step 15), 800 µL of sample supernatant is added to 3.2 mL of 2% HNO₃. This is a 1:5 dilution, and this factor 5 must then be applied to the preliminary dilution factor [pre-DF] to get the final dilution factor [DF].
Acknowledgements
We thank Maike Grusch for her dedicated technical support and her contribution to the experimental groundwork for this protocol. We would like to thank Prof. Petra Bauer of the Heinrich-Heine-University for recommending protocols.io to us.
This work is supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Collaborative Research Centre TRR341 on ‘Plant Ecological Genetics’ - project ID 456082119 - and the Germany´s Excellence Strategy - EXC-2048/1 - project ID 390686111 (CEPLAS).