Mar 30, 2026
P1.5 Biomass Digestion, ICP-OES Operation, and Data Analysis-BioCirV
- Maria F. Duran1,
- James Gardner1,
- Xihui Kang1
- 1Lawrence Berkeley National Laboratory
Protocol Citation: Maria F. Duran, James Gardner, Xihui Kang 2026. P1.5 Biomass Digestion, ICP-OES Operation, and Data Analysis-BioCirV. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwnjy7vmk/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 09, 2026
Last Modified: March 30, 2026
Protocol Integer ID: 238362
Keywords: Biomass ash digestion, ICP-OES, Elemental analysis, Nitric acid digestion, digestion of agricultural waste biomass ash, agricultural waste biomass ash, biomass ash, including biomass ash, sample digestion, elemental analysis at abpdu, plasma optical emission spectrometry, agricultural residue, elemental analysis, coupled plasma optical emission spectrometry, nitric acid, subsequent elemental analysis, preparation for icp, digestion, sample preparation, biocirv this procedure
Funders Acknowledgements:
A gift from Schmidt Sciences' Virtual Institute for Feedstocks of the Future
Grant ID: N/A
Abstract
This procedure describes the digestion of agricultural waste biomass ash using 70% nitric acid for subsequent elemental analysis by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The procedure covers sample digestion, dilution, centrifugation and filtration, and preparation for ICP-OES measurement. It is intended for personnel involved in sample preparation and elemental analysis at ABPDU. This protocol is applicable to a range of sample types, including biomass ash derived from agricultural residues (e.g., tomato, almond, and wheat straw) and aqueous matrices containing dissolved elements.
Guidelines
**Responsibilities**
- Operator**: Complete required training; ensure all mandatory modules are finished. Confirm inclusion in WPC activity; verify that the task is listed and aligned with WPC requirements. Follow SOP guidance; perform activities in accordance with established procedures.
Materials
**Chemicals**
- 70% Nitric acid (HNO3)
- Distilled water (DI water)
**Supplies**
- Biomass (agricultural waste, ash)
- 20ml glass digestion tubes with loose caps
- 10ml glass pipette
- 1ml pipette tips (modified with cut opening)
- 15ml/50ml Falcon tubes
- 0.22um syringe filters
**Equipment**
- Chemical fume hood
- Benchtop Centrifuge (capable of accommodating 15ml Falcon tubes)
- Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES)
Troubleshooting
Safety warnings
**CAUTION! Handling 70% Nitric Acid: Do not open or handle 70% nitric acid outside of a chemical fume hood. Nitric acid is a strong acid and oxidizing agent that can cause severe burns, respiratory problems, and other health hazards. Always wear personal protective equipment (PPE) including gloves, safety goggles, and a lab coat when handling nitric acid. Ensure that the fume hood is functioning properly and that the area is well-ventilated to prevent inhalation of fumes.
**PPE**
- Long Pants
- Closed-toed shoes
- Safety glasses - must be worn at all times in the lab space
- Lab coat - must be worn when spending extended time in lab space or any time when doing hands on work in the lab
_For handling 70% Nitric Acid:_
- Wear butyl rubber Gloves and Ansell gloves (common nitrile gloves are NOT appropriate for handling 70% Nitric Acid)
- Wear a face shield to protect against splashes when a fixed shield is not in place
**Ergonomics**
- Reach out to LBL Ergonomics team with any specific issues or questions as they arise while performing the below protocol
**High Hazard Operations**
- STOP WORK if things feel off-normal.
- Nitric acid is a strong acid and oxidizing agent, handle with care!
- Avoid at all cost skin contact and inhalation of fumes
**Chemical safety**
- WARNING! Handling 70% Nitric Acid: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before storing, handling, or working with 70% nitric acid.
- Minimize inhalation of nitric acid fumes by working in a chemical fume hood.
- The exposure limits for 70% nitric acid are an 8-hour TWA of 2 ppm and a 15-minute STEL of 4 ppm. These limits help protect workers from harmful effects of nitric acid vapors during both long-term and short-term exposures.
- Do not leave 70% nitric acid containers open, and check regularly for leaks or spills.
- Inspect nitric acid containers. Before using nitric acid, carefully inspect the container for any signs of damage, such as dents, bulges, cracks, or other unusual deformations. Do not use the container if any damage is observed**, and report it to the appropriate supervisor or safety personnel.
- Ensure that all chemicals and waste are placed in secondary containers, and that empty containers are sealed with their provided caps to prevent leakage and contamination.
**Emergency Procedures**
- Spills**: In case of a nitric acid spill, immediately neutralize the area with a base such as sodium bicarbonate. There is one acid spill kit located in Room 3215, near the fermentation sink, for immediate response to acid spills. Wear PPE and contain the spill to prevent further spread.
- Inhalation**: In case of inhalation of nitric acid fumes, evacuate the area and seek fresh air. If symptoms persist, seek medical attention and notify your supervisor.
- Skin and Eye Exposure**: In case of skin or eye exposure to nitric acid, immediately flush with water and seek medical attention.
**Administrative Controls**
- Training**: All personnel involved in this procedure must receive training on handling nitric acid and ICP-OES analysis. Training records must be kept and updated regularly.
- Standard Operating Procedures (SOPs)**: This SOP must be followed for all biomass digestion and ICP-OES analysis procedures. Deviations from this SOP must be approved by a supervisor or laboratory manager.
- Quality Control (QC)**: QC samples must be included in each batch of samples to ensure accuracy and precision of results.
Before start
**AUTHORIZATION REQUIRED: Handling 70% nitric acid for biomass digestion requires proper training and authorization. Ensure that you have received the necessary training and have been authorized to perform this procedure before proceeding.
PICTOGRAM
Nitric Acid Digestion of Biomass Ash for ICP Analysis
MATERIALS AND REAGENTS
Chemicals
70% Nitric acid (HNO3)
Supplies
Biomass (agricultural waste, ash)
50 mL conical Falcon tubes
15 mL conical Falcon tubes
2ml glass pipettes
Centrifuge filter tubes 0.45um
Equipment
Chemical fume hood
Bench-top centrifuge (capable of accommodating 5ml Falcon tubes)
Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES)
SAFETY INFORMATION
Safety information
**CAUTION! Handling 70% Nitric Acid: Do not open or handle 70% nitric acid outside of a chemical fume hood. Nitric acid is a strong acid and oxidizing agent that can cause severe burns, respiratory problems, and other health hazards. Always wear personal protective equipment (PPE) including gloves, safety goggles, and a lab coat when handling nitric acid. Ensure that the fume hood is functioning properly and that the area is well-ventilated to prevent inhalation of fumes.
The recommended flow rate for a chemical fume hood is typically between 80 to 120 feet per minute (fpm), with an optimum face velocity of around 100 fpm at a sash position of 18 inches.
PPE
Long Pants
Closed-toed shoes
Safety glasses
Lab coat
Butyl rubber Gloves and Ansell gloves (common nitrile gloves are not compatible)
Face shield to protect against splashes when a fixed shield is not in place
Chemical safety
WARNING! Handling 70% Nitric Acid: Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before storing, handling, or working with 70% nitric acid.
Minimize inhalation of nitric acid fumes by working in a chemical fume hood.
The exposure limits for 70% nitric acid are an 8-hour TWA of 2 ppm and a 15-minute STEL of 4 ppm. These limits help protect workers from harmful effects of nitric acid vapors during both long-term and short-term exposures.
Do not leave 70% nitric acid containers open, and check regularly for leaks or spills.
Inspect nitric acid containers. Before using nitric acid, carefully inspect the container for any signs of damage, such as dents, bulges, cracks, or other unusual deformations. Do not use the container if any damage is observed, and report it to the appropriate supervisor or safety personnel.
Ensure that all chemicals and waste are placed in secondary containers, and that empty containers are sealed with their provided caps to prevent leakage and contamination.
Emergency Procedures
Spills:
In case of a nitric acid spill, immediately neutralize the area with an Acid Handler Spill Powder. Identify the location of the nearest acid spill kit BEFORE beginning the procedure.
Inhalation:
In case of inhalation of nitric acid fumes, evacuate the area and seek fresh air.
If symptoms persist, seek medical attention.
Skin and Eye Exposure:
In case of skin or eye exposure to nitric acid, immediately flush with water and seek medical attention.
PROCEDURE
2d 0h 10m
Fume Hood Setup and Spill Containment
Safety information
AUTHORIZATION REQUIRED: Handling 70% nitric acid for biomass digestion requires proper training and authorization. Ensure that you have received the necessary training and have been authorized to perform this procedure before proceeding.
Set up your workstation by preparing the fume hood to ensure adequate space for safe sample handling. Place a pig mat inside the fume hood, and position the sample rack on top of the pig mat to securely hold the samples during manipulation.
Sample Preparation
Weigh 0.10 g of biomass ash into individual 50 mL Falcon tubes. Process no more than 10 samples per batch (e.g., 5 samples and 5 duplicates) to ensure safe handling and consistency.
Addition of 70% Nitric Acid
In the fume hood, dispense 20 mL of 70% nitric acid into a 50 mL glass reservoir bottle. Using a 1–2 mL glass pipette, transfer 1 mL of 70% nitric acid from the reservoir into each ash sample tube. Immediately place the cap loosely on each tube after acid addition to minimize evaporation, splashing, or fume release while preventing pressure buildup.
Note
Each time the 70% nitric acid bottle is accessed, open and close it immediately to minimize vapor exposure.
Use a 1–2 mL glass pipette to reduce dripping and minimize exposure risk.
Biomass Digestion
Place the rack containing the capped sample tubes into a secondary containment vessel. Incubate the samples at room temperature in the fume hood for 48:00:00 to allow complete digestion. Clearly label the secondary container with appropriate hazard and sample identification labels.
Note
Process-specific safety labels - 70% Nitric Acid
Process-specific safety labels - 70% Nitric Acid.pdf 2d
Sample Dilution
After incubation, add 34 mL of deionized (DI) water to each tube to dilute the digest to a final nitric acid concentration of 2%. Maintaining the samples in 2% nitric acid preserves dissolved metals, prevents adsorption and precipitation, and ensures matrix matching between samples and standards for accurate ICP analysis.
Centrifugation
After dilution, transfer approximately 10 mL of each sample into 0.45 um centrifuge filter tubes and securely tighten the caps. Place the filter tubes into centrifuge bucket racks while still inside the fume hood. Close the buckets securely, then transport them to the bench-top centrifuge. Centrifuge, 4000 rpm, 22°C, 00:10:00
0.45 um Centrifuge Filter Tube
Closed centrifuge bucket loaded with 0.45 µm centrifuge filter tubes for sample centrifugation. Buckets should be securely closed before transport to and use in the bench-top centrifuge.
Note
Collect all waste generated during this protocol, which contains 2% nitric acid, in a properly labeled, acid-compatible container.
10m
Final Sample Collection for ICP
Collect 10 mL of the filtered supernatant into 15 mL Falcon tubes for ICP analysis.
Waste Handling and Disposal
At the completion of the procedure, all waste containing 2% nitric acid must be handled with extreme care. Filter the waste through cheesecloth to separate liquid and solid fractions.
Liquid waste should be collected in a clearly labeled, acid-compatible container and disposed of by submitting a waste requisition through Waste Management Services.
Note
Waste containing nitric acid must be evaluated before disposal. If the acid concentration exceeds 5% by weight (approximately 0.8 M or pH < 1), or if any organic constituents are present, the waste must be neutralized prior to disposal. Follow established laboratory procedures to safely neutralize the waste, ensuring that pH and composition meet regulatory and institutional disposal requirements.
Solid material retained on the cheesecloth should be thoroughly rinsed with water to remove residual acid, placed in a sealed Ziploc bag, and discarded in a designated biohazardous waste container.
Collect solid waste materials, such as empty tubes, pipettes, and pipette tips that have been in contact with 2% nitric acid, in a designated acid-compatible waste container and submit them through the appropriate waste requisition process.
ICP OPERATION (Adapted from the Molecular Foundry ICP Standard Operating Procedure)
35m
Instrument Setup
- Open the ICP Expert software and confirm the instrument is connected
- Verify purges are complete
- Install, rinse, and outlet tubing correctly
- Start rinsing the autosampler and instrument
- Turn on the chiller, wait for -38 °C , then ignite the plasma
- Let the plasma stabilize for about 00:15:00
15m
Method Setup
In the software:
- Open an existing worksheet/template or create a new quantitative worksheet
- Select elements and wavelengths
- Assign axial or radial viewing conditions
- Define calibration standards and blanks
- Set QC options such as the continuing calibration blank (CCB), the continuing calibration verification (CCV), and the initial calibration verification (ICV).
Run Setup and Analysis
- Arrange standards, QC solutions, and samples in the autosampler racks
Example S Rack configuration for ICP analysis. The center autosampler rack (S Rack) includes fixed positions for key quality control solutions: blank (CCB) in S1:3 and standard (CCV) in S1:4. Staff-use positions are reserved and should not be disturbed.
- Assign rack and tube positions in the sequence
- Check that all samples to be analyzed are selected
- Start the run and confirm the first standards and samples run correctly
- Review spectra and confirm suitable analytical wavelengths during the run
Shutdown and Cleaning
- Rinse the torch with rinse solution for 00:10:00 while plasma remains on
- Turn off the plasma, then turn off the chiller
- Rinse the tubing system for 00:10:00
- Flush all lines dry with air
- Park the autosampler
- Release and unclip tubing as required
- Remove samples and dispose of waste properly
20m
Quality Assessment and Data Acceptance
- Assess data quality by using only reliable wavelengths with strong, well-resolved peaks and minimal interference.
Example of good and poor ICP spectral peak quality. A good analytical peak is sharp, symmetrical, and clearly separated from the background, with strong signal intensity and minimal nearby interference. A poor peak is weak, broad, noisy, or overlapped by neighboring signals, which increases uncertainty and makes the reported concentration less reliable.
Confirm that peaks remain sharp and symmetrical at high standard concentrations, and re-center spectral markers as needed. Review unknown spectra, replicate consistency, calibration curves, and final data tables for agreement and reasonable trends. Percent RSD is a critical precision check; for our work, acceptable results must have ≤10% RSD.
ATTACHMENTS
Quick-Reference Table for Nitric Acid Biomass Digestion
Quick-Reference Table for Nitric Acid Biomass Digestion.pdf Safety Data Sheet - 70% Nitric Acid
SDS of nitric acid.pdf Protocol references
Thompson, M. & Walsh, J. N. Handbook of inductively coupled plasma spectrometry. (Springer US, 1989). doi:10.1007/978-1-4613-0697-9.
Acknowledgements
The authors thank Rosanne Boudreau, Biosciences Division Safety Coordinator, Karla Arredondo, Occupational Safety Specialist, Jeremy Hamilton, Industrial Hygienist, and Julie Zhu, Industrial Hygienist, for their support in evaluating hazards, assessing fumes, and helping develop a safe procedure for this work. They also thank Rachel Alvelais for providing the Molecular Foundry ICP Standard Operating Procedure, which informed the ICP operation section of this protocol.