Dec 18, 2024
Routine Operation of a Triple Quadrupole Inductively Coupled Plasma Mass Spectrometer (ICP-MS/MS) for Determining the Elemental Composition of Various Cell Types.
- 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA;
- 2Quantitative Biosciences Institute, University of California, Berkeley, California 94720, USA
- Merchant Lab UC Berkeley
Protocol Citation: Dimitrios J. Camacho, Charles Perrino, Sabeeha S. Merchant 2024. Routine Operation of a Triple Quadrupole Inductively Coupled Plasma Mass Spectrometer (ICP-MS/MS) for Determining the Elemental Composition of Various Cell Types.. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lywebevx9/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: July 17, 2024
Last Modified: December 18, 2024
Protocol Integer ID: 103547
Funders Acknowledgements:
National Institutes of Health (NIH): Nutritional Copper Signaling and Homeostasis
Grant ID: GM 042143
Molecular Basis of Cell Function T32 Training Grant
Grant ID: 5T32GM007232-44
US Department of Energy (DOE), Office of Biological and Environmental Research (BER): Systems Engineering of Auxenochlorella protothecoides: from Photosynthesis to Biofuels and Bioproducts
Grant ID: DE-SC0023027
University of California, Berkeley, Chancellor’s Fellowship
Grant ID: N/A
Molecular Genetic Dissection of Chloroplast Fe Homeostasis
Grant ID: DE-SC0020627
Abstract
This protocol describes a method for the routine operation of the Agilent Triple Quadrupole Inductively Coupled Plasma Mass Spectrometer 8900 (ICP-QQQ) for the elemental analysis of digested cells, spent media, and reagents. This protocol will outline the formulation of needed solutions, startup checklist, MassHunter software operation, performance checks, tuning, calibration, sample introduction, data acquisition, data analysis, and shut down. The parameters used in this protocol enable the efficient and accurate quantitation of Na, Mg, P, S, K, Ca, Fe, Mn, Co, Ni, Cu, Zn, Se, Mo, Ag, and Cd in algal cells, the growth media used for culture, and spent media. Cells digested in 70% nitric acid or the media are separately diluted to a final matrix of 2% nitric acid. Samples are mixed with on-line addition of internal standards Sc, Y, and Re. Three gases are used sequentially in the collision reaction cell to minimize interferences. Helium is used in collision mode for the removal of polyatomic ions through kinetic energy discrimination. Hydrogen and oxygen are used in reaction mode for the removal of mass shifted contaminants and the selection of mass shifted ions, respectively. The linear dynamic range of the instrument using this set up spans 4 orders of magnitude with a detection limit in the parts per trillion range for trace elements such as Cu, Zn, Mn, Se, Mo, Ag, and Cd.
Attachments
Guidelines
Read the entire protocol before attempting to operate the instrument. In some steps, detailed instructions and things to watch out for are listed after the general idea of the step is stated first.
Purity of reagents
The sensitivity, accuracy, and precision of the instrument is contingent on the purity of the reagents used. Use only ultra-high purity chemicals that have a certificate of analysis indicating the level of impurities, as assayed by another ICPMS or ICP-OES. Verify that your calibration standards, internal standards, and tuning solutions have not expired. Use ICP-MS grade ultrapure H2O only. Our lab’s supply of ultrapure H2O is produced by the Milli-Q Advantage A10 Water Purification System. In addition to the standard filtration units of the system (A10 UV lamp, Q-GARD T2 pack filter, Quantum TIX Ultrapure Cartridge), our system employs an additional filtration step using a Q-POD Element that is equipped with a Quantum ICP filter. If you are using a similar system, verify that the resistivity is 18.2 MΩ.cm and that the total organic carbon level is <10 ppb.
Common sources of contamination
We have found detectable levels of Zn in serological pipettes when they are not pre-wetted. We recommend rinsing the pipet by filling it with Milli-Q H2O and dispensing the water fully before pipetting samples or solutions. Avoid using colored plastics. HNO3 and HCl will discolor plastics and may digest dyes. Keep tubes and solutions capped as much as possible to avoid contaminants from dust particles. Using powdered latex gloves will introduce Zn contamination into samples and solutions. We recommend using nitrile gloves. 15 mL and 50 mL plastic sample tubes often contain detectable levels of trace metals. Our lab has tested 15 mL and 50 mL tubes from various suppliers and found Globe Scientific’s Diamond Max centrifuge tubes (Lot# 15CG1139B and 15CG1140B) suitable for use with our protocols. All tubes must be washed with 12 M HCl. See step 1.
Materials
Equipment
Agilent 8900 Triple Quadrupole Inductively Coupled Plasma Mass Spectrometer
Agilent SPS4 Autosampler
Agilent MS40S Foreline pump
Agilent G3292A Recirculating Chiller
Pipette 1000 μL, Rainin, (Cat. no. P1000)
Pipette 200 μL, Rainin, (Cat. no. P200)
Pipette 20 μL, Rainin, (Cat. no. P20)
Pipette 2 μL, Rainin, (Cat. no. P2)
Shaker water bath, 65 °C - 80°C , New Brunswick Scientific, (Cat. no. G75)
Ultrasonic bath 2.8 L with heater, Fisher, (Cat. no. CPX2800)
Fume hood, Fisher Hamilton, (Cat. no. 76RM)
Centrifuge, model JXN-26, Beckman Coulter Avanti (with JA14.50 fixed angle rotor)
Centrifuge, model 5810 R, Eppendorf, (with A-4-81 15/50 mL swinging bucket rotor)
Centrifuge, model 5424R, Eppendorf, (with FA-45-24-11 2 mL fixed angle rotor)
Analytical balance, Mettler Toledo, (model: MS303TS/00, max = 320 g, d = 0.001 g)
Analytical balance, Mettler Toledo, (model: MS3002TS/00, max = 3,200 g, d = 0.01 g )
Serological pipettor, S1 Pipet filler,Thermo Scientific, (Cat. no. 14-387-042)
Drill, Ryobi, (Cat. no. P207)
1.7 mm drill bit (1/16"), Ryobi, (Cat. no. A971503)
Personal Protective Apron,Tychem ThermoPro , DuPont (Cat. no. J10090)
Vise Grip Irwin, (Cat. no. 10CR)
Gas wrench, Non-Sparking, 1-1/8", CS Unitec (Cat. no. EX206-100UA)
Consumables
Torch, quartz, 2.5 mm i.d., Agilent, (Cat. no. G3280-80053)
Nebulizer, MicroMist, Agilent, (Cat. no. G3266-80005)
PTFE tubing, sample 1.02 mm, Agilent (Cat. no. 5005-0020)
PTFE tubing, internal standard 0.25 mm, Agilent (Cat. no. 5005-0021)
PTFE tubing, waste 1.52 mm, Analytical West, (Cat. no. PT-3220SA)
Sampling cone, nickel, Agilent, (Cat. no. G2380-67040)
Skimmer cone, nickel, x-lens, Agilent, (Cat. no G8400-67200)
5 mL serological pipette tips, Thermo Scientific, (Cat. no. 170366N)
10 mL serological pipette tips, Thermo Scientific, (Cat. no. 170356N)
25 mL serological pipette tips, Thermo Scientific, (Cat. no. 170357N)
Note
We have found traces of Zn in solutions handled using the listed serological pipettes.
Before handling liquid samples or reagents, rinse each pipette with Milli-Q H2O. Aspirate more Milli-Q H2O than the volume of sample you intend to pipette. Dispense and discard the water before pipetting any samples or reagents.
Metal free 50 mL tubes, NUNC, Thermo Scientific, (Cat. no. 339653)
Nitrile gloves (Do not use latex powdered gloves), Layer 4 Rapidon, Size L, USA Scientific, (Cat. no.3925-4400)
Coolant for chiller, Agilent, (Cat. no 5799-0037)
Argon filter, Big Universal Trap, Agilent, (Cat. no. RMSA-2)
Gas filter for Helium and Hydrogen, Agilent, (Cat. no. CP17973)
Helium gas , 99.999% Ultra High Purity 5.0 Grade, Size G Cylinder, Linde, (Cat. no. HE 5.0UH-G)
Hydrogen gas, Ultra High Purity 5.0 Grade , Size 35 Cylinder, Airgas, (Cat. no. HY UHP35)
Oxygen gas, Research 5.0 Grade, Size 80 Cylinder, Airgas, (Cat. no. OX80)
Argon liquid dewar, Linde, (Cat. no. AR UHP180LT230)
Reagents
Environmental calibration standard, Agilent (Cat. no. 5183-4688)
Phosphorous calibration standard, Inorganic Ventures (Cat. no. CGP1)
Sulfur calibration standard, Inorganic Ventures (Cat. no. CGS1)
Scandium internal standard, Agilent (Cat. no. 5190-8517)
Yttrium internal standard, Agilent (Cat. no. 5190-8555)
Rhenium internal standard, Agilent (Cat. no. 5190-8507)
Tuning solution, Agilent (Cat. no. 5185-5959)
Nitric acid, Optima grade 70%, Fisher, (Cat. no. A467-500)
Nitric acid, Trace Metal grade 70%, Fisher, (Cat. no. A509P212)
MS40S Foreline Pump Oil, Agilent, (Cat. no. X3760-64004)
Safety warnings
Read the entire protocol before attempting to operate the instrument. Always read the entire next step before proceeding. Important safety information may be listed later in each step.
Chemical Hazards
Download and review the Safety Data Sheets (SDS) for all reagents and compressed gases used in this protocol.
Engineering controls
Proper engineering controls must be implemented to ensure safety of personnel and equipment. Do not start unless all engineering controls have been met.
A “clean room” is not required however the room in which the ICP-MS is situated needs to be kept extremely clean. Do not operate the instrument in a room with deteriorating paint. Avoid dust accumulation.
Argon dewars may vent extremely loudly when over pressurized and may cause hearing damage. If possible store argon dewars away from lab benches and desks. When venting occurs, leave the room or wear hearing protection. Venting of argon may also cause asphyxiation and death if the room is not properly ventilated.
The room will become noticeably warmer when the plasma is ignited due to the heat radiating from the exhaust duct above the instrument. This is normal. Excessive heat however will cause the cryogenic liquid argon to evaporate and vent more quickly.
Compressed gas storage and handling
Please read the safety data sheet for H2, Ar, He, and O2 compressed gases. Make sure to take your institution’s compressed gas safety course or training before handling compressed gases. Only properly trained personnel should handle compressed gases. All compressed gas cylinders should be securely restrained to a fixed housing or wall and positioned so that their valves are easily accessible. Cylinders should be restrained at 1/3 and 2/3 of the cylinder’s height. Use a dedicated cylinder cart to transport capped cylinders. All gas cylinders, gas lines, and regulators should be properly labeled.
Hydrogen gas is flammable and potentially explosive. It should be stored in a fireproof cabinet and separated from the oxygen gas cylinder. Use a bronze wrench to avoid electrostatic discharge and possible ignition. Right hand regulator threads are used for flammable gases.
Take care not to cross cylinder to regulator threads. If leaks occur between the cylinder and the regulator, contact the gas supplier and request another cylinder. Never use teflon tape to seal the connection between the cylinder and the regulator. Fragments of teflon tape may damage the regulator.
Flammable gas cabinet. Helium is inert but is stored with hydrogen for convenience.
Oxygen gas may cause normally noncombustible material to become combustible. Oxygen cylinders (oxidizers) must be stored separately from hydrogen and other flammable gas cylinders. Open the oxygen cylinder valve very slowly. A rapid compression of oxygen may cause ignition in a process called adiabatic compression. Do not use oil or grease to lubricate any O2 gas fittings because they may cause explosion.
Argon is typically supplied in a low-pressure cryogenic liquid dewar outfitted with a pressure relief valve set at 230 psi. When the argon gas pressure exceeds 230 psi, the pressure relief valve will open and vent argon into the room. Use ear protection when argon gas vents loudly. Argon is an inert gas but may cause asphyxiation if it displaces oxygen in the room. Do not accompany argon dewars in the elevator. Never open the liquid port and use cryogenic gloves to ensure that it is tightly closed. The liquid port valve tends to loosen as frost builds up on the valves and level indicators of the dewar. Exposure to cryogenic liquid argon may cause frostbite. As the argon is depleted, a ring of frost will appear around and on top of the dewar. This is normal but may pose a slip hazard when the frost melts.
Frost on liquid argon dewar when plasma is ignited and 15-17 L/ min of argon gas is consumed.
Main Instrument
A ventilation duct with an extraction flowrate of 5 – 7 m3 / min is needed for the main instrument. A smaller duct is routed to the autosampler for removal of HNO3 fumes. Check the status of the building’s air handling units before operation. The instrument will overheat and shut down if the exhaust flowrate is too low. HNO3 fumes are hazardous and will damage the autosampler’s electronic components if not exhausted properly.
A 200 – 240 VAC, 50/60 Hz, 30 A, single phase power source with protective earth contact is required. Connection to a power source without a protective earth contact may result in electrical shock to the operator and damage to the instrument.
ICP-MS instrument workstation.
Dress code
Please wear safety glasses, gloves, a lab coat, closed toe shoes, ear protection, and pants.
Acids
Hydrochloric (HCl) and nitric acid (HNO3) are used in this protocol. Both acids are hazardous and extremely corrosive to skin and metals. HCl and HNO3 will cause severe skin burns and eye damage. Perform all work in a fume hood to avoid inhalation of fumes. Always use a secondary container. HCl fumes are invisible, whereas HNO3fumes may be brown in color. Concentrated 70% HNO3 may burn through nitrile gloves in a matter of minutes. Released oxygen from HNO3 fumes may intensify fires.
Radio frequency radiation
Exposure to strong radio frequency radiation from the RF power supply may interfere with pacemakers and other medical devices. Electrical appliances should not be installed within 5 cm of the ICP-MS. Magnetic interference from the cool water inlet valve (left of the instrument) may cause damage to electrical appliances in proximity.
Before start
1. Read the entire protocol before you attempt to operate the instrument.
2. Make sure that the sampling and skimmer cones are properly conditioned prior to the run. If the sample and skimmer cones are brand new, or if they have just been cleaned, condition them with a matrix like that of your samples for at least two hours or until the internal standard recovery is stable across replicate measurements of a conditioning solution.
3. Make sure that all gases are pure and that gas lines have been purged of contaminants. Check H2 and He filters for moisture and oxidation.
4. Replace filters if necessary and purge each carrier gas for at least two hours at 12 mL / min whenever the gas lines are opened or depressurized.
5. Make sure that the MS40S vacuum pump has been turned on and the machine has been set to standby mode for at least 24 hours before the run. The longer the vacuum pump has been shut off, the longer it will take to achieve the required vacuum pressure again.
6. Inspect all peristaltic pump tubing and replace if necessary. Replacement tubing needs to be soaked in 5% HNO3 for two to three days prior to installation.
7. Make sure that samples are properly digested. See ICPMS sample preparation protocol (Camacho et al., 2024) for detailed instructions.
Sample digestion may take 16-18 hours.
8. Prepare the ISTD and probe rinse caps.
Place the caps of the 1 L ISTD HDPE bottle and 1 L probe rinse HDPE bottle on a spoil board.
Use a vise grip or a clamp to clamp a cap in place.
Drill a 1.7 mm hole into the center of the caps of the ISTD and probe rinse bottles.
Clean the plastic debris from the caps.
Make sure to wash the caps, and especially the drilled orifices in step 4.
9. Check the SPS4 auto sampler (see step 22). The microcontroller chip in the autosampler is prone to corrosion due to long-term exposure to HNO3 fumes in the autosampler cabinet. Make sure that the exhaust duct connected to the autosampler is working.
Preparation of solutions
Preparation of solutions
Acid wash all sample tubes with 12 M HCl.
Safety information
HCl and HNO3 are highly corrosive and hazardous. Wear gloves, eye protection, a lab coat, a plastic apron, pants, and closed toe shoes. Perform acid washing within a secondary container inside a clean fume hood.
Acid-wash all sample tubes with 12 M HCl.
All samples will be measured and stored in 15 mL tubes.
Begin by filling one tube with roughly 7 mL of 12 M HCl.
Cap the tube securely and slowly invert to mix.
Ensure all surfaces within the tube have been washed.
Pour the used HCl into the next tube and repeat until all tubes have been washed.
Pour used HCl into a capped and labeled waste container.
Place the waste container into a secondary container and store it in the hood until the neutralization in step 3.
Rinse the tubes with ICP-MS grade Milli-Q H2O at least three times.
Wash the exterior of the capped tubes to wash away residual HCl.
Dry the exterior of the capped tubes with a Kimwipe.
Centrifuge the empty tubes for 30 s at 3000 ×g using the A-4-81 15/50 mL swinging bucket rotor in an Eppendorf centrifuge, model 5810 R.
Use a P200 to remove any remaining liquid in the tube.
Neutralize used 12 M HCl by first diluting to 3 M HCl.
Fill a 4 L beaker with 1.5 L of H2O. Place the beaker into a larger secondary container and set it in the fume hood.
Slowly and carefully add up to 500 mL of 12 M HCl to the H2O.
The solution may heat up. Always add acid to water and never water to acid.
Slowly add NaHCO3 (Arm & Hammer pure baking soda) scoop by scoop (< 25 g / scoop) until no foam is formed.
The reaction will become more violent towards the end so take your time to ensure that no acid is splashed.
Use a pH indicator strip to verify that the acid is safely neutralized (pH 7). Refer to your institutional and municipal guidelines for disposing liquids with high concentrations of NaCl.
Acid-wash calibration standard tubes and all reagent bottles with 5% HNO3.
Obtain three 500 mL and nine 1 L clean HDPE bottles with caps.
500 mL bottles will store:
(x3) 2% HNO3 washing solutions
1 L bottles will store:
(×1) 5% HNO3 washing solution
(×2) 2% HNO3 washing solutions refill
(×1) 2% HNO3 probe rinse solution (with predrilled cap)
(×2) Supernatant diluent (2.8% and 2.1% HNO3)
(×2) ICPMS grade Milli-Q H2O
(×1) Internal standard (ISTD) solution containing Sc, Y, and Re (with predrilled cap)
Measure and record the mass of each empty (dry) bottle with the cap before acid washing. Make sure to weigh the ISTD and probe rinse bottles and caps after the cap has been drilled (see the "before you begin" section for instructions on drilling holes into caps).
Add 65 mL of Milli-Q H2O to one 500 mL HDPE bottle.
Place all bottles in a secondary container, in the fume hood.
Slowly add 5 mL of 70% HNO3 to the water in the 500 mL bottle. The solution may heat up, so add the acid slowly.
Cap the bottle securely and slowly invert to mix three times.
Transfer the 5% HNO3 acid solution to the next 500 mL bottle. Swirl and invert to rinse the bottle.
Repeat this process until all 500 mL and 1 L bottles have been rinsed with 5% HNO3.
After the last bottle has been rinsed, pour the 5% HNO3 into a dedicated 4 L beaker used for HNO3 neutralization (see next step).
Make 1 L of 5% HNO3 by adding 928 mL of Milli-Q H2O to a 1 L bottle and slowly add 72 mL of 70% HNO3.
Repeat the process to fill each bottle with new 5% HNO3.
Leave the capped bottles in a secondary container within the fume hood overnight.
Pour used 5% HNO3 into a waste container or store it safely to reuse it for cleaning other dirtier components.
Rinse all bottles at least three times with Milli-Q H2O.
The bottles should always be capped to avoid dust from entering and contaminating the solutions.
It is OK to leave residual water in the bottles.
Calibration standards will be measured and stored in 17 × 50 mL tubes.
Prepare 24 × 50 mL tubes and 4 × 15 mL tubes.
Measure and record the mass of each tube using an analytical balance (model: MS303TS/00, max = 320 g, d=0.001 g). Always make sure that you do not exceed the mass limit for each balance. The analytical balance referenced above is limited to 300 g and has milligram precision.
Add 25 mL of 5% HNO3 to the first 50 mL tube. Slowly invert to rinse the tube. Pour the 5% HNO3 into the next tube and repeat until all 50 mL tubes are rinsed.
Rinse 15 mL tubes with 10 mL of 5% HNO3.
Pour the acid into a neutralization container.
Fill each tube with 5% HNO3 and leave them in the fume hood overnight.
Pour used 5% HNO3 into a waste container or store it safely to reuse it for cleaning other dirtier components.
Rinse the interior of the tubes with Milli-Q H2O three times. Rinse the exterior of the tubes to prevent exposure to residual HNO3. Dry the exterior of each tube with lint free Kimwipes.
Centrifuge the empty tubes for 30 s at 3000 ×g using the A-4-81 15/50 mL swinging bucket rotor in an Eppendorf centrifuge, model 5810 R.
Use a P200 to remove the residual water.
Keep tubes capped at all times.
Neutralize 5% HNO3
Neutralize 5% HNO3 by diluting to 2.5% HNO3 in H2O. Do not mix HNO3 with HCl for neutralization!
Fill a 4 L beaker with 1 L of H2O. Place the beaker into a larger secondary container and set it in the fume hood.
Slowly add up to 1 L of 5% HNO3 to the H2O.
Slowly add NaHCO3 (Arm & Hammer pure baking soda) scoop by scoop (< 25 g / scoop) until no foam is formed.
Use a pH indicator strip to verify that the acid is safely neutralized (pH 7).
Refer to your institutional and municipal guidelines for disposing liquids with high concentrations of salt.
Prepare the 2% HNO3 wash solutions.
Three wash solutions will be stored in 500 mL acid washed bottles.
Two additional 1 L bottles will contain back up 2% HNO3 rinse solutions.
The probe rinse solution will be stored in a 1L bottle with a drilled 1.7 mm hole in the cap.
Place an acid washed 1 L bottle (with predetermined dry weight) on a balance (model: MS3002TS/00, max = 3,200 g, d = 0.01 g).
Fill the bottle with 971.4 g of Milli-Q H2O.
Cap the bottle (use cap without drilled hole) and move to a clean fume hood.
Very slowly and carefully, add 28.6 mL of 70% HNO3 to the bottle of H2O. The solution may heat up. Cap the bottle and let it cool.
Slowly invert the bottle to mix at least 10 times.
Repeat this process to fill all 2% HNO3 wash solution bottles including the probe rinse.
Place three 500 mL wash solutions into the Autosampler in positions 1, 2, and 3.
Place the probe rinse solution behind the Autosampler and in a secondary container. Put the probe rinse tube inside the hole of the cap and into the 2% HNO3 probe rinse solution.
Prepare 2.8% and 2.1% HNO3 diluent solutions.
To make a 2.8% HNO3 diluent solution, place an acid washed 1 L bottle on an analytical balance (model: MS3002TS/00, max = 3,200 g, d = 0.01 g).
Fill the bottle with 960 g of Milli-Q H2O.
Cap the bottle and move it to the fume hood.
Very slowly and carefully, add 40 mL of 70% HNO3 to the bottle of H2O.
Cap the bottle, let it cool and slowly invert the bottle to mix at least 10 times.
To make the 2.1% HNO3 solution, follow the steps above but add 970 g of Milli-Q H2O and 30 mL of 70% HNO3.
Prepare the Internal standard (ISTD) solution.
The 250x ISTD stock solution will be stored in an acid-washed 50 mL tube and will contain 20 µg / mL Sc, 10 µg / mL Y, and 10 µg / mL Re.
The diluted 1x ISTD solution will be stored in an acid-washed 1 L bottle and will contain 0.08 µg / mL Sc, 0.4 µg / mL Y, and 0.4 µg / mL Re.
To prepare a 250x ISTD stock solution, obtain the following solutions:
Scandium [1000 µg / mL]
Yttrium [1000 µg / mL]
Rhenium [1000 µg / mL]
Place the tube on a balance (model: MS303TS/00, max = 320 g, d=0.001 g) and add 23.36 g of Milli-Q H2O.
Cap the tube and move it to a clean fume hood.
Add 640 µL of 70% HNO3 to the tube.
Add 500 µL of 1000 µg / mL Sc.
Add 250 µL of 1000 µg / mL Y.
Add 250 µL of 1000 µg / mL Re.
To dilute the stock solution to 1x, place an acid washed 1 L bottle on a balance (model: MS3002TS/00, max = 3,200 g, d = 0.01 g) and fill with 967.4 g of Milli-Q H2O. Cap the bottle and move to a clean fume hood.
Slowly add 28.6 mL of 70% HNO3.
Slowly add 4 mL of 250x ISTD stock solution.
Use parafilm to cover the 1.7 mm drilled hole and invert slowly and carefully to mix above a secondary container. Place the 1x ISTD solution near the Autosampler in the secondary containment tray. Insert the ISTD’s PTFE tubing from the peristaltic pump into the drilled 1.7 mm hole in the cap and make sure that the end of the tube is at the bottom of the bottle and has not curved upwards. Create a <85-degree bend in the PTFE tube where the tube meets the cap to avoid the tube from slipping further into the bottle. Do not constrict flow. Tape the PTFE tube to the outside of the cap.
Prepare the tuning solution.
The tuning solution contains 1 µg / L of Ce, Co, Li, Mg, TI and Y.
Place the tuning solution into position 5 of the Autosampler. Please ensure that the tuning solution is properly capped after the tuning steps to prevent evaporation and improper tuning.
Ensure that the liquid waste container (containing 2% HNO3 that drains from the spray chamber and probe rinse) is empty. Longer runs exceeding 24 hours may require neutralization of liquid waste during the run. See step 5 for neutralization of HNO3. 2% HNO3 does not need to diluted before neutralization.
Prepare the calibration standards
Prepare the calibration standards
Label the 50 mL calibration standard tubes that were washed in step 4 by level (1-16).
Label two 50 mL tubes as rinses.
Dilute Environmental standard.
The Environmental standard contains 1000 µg/mL of more abundant elements such as Ca, Fe, K, Mg, Na and 10 µg/mL of less abundant trace elements such as Ag, Al, As, Ba, Be, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, Sb, Se, Th, Tl, U, V, Zn. Two concentrations are used to describe the Environmental standard in the subsequent steps. For example, the concentration of the undiluted Environmental standard is written as 1000/10 µg/mL.
Place an empty acid washed 15 mL tube in a beaker (use a lightweight beaker not exceeding 250 g) on an analytical balance (model: MS303TS/00, max = 320 g, d=0.001 g).
Add 9.90 g or 9.90 mL of Milli-Q water to the tube. If there is residual water left in the tubes from washing, subtract the mass of the empty tube (recorded in step 4) from the mass of the tube with water (tare with beaker) to calculate how much water was added.
Dilute the environment standard 100 fold from 1000/10 µg/mL to 10/0.1 µg/mL by adding 100 µL of the Environmental standard to the tube containing 9.90 g of MilliQ water. Cap the tube, and invert it to mix.
Dilute P and S standards.
The phosphorous and sulfur calibration standards are purchased separately and are supplied at a concentration of 1000 µg/mL. Dilute each standard 100 fold to 10 µg/mL in separate acid washed 15 mL tubes.
Place an acid washed calibration standard 50 mL tube in a light weight beaker on an analytical balance (model: MS303TS/00, max = 320 g, d=0.001 g). Each calibration standard will contain specific volumes of Environmental standard, phosphorous standard, sulfur standard, Milli-Q H2O, and 70% HNO3. Use the analytical balance to precisely measure the mass of water and other reagents with required volumes that are difficult to pipette precisely.
Refer to table 1. for the volumes of each solution required for each calibration standard.
Table 1. Final concentrations of calibration standards and volumes of each component in each level.
Example: preparation of level 16 standard
Tare the 50 mL tube labelled "16".
Add 41.61 g of MilliQ water to the tube by reading the mass on the balance.
Add 2.5 mL of the 1000/10 µg/mL Environmental standard.
Add 2.5 mL of the 1000 µg/mL Phosphorus standard.
Add 2.5 mL of the 1000 µg/mL Sulfur standard.
Add 0.89 mL of Fisher Optima‱ grade 70% nitric acid.
Cap the tube and invert five times to mix.
Note
The density of water, the phosphorous standard, and the sulfur standard is 1.00 g / mL.
The density of 70% HNO3 is 1.42 g / mL.
The environmental calibration standard contains 5% HNO3 and has a measured density of 1.05 g / mL.
Store standards at room temperature in a hood designed to extract HNO3 fumes.
Checking the instrument status using the MassHunter software
Checking the instrument status using the MassHunter software
Directions in this protocol are for the MassHunter 4.4 (vC.01.04 Build 544.8) software only. If you are using a different version of MassHunter, the general idea and workflow will be similar but refer to your specific version’s manual for more information.
Check the early maintenance feedback page.
Open the early maintenance feedback page to view required and recommended maintenance.
Navigate to the hardware pane.
Right click on the mainframe and choose the early maintenance feedback option.
Ensure that all necessary maintenance has been performed.
Please also refer to the 8900 maintenance manual for required and recommended maintenance.
Early maintenance feedback panel with status bars.
To view the status of the instrument in greater detail, you may view up to 5 meters at a time which display real time information from various sensors onboard the instrument. Click on the view tab on the top left of your screen. Select meters from the drop-down menu. Select up to 5 meters.
During the startup procedure we recommend selecting the meters for the IF/BK pressure, analyzer pressure, forward power, reverse power, and plasma gas flowrate.
A table of typical ranges for each meter during shut down mode, standby mode, and analysis mode can be found in the help tab on the top of your screen. Click on the help tab. Type "instrument status" in the search bar and add it to your favorites tab. You may print it out as well.
Table of typical meter values during different instrument modes, which can be accessed in the help section of Mass Hunter v4.4.
Check the status of the vacuum.
Ensure the instrument is in standby mode and not in shut down mode. If the instrument is in shut down mode it means the vacuum pump is shut off.
You will need to put the instrument in standby mode and let the vacuum pump run for a few hours even after the typical standby vacuum pressures (IF/BK pressure: 0.3-5 Pa, analyzer pressure: 1 × 10−5 - 7 × 10−4 Pa) been reached. If the vacuum pump has been shut off for longer than 10 hours or if the vacuum seal was broken, you may need to run the vacuum pump overnight.
Check the oil level on the vacuum pump. If it is dark in color, replace it. If it is below the fill line, fill it. Please see the maintenance section for more details.
MS40S foreline vacuum pump.
Check the error logs.
Check the error logs to make sure that no new errors have been reported. The error log will have a timestamp of when the error was detected, an error code, and an error description. If you unfamiliar with the error and its cause, you can read more about the specific error by searching the error code in the help tab.
Note
On our system, error code 2416, "Option gas flow rate is out of allowable range" sporadically appears. Since this method does not use an option gas, the error can be ignored.
Error log history drop down bar. The log contains the date, time, error code, and error description.
If all required maintenance has been performed and both the IF/BK and analyzer pressures are within the typical ranges in standby mode, you may proceed.
Inspect the sample introduction and on-line internal standard addition components
Inspect the sample introduction and on-line internal standard addition components
Initialize the Autosampler.
Test the Autosampler by clicking on the Autosampler drop down menu beneath the hardware pane.
Click on initialize.
The Autosampler should move the probe to the four corners of the Autosampler.
Make sure that the Autosampler is connected to the exhaust duct so that harmful HNO3 fumes can be safely extracted.
Inspect the peristaltic pump tubing located behind the Autosampler that provides 2% HNO3 to the probe rinse station. Replace the tubing if it is damaged or discolored.
Inspect sample introduction tubing.
Inspect the sample, ISTD, and waste peristaltic pump tubing for cracks, flat spots, or interior damage.
If the peristaltic pump tubing was left clamped to the peristaltic pump, you may have to replace it. Replace the peristaltic pump tubing frequently. Most issues in the analysis can be traced back to the peristaltic pump tubing and sample introduction components. Check each of the rollers of the peristaltic pump to ensure that they roll smoothly.
Install the sample, internal standard, and drain peristaltic pump tubing to the peristaltic pump.
Observe the rotation of the peristaltic pump and always hang the upstream stopper first.
The sample tube (white/ black stoppers, 1.02 mm i.d.) goes on the left track.
The internal standard tube (orange/blue stoppers, 0.25 mm i.d.) goes on the middle track.
The drain tube (yellow/blue stopper, 1.52 mm i.d.) goes on right track. The upstream tube of the drain tubing should connect to the spray chamber. Do not reverse the tubing.
Extend the tubing over the rollers and hang the downstream stopper.
Clamp the tubes by closing their respective levers.
Re-check the direction of flow for all tubing.
Verify that the internal standard is connected to the connector block downstream.
Peristaltic pump and tubing.
Set the proper tension on the peristaltic pump clamp levers.
Click on the peristaltic pump icon beneath the hardware pane.
Set the speed to 0.1 revolutions per second (rps).
Click on the Autosampler icon beneath the hardware pane.
If the probe was dry, move the probe to a rinse solution. If the probe has liquid in it, move it to the home position (no liquid) and then back to a rinse solution to introduce an air bubble.
Observe the bubble as it enters the probe and travels through the sample tubing.
Loosen the clamp tension knob until the bubble does not move.
Slowly tighten until the bubble moves (it may be pulsating).
Tighten the knob an additional 1 to 2 full turns and ensure that the bubble travels without a jerking or pulsating motion.
Repeat this process for the internal standard.
For the drain tubing, observe bubbles in the drain tubing leaving the spray chamber. Alternating liquid and gas discharge is normal.
Loosen the clamp tension until the bubbles do not move.
Slowly tighten until the bubbles start to move.
Tighten an additional 2 to 3 full turns.
Check tubing for leaks.
Using the Autosampler control window, put the probe into a rinse solution.
Make sure that the internal standard tube is in the internal standard solution.
Set the peristaltic pump speed to fast (0.5 rps) for 5 min to let all the bubbles flow through the nebulizer. When all expected bubbles in the tubes have made it past the connector block and into the nebulizer, observe the connector block and nebulizer tubing for 1 min.
Make sure there are no unexpected bubbles introduced into the tubing. If bubbles are present, tighten all PTFE nuts on the connector block and make sure that the probe and internal standard tubes are in the solutions. Check the tubing connections where the firm PTFE tubing meets the soft peristaltic pump tubing for both the sample and internal standard tubes.
Disconnect the internal standard before plasma ignition and performance checks.
Stop the peristaltic pump and disconnect the internal standard from the connector block. Keep the tip (PTFE connector) of the internal standard tubing clean and make sure that it does not drip. Wrap the tip in a Kimwipe and secure it in an elevated position to avoid gravity flow.
Use a threaded white plug to replace the internal standard connector. Remove the internal standard peristaltic tubing from the peristaltic pump.
Set the peristaltic pump speed to normal and check for leaks by observing the nebulizer tubing for 1 min to make sure that bubbles are not introduced at the connector block.
Chiller and compressed gases
Chiller and compressed gases
Turn the chiller on.
Ensure that the coolant is filled to the proper level before powering the unit on. The coolant level indicator is located behind the machine and can be easily checked by holding a flashlight to the reservoir from the side and through the metal screen. The chiller should be powered on before the plasma is ignited and should remain on at least 10 minutes after the plasma has turned off. To assess the performance of the chiller, navigate to meters tab and monitor the meters for water RF/WC/IF, Water temperature, inlet temperature, and internal temperature. Please note that the exhaust flowrate also influences the internal temperature.
G3292A recirculating chiller
Check hydrogen, helium, and oxygen gas lines for leaks.
Do not open any valves (cylinder and regulator) until you have verified there are no leaks in the gas lines. See steps below to determine if a leak is present with the valves closed. Verify that the exhaust is working properly and that the room is properly ventilated. Make sure that the regulators are connected properly and safely. Only trained personnel should handle compressed gases.
If the cylinder valves were closed, check the delivery pressure gauges on all regulators to ensure that all gas lines are pressurized before you open any cylinder valves. The collision reaction gas (O2, He, and H2) lines should remain pressurized even if the cylinder valve is closed. If a line is not pressurized, then you may have a leak and you will need to purge that line. See details below.
Safety information
If gas lines are depressurized, use the regulator pressure control knob to close the regulator. Most regulator pressure control knobs are counter-intuitively turned to the left to close the regulator. Read the specific regulator's instruction manuals. The knob should feel loose. Failure to close the regulator prior to opening the cylinder valve may cause the regulator's diaphragm to rupture, causing an uncontrolled release of gas, potential over pressurization, and explosion.
Check for leaks using a leak detector or pressure test. H2 and He gases are prone to leaks.
Inspect the H2 and He gas filters for moisture and oxidation. You may need to change a filter(s) before analysis. Purge the gas lines for 2 hours at 12 mL/ min, whenever a cylinder or filter is replaced.
Side-by-side comparison of an old and new carrier gas filter. A brown moisture indicator signifies moisture saturation. A black oxygen indicator represents oxygen saturation. Both indicators should be light green. The old carrier gas filter in the image above is saturated with moisture.
Open the argon dewar gas valve (Do not open the liquid or vent valves).
If the gas line is not pressurized, close the regulator prior to opening the gas valve on the dewar.
Position yourself with the dewar between you and the regulator.
Keep your hands free of the regulator and look away as you slowly open the argon cylinder valve.
Verify that the argon delivery pressure is set to 90 psi.
Verify that the argon gas pressure of the dewar is above 100 psi. If not, you will have to open the pressure building valve. The pressure building valve controls the flow of liquid argon through an uninsulated circuit, which causes it to boil into a gas. You will need to monitor the pressure as it builds. Do not leave the pressure building valve open unattended. Close the pressure building valve before the gas pressure reaches the venting pressure (usually 230 psi). The plasma will shut off if the delivery pressure drops below approximately 70 psi.
Argon dewar valves and pressure building circuit.
Carefully open cylinder valves for the collision reaction cell gases (read entire step first).
Open the H2 and He cylinder valves very slowly. Do not look at the regulators while opening the cylinder valves. Verify that the H2 and He delivery pressures are set to 12 psi.
Take extra caution when opening the oxygen cylinder valve. Open the valve very slowly. Rapid adiabatic compression of oxygen gas can cause auto-ignition and explosion.
Purge argon gas.
If you have just replaced your argon dewar, there will be air in your argon line.
Purge the argon gas for 5 min.
View the “Plasma Gas” meter to determine if argon is flowing.
Right click on the plasma icon in the middle of the screen. Select “Gas Purge at Standby”
Click on “Gas Purge at Standby” on the left of the screen and the icon should turn orange when it is purging.
Verify that the Plasma Gas meter reads 15-17 L / min.
After 5 min, make sure to close the purge valve by clicking on the same icon.
Plasma ignition
Plasma ignition
Right click on the “plasma” tab and click “plasma on”.
The plasma will ignite, and the system will transition from standby mode to analysis mode. The status light on the main instrument will turn green. You should see the plasma ignite through the tinted cover.
Verify that the forward power is between 600 and 1600 W.
The reflected power should drop to below 20 W within the first few minutes.
Verify that the plasma gas flowrate is around 15-17 L / min.
Monitor the internal temperature.
The instrument will warm up for 30 min and will automatically add the rest of the plasma ignition sequence from the plasma tab to the Queue. During this sequence, the instrument will use a tuning solution to perform a standard tune.
As the instrument warms up, uncap the tuning solution and all 2% HNO3 rinse solutions in the Autosampler.
Click on the “Queue” tab to view the status of the standard tuning steps.
At the end of the standard tune, a performance report is generated.
The most important thing to look at are the counts for channel 1, 2, and 3. Look at the performance report in history mode to see the how the counts for each channel have changed throughout time. A significantly lower count in all channels usually indicates that maintenance is needed.
Creating a batch file from an existing batch
Creating a batch file from an existing batch
The easiest way to run a routine analysis is to create a new batch file from an existing batch file.
This will copy the acquisition method, data analysis method, calibration method, and sample list from the old batch. You may only need to change the sample names.
Note
We have attached a compressed batch folder to this protocol so that you may use it as a template (file: ICPMS_Batch_Template_v01_20241210.b.zip).
Decompress the folder before creating a new batch.
Choose the option to create from “existing batch”
Type in the file path or click “select…”
Choose the batch you wish to use as a template.
Click open
Select acq method, DA method, and Sample List. Verify that the tune modes are correct and press OK.
Navigate to the folder in which you wish to save your batch and name your batch with the date, your initials, and a short description.
Navigate to the sample list tab. In column 1, specify the sample type.
Choose the location position of your sample in the Autosampler. Use the fill down function.
Autotune all tune modes of the batch.
After setting up the batch, perform a batch tune in all three tune modes (collision reaction cell gas modes).
Access the “tune” tab, then click “autotune”
Click “Tune to all modes”
The autotuning step will take approximately 30 min.
When tuning is complete, cap the tuning solution to avoid evaporation.
A tune report is automatically generated and saved in the batch folder.
Read the tune report and verify
- RSD values for the monitored tuning masses
- Peak shapes
- Doubly charged oxides
Connect the internal standard when batch tuning is complete.
Stop the peristaltic pump and install the internal standard peristaltic pump tubing to the pump.
Remove the white threaded plug on the connector block.
Screw the internal standard line into the connector block tightly.
Inspect and wipe the conductive ground connector and ground clamp if wet or dusty.
Place the connector block back into the ground clamp.
Set the peristaltic pump speed to fast and put the Autosampler probe into a rinse solution.
Click on the signal monitor and make sure that yttrium (atomic mass = 89) is selected.
Observe the connector block for 5 min. If there is air in the internal standard line, there will be bubbles in the connector block and the Y89 signal will be unstable. Make sure that the internal standard line is at the bottom of the solution in the bottle. Wait until the signal for Y89 is stable and there are no bubbles in the connector block.
Adding samples
Adding samples
Uncap calibration standards and samples.
To see a specific example of ICPMS sample preparation, refer to the protocol outlined by Camacho et. al., (2024), which details the processing of Auxenochlorella protothecoides cells and spent medium for ICPMS and total organic carbon (TOC) analysis.
Place tubes in the correct positions of Autosampler trays.
Minimize the amount of time samples and calibration standards are uncapped to avoid:
-Contaminating particles from entering the tubes
-Release of toxic and corrosive HNO3 fumes
-Evaporation and alteration of analyte concentrations.
Click “add to queue” to add the batch to the queue.
The Agilent ICP-MS Analysis software will launch automatically.
Data Acquisition
Data Acquisition
As the calibration standards and samples are measured, they will populate the table.
Choose the internal standard and tune mode of interest to view the calibration and calculated concentrations.
The atomic mass of Sc is closer to most elements that our lab is interested in so we prefer to use Sc to normalize the data. To account for drift and variations in sensitivity throughout a run, analyte counts are normalized against internal standard counts.
Monitor the recovery of the internal standard's masses in all tune modes. A graph is displayed and is updated after every sample that is analyzed. The internal standard recovery should be stable at 100%.
Pay attention to the Background equivalent concentration and detection limits displayed for each analyte during the calibration.
It is important to monitor the first few calibration standards of low concentrations to determine if the calibration standards have been contaminated or if the concentrations may have changed due to evaporation. If the background equivalent concentration (BEC) and the detection limits (DL) are high, then you may need to stop the run and remake fresh calibration standards.
Pay attention to the R2 values of the calibration graph which plots the calculated concentration of the calibration standard against the expected concentration. You may reject individual calibration standards for each analyte if the calculated concentration deviates from the line of best fit.
As soon as the calibration is complete, cap your calibration standards. Do not wait until the end of the run to cap your calibration standards.
Pay attention to the RSD values of the replicate measurements for each analyte. Reject replicate measurements where the RSD of all replicates exceeds 5%. If the RSD values are consistently high in one tune mode, then you may have an issue with the purity of the cell gas or stabilization time.
Export the data.
Shutting down
Shutting down
We recommend running a few fresh blanks at the end to verify that there are no residual ions (memory effects) in the sample introduction system.
You can choose what the instrument will do once all items in the queue have been completed. You can choose to turn the plasma off at end or to keep the plasma on and aspirate a wash solution.
We recommend "pausing at end" and aspirating the wash solution to wash the sample introduction system and to keep the peristaltic pump running so that the peristaltic pump tubing can be reused.
When you are satisfied with the run and are ready to put the instrument in standby mode, return the probe to the home position.
Remove and cap all your samples.
Cap all wash solutions.
Right click on the plasma icon and select "plasma off".
Stop the peristaltic pump and remove the peristaltic pump tubing from the pump so that no flat spots are created.
Neutralize waste 2% HNO3 following the directions from step 5.
Protocol references
Dimitrios J. Camacho, Charles Perrino, Sabeeha Merchant 2024. Sample Preparation for Elemental Analysis of Auxenochlorella protothecoides (UTEX 250) Cells and Spent Media by Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS) and Total Organic Carbon (TOC).. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn69wml5d/v1
Acknowledgements
We thank Dr. Anne G. Glaesener and Dr. Stefan Schmollinger for their guidance and instruction.