Acid digestion of samples using a Multiwave GO Plus microwave system

1. Introduction
This standard operating procedure (SOP) describes a procedure for acid digestion of samples using the Multiwave GO Plus microwave system. A known weight of the sample is placed in the digestion vessel and digestion acid added. The digestion vessel is capped and subjected to microwave heating within a sealed pressurized chamber to achieve a high digestion temperature, then cooled down to room temperature. The digest liquor is quantitatively transferred into a plastic test tube and diluted with water to a known volume.

2. Scope
This procedure may be applied to any organic sample that is amenable to acid digestion prior to analysis by inductively coupled plasma-optical emission spectrometry. This includes several commodities like coffee, soy and cocoa beans.

3. Materials

Chemical/Solution	Description	Storage Location
Sample	Organic sample of commodity that will be analysed	/
HNO3 (65%)	Highly concentrated Nitric acid (see Safety!) to digest sample	Room 227 ‘’Stock room’’, D2
Neutralizing agent (Chemizorb or NaHCO3)	Sodium bicarbonate (baking soda) or Chemizorb as neutralizing agent against accidental spills	Room 218b ‘’Main lab’’, A5
MilliQ water1	Water of Millipore grade or equivalent used for general cleaning and more. Always see that several jerry-cans of Millipore water are available as a lot of it is used during the procedure.	Room 218b ‘’Main lab’’, A17

4. Equipment

Equipment	Description	Storage Location
PPE	Personal Protective Equipment, which includes safety glasses and a laboratory coat	Room 218b ‘’Main lab’’
Analytical Balance	Analytical Balance Sartorius CP 224 S that weighs samples accurately up to 4 decimal places	Room 218b ‘’Main lab’’, A7
Plastic spoon	Plastic spoon to transfer sample (if milled into powder sample) quantitatively into pressure vessels	Room 218b ‘’Main lab’’, A8
Pressure Vessels with vessel caps	PTFE Pressure Vessels and appropriate PTFE caps included with the Multiwave GO Plus microwave system	Room 218b ‘’Main lab’’, A8
Laminate gloves and acid-resistant sleeves	Safety gloves made of neoprene or better (e.g. butyl rubber). Standard nitrile gloves do NOT suffice to handle highly corrosive acids ( HNO3, >65%)	Room 218b ‘’Main lab’’, AV
Protective apron	Apron to add an extra layer of protection against splashing and spilling of highly corrosive acid	Room 218b ‘’Main lab’’, AV
Microwave system	A Multiwave GO Plus microwave system by Anton Paar (Figure 1)	Room 218b ‘’Main lab’’, A4
Dispenser	Acid resistant dispenser intended to efficiently dispense a correct volume of HNO3	Room 218b ‘’Main lab’’, A5
Volumetric flask(s), 50 mL	Volumetric flask (50 mL) to dilute the digest after acid digestion.. This dilution is usually a prerequisite to analyze the digest with ICP-OES.	Room 218b ‘’Main lab’’, A15
Glass funnel	Glass funnel to catch rinse water	Room 218b ‘’Main lab’’, A15

5. Safety

5.1 H/P phrases HNO3 (65%)

5.2 Safety Summary: HNO3 (65%)
Inhalation
- Toxic if inhaled
- Corrosive to the respiratory tract
\=> work in fume hood
Skin
- Causes severe skin burns
\=> Wear laboratory coat, protective apron and safety gloves (laminate or neoprene)
Eyes
- Causes serious eye damage
\=> Wear safety glasses
Fire
-Oxidiser
\=> Keep away from heat sources
! Keep a neutralizing agent (Chemizorb or NaHCO3) nearby in case of accidental spills

5.3 Before starting digestion

5.3.1 Checklist

• Always wear goggles, protective gloves and appropriate protective clothing. Set a perimeter around the fume hood in which individuals are always wearing the appropriate protective wear (e.g. safety glasses)
• Check if pressure vessels aren’t directly next to Multiwave GO Plus, keep a distance of at least 1m
• Check if the fume hood is turned on and running
• Check if no containers with flammable reagents or other ignitable materials (e.g. soaked cleaning paper) are close to the instrument or on the instrument
• Check the mains cable for any signs of damage prior to connecting the instrument to the mains supply
• Clean the surface of the instrument with damp cloth if contaminated
• Clean the display with a wet, lint-free cloth if contaminated
• Check the pressure vessels for deformation or damage and especially scratches in the ceiling areas. Check the vessels for whitish spots, which are signs of thermal damage. Inspect the vent hole of the screw cap of the pressure vessels
• Check the rotor and the rotor lid for deformation or damage and especially scratches
• Check for impurities on vessel or rotor parts as this can lead to local overheating and result in damage of these parts
• Check the IR sensor foil on stubborn stains, wrinkles or scratches and replace the foil if necessary. After replacing the IR sensor foil, the IR sensor needs to be calibrated (See Cleaning and Maintenance)
• Check microwave chamber for spills, deformations or damage and the perforated plate in the middle of the chamber for damage/corrosion as this can cause microwave radiation leakage (!)

5.3.2 Place exhaust hose
The exhaust hose deducts the produced acid vapors safely from the instrument to your exhaust system. Multiwave GO Plus has to be connected to a fume hood with a minimum exhaust capacity of 100 m3/h. Make sure that the clip of the hose is tightened properly. To do so, fix the exhaust hose with the supplied hose clip to the flange of the exhaust of Multiwave GO Plus. Install the exhaust hose in a U shape (Figure 2, left) to avoid reflux of condensed acids. Carefully fix the open end of the exhaust hose with the delivered clip in the fume hood. Unscrew the metal rod and attach the exhaust hose (Figure 2, middle & right). While the instrument is working do not perform labor work in the same fume hood which is used for the exhaust hose of the instrument.

Figure 2: Correct attachment of exhaust hose to the fume hood in U shape

5.4 During digestion

5.4.1 General safety precautions
Do not lean against the instrument. Instead, it is recommended to keep a safe distance of approx. 1m from Multiwave GO Plus during operation.

During operation (microwave energy "ON") an electromagnetic field is generated by two transformers. People with pacemakers should keep a distance of at least 1m to the instrument during operation to ensure maximum safety. Keep the following items away from Multiwave GO Plus: credit cards or other cards with a magnetic read-write band. These might be damaged (e.g. if carried in the operator's pocket).

5.4.2 Power Failure
In case of power failure during the operation, please refer to the Instruction Manual and Safety Information or the Reference Guide of Multiwave GO Plus.

6. Sample Digestion

6.1 Preparing the pressure vessel HVT50

1. Make sure the pressure vessels are completely dry and clean
2. Weigh the raw sample (or sample powder from ball mill²) into the vessel on an analytical balance (don’t forget to tare!):

• For unknown samples with high reactivity: start with 100 mg and slowly increase to max. 500 mg
• For unknown samples with normal reactivity: start with 300 mg and slowly increase to max. 1 g

• For coffee beans, soybeans and timber: weigh 0.5 g -1 g (Figure 3)

  Figure 3: Weighing ball milled sample powder (right) directly into the pressure vessel

3. Repeat the weighing process for every vessel
4. Add 2 mL MilliQ water to each vessel
5. Place a warning sign at the door of the lab so other laborants know that pure acids will be handled
6. Put on acid-resistant neoprene or laminate gloves AND sleeves.
7. Add HNO3 to each vessel via a dispenser that is set to the correct volume. If using the generic method Organic A, add a volume of HNO3 according to Table 1. For a small number of vessels (1 or 3) use an acid amount of not less than 6 mL to provide enough mass for microwave absorption. For other acids, see Appendix I

Table 1: volume of HNO3 to be added when using Organic A on the Multiwave GO Plus

Sample amount (g)	Acid volume (mL)
0-0.6	6
0.7	7
0.8	8
0.9	9
1	10

8. Ensure that all vessel parts are completely dry: remove any acid or water droplets from the vessel surface. Ensure that no residues of the sample are sticking on the sealing and screwing area of the vessel body
9. Place the seal onto the vessel and close the screw cap with your fingers until the spring holder and the spring holder insert are on the same level. The screw cap must be closed without overlap at the vent hole otherwise the rotor can be hot, even if cooling has supposedly finished and the lid lock has been released (Figure 4)

Figure 4: Correct way of closing the pressure vessel

10. Place the close vessel into the rotor as described in 6.2
11. Repeat for the other vessels

6.2 Loading the rotor 12HVT50

1. Multi-vessel digestion
   Enter the vessels into the rotor. To ensure uniform temperature distribution during the run, the rotor has to be loaded symmetrically³. Follow the load patterns as described in Figure 5. If samples and blanks will be digested in the same run, mix them alternating in the rotor. If a spark error (0x0024) occurs, check if acid venting took place, increase the amount of reagents, or increase the number of used vessels.

Uni-vessel digestion
If only one pressure vessel, activate the single vessel mode in the run settings and load the vessel on position 1 (Figure 5). If a spark error (0x0024) occurs, check if acid venting took place, increase the amount of reagents, or increase the number of used vessels.

Figure 5: load patterns for the rotor

2. Put the rotor lid onto the rotor
3. Open the lid of Multiwave GO Plus and place the rotor onto the drive ring
4. Close the lid of Multiwave GO Plus (you should hear a click sound)

6.3 Running an experiment
The recommended temperature program for organic samples is ‘’Organic A’’. To create a new program, please refer to the Instruction Manual and Safety Information or the Reference Guide of Multiwave GO Plus

1. Make sure you remove your protective gloves so the machine will not be contaminated
2. Switch on the instrument via the power button on the back of the machine. The Multiwave GO Plus automatically displays the start screen (Figure 6). Operate the touchscreen with gentle fingertip pressure only to ensure a long lifetime of the touchscreen.

Figure 6: Start screen

3. Tap \< > "Create Run" on the start screen.

4. Define the following settings:
   1) Enter a "Run Name"
   2) Select a "Method" e.g. preinstalled method ‘’Organic A’’. Move the desired list item into the red selection bar and tap \<Select>.
   3) Optionally enter a "Run Note"

5. Tap \< > to confirm your settings.

6. Check if Multi vessel (or Single vessel) mode is selected under \<>
7. Tap \<Start> to start the run. Multiwave GO Plus starts processing the chosen method
   (Figure 7).

Figure 7: Run screen

8. Wait until the run has finished. The results of the experiment are stored and can be viewed and/or exported onto a USB stick via the "Data Memory" dialog

6.4 Viewing and exporting experiment results

1. Tap \<Menu> on the start screen and select "Menu > Data Memory". The data memory with all processed runs opens. Select a run by scrolling the red frame (Figure 8).

Figure 8: How to open Data Memory and select your desired run

2. Tap \<Details> to open detailed information of your run
3. Scroll the "Details" window to navigate through the run details.
4. Tap \<Export> to export the run to a USB device

6.5 Unloading the rotor

1. After the cooling has finished take the rotor from the oven (be careful: might still be hot!) and place it into a fume hood.
2. Take off the lid and clean any traces of liquid on the vessels’ surface and the inside of the lid
3. The vessels may still be under overpressure, thus, when opening the vessel in a fume hood point the vessel away from the body. Carefully and slowly open the vessel to release the residual pressure. Take care of possible foaming due to dissolved gases.
4. Remove the seal from the pressure vessel. If the seal sticks in the pressure vessel, tilt the seal carefully into the cap to remove it easily.
5. Transfer the content of the pressure vessel into a volumetric flask (50 mL). You can use a stand to hold the volumetric flask upright.
6. Rinse the seals and vessels with MilliQ water and catch the rinse water using a glass funnel. Wash the funnel and dilute the digest to 50 mL with MilliQ water. Don’t forget to homogenise from time to time. You can use a piece of parafilm to close the flasks for homogenizing.
7. Transfer the diluted digests into the labeled ICP-OES sample tubes and proceed with your task at hand.
8. Turn off the microwave system and put the exhaust hose as shown in Figure 9.
9. Clean and dry the rotor, vessels and instrument and inspect all parts for damage as described in 7. Cleaning, maintenance and storage.

10. Dry the lid and keep it separately from the rotor in the venting hood. The lid should not stay on the rotor, as condensed acid can corrode the top of the rotor.

    Figure 9: Proper way of putting away the exhaust

7. Cleaning, maintenance and storage
Before you clean the instrument switch off the instrument and disconnect the mains plug.

• Clean the rotor lid with paper if there is condensation. Store the lid separately from the rotor.
• Clean the pressure vessels, screw caps and the seal plugs after every run + check pressure vessels for deformation or damage and especially scratches in the ceiling areas:

(To remove oily residues, use alcohol/acetone but do NOT clean the screw caps in solvents like acetone! The O-rings will swell and can be destroyed)

1. Rinse the vessels, screw caps and seals thoroughly with deionized water.
2. If different types of samples are used, perform a cleaning run to avoid memory effects. A cleaning run goes as follows:
   1) Fill each vessel with at least 6 mL of a diluted digestion reagent mixture (e.g. 3 mL HNO3 and 3 mL H2O).
   2) Place the vessels into the rotor and place the rotor onto the drive ring
   3) Close the lid of Multiwave GO Plus and perform a cleaning run.
3. All parts must be completely dry before they are reused again. Note that seals should always be stored with the same screw caps. Store pressure vessels on the drying rack and unscrewed, as the thread of the cap could be harmed.
   - Clean rotor with damp cloth after every run + check the sealing foam of the rotor for deformation or damage:
4. Remove visible contaminations (e.g. reagent spills) with a damp cloth.
5. Parts must be completely dry before they are reused again.
6. Store the rotor outside of the microwave without the lid.
   - Clean the table top and water bottles as these might have been in contact with contaminated gloves. Don’t forget to clean your hands at the end too!

• Intense cleaning of pressure vessels after several runs:
  1. Remove any visible impurities from the vessel body with water, a lab cleaning detergent and a soft brush.
  2. Perform a cleaning run (see earlier).
  3. Place the liner and vessels into a drying oven at 200 °C to 250 °C for at least 4 hours, or overnight. Any residues, which are not removed before heating the vessels in the drying oven will be burned-in and can be hardly removed afterwards.
  4. Let the vessels cool down prior to first use.

• Clean microwave chamber with damp cloth after each working day:
  1. Remove the drive ring and the drip cup from the microwave cavity and clean with a damp cloth.
  2. Clean the lid in the same way, also the contacting surfaces between lid and instrument.
  3. Reassemble the parts and make sure they are completely dry before they are reused again.

• Exchange IR sensor foil (if contaminated) + recalibrate IR sensor every month:
  1. Remove the drive ring and drip cup from the cavity.
  2. Remove the old IR sensor foil from the cavity (if contaminated). Remove the protective paper from the sticking area of the new IR sensor foil and align correctly (the ear of the foil should point inwards)
  3. Stick the IR sensor foil directly onto the recess of the IR-sensor
  4. Recalibrate:
  1) Open the lid of Multiwave GO Plus
  2) Remove the drive ring
  3) Place the calibration unit into the two bores of the microwave cavity and connect the plug on the rear side of Multiwave GO Plus (Figure 10)

Figure 10: Correct placement of calibration unit

4) Tap ‘’Menu > Service > T-Calibration". The calibration is now performed.
5) Remove the calibration unit from the instrument.

• Replace sealing foam of the rotor every 1-2 months

• Cleaning after a vessel breakage \=> refer to Multiwave GO Plus Reference Guide

8. Waste disposal

• Organic waste that has NOT been contaminated with HNO3 may be disposed of into the drain.
• All HNO3 containing waste must be disposed of into glass waste containers that are exclusively used for HNO3 containing waste (highly reactive!) and with labels that indicate its content. Frequently check the content level of the waste containers so it does not exceed the threshold line.

9. Appendix

1. Different acids
   IMPORTANT: It is not recommended to digest samples with different acid mixtures in the same run. HNO3, HCl and H2O2 are suitable for digesting most environmental, agricultural and food samples. HF is required for total digestion with subsequent determination of the concentration of certain recalcitrant elements such as SI and Al, whereby the remaining HF can be complexed with H3BO3. H2SO4 and H2SO4 are only used for open-vessel digestions.

Acid	Volume Limitations	Advantages	Disadvantages
HNO3	Between 3 mL and 15 mL	- Dissolves most metals	- Noble metals (Au, Pt, …) are not oxidized - Some metals (Al, Cr, …) are passivated
HCl	Always add HNO3 to pure HCl. Aqua Regia is commonly used (HNO3 3:1 HCl).	-Complexation of several metals such as Fe(II), Fe (III), Sn (IV) and Ti (III) - Powerful in combination with HNO3 (Aqua Regia) - Adding HNO3 improves energy absorption - Stabilizes several elements	- Corrosive to vessels - AgCl, HgCl & TiCl are insoluble - PbCl2 is slightly soluble - absorbs microwave energy inefficiently
H2O2	Max. 6% of the reagent mixture + max. 2 mL per vessel	- Oxidizing power increases	- Can react explosively with organics
HF	Max. 0.5 mL HF per 100 mg expected SiO2	- Can dissolve Si - Complexation with H3BO3 (6 mL per 1 mL HF) resolubilizes insoluble elements + conserves ICP glassware	- Alkaline earth metals, lanthanides and actinides are insoluble - Toxic ! - Damages ICP glassware and Teflon
H2SO4	Max. 20% of the reagent mixture	- Increases reaction temperature at low pressure	- Stains + corrosion on vessels - BaSO4, SrSO4, PbSO4, CdSO4,... are insoluble
H3PO4	/	- Increases reaction temperature at low pressure	- Stains on vessels - MgPO4, CaPO4, … are insoluble

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