Preparation of Food Samples Using Homogenization and Microwave-Assisted Wet Acid Digestion for Multi-Element Determination with ICP-MS

Summary

The presented protocol describes sample homogenization with a laboratory mixer, acid digestion of food samples using a mixture of 68 wt% HNO₃ and 30 wt% H₂O₂ via microwave-assisted wet acid digestion, and multi-element determination performed with inductively coupled plasma mass spectrometry.

Abstract

Sample preparation is crucial for elemental determination, and various techniques are available, one of which involves homogenization followed by acid digestion. Special care is required during sample handling in the preparation stage to eliminate or minimize potential contamination and analyte loss. Homogenization is a process that simultaneously reduces particle size and uniformly distributes sample components. Following homogenization, the sample undergoes acid digestion, wherein it is digested with acids and auxiliary chemicals at elevated temperatures, transforming solid samples into a liquid state. In this process, metals in the original sample react with acids to form water-soluble salts. Samples prepared through acid digestion are suitable for elemental analysis using techniques such as inductively coupled plasma mass spectrometry, inductively coupled plasma optical emission spectroscopy, atomic absorption spectroscopy, electrochemical methods, and other analytical techniques. This work details the preparation of food samples for multi-element determination using inductively coupled plasma mass spectrometry. The step-by-step procedure involves the homogenization process using a laboratory-sized mixer with ceramic blades, followed by acid digestion in closed vessels using microwave-assisted wet acid digestion. A mixture of 5.0 mL of 68 wt% HNO₃ and 1.0 mL of 30 wt% H₂O₂ serves as an auxiliary reagent. This guide provides an explanation of the processes involved in both stages.

Introduction

Elemental analysis is an analytical process for determining the elemental composition of various samples. It can be used to control the intake of metals into human bodies (especially heavy metals¹) since their high concentrations may cause unwanted health problems. Heavy metals are also one of the main environmental contaminants, therefore, control of their presence in the environment is necessary². Moreover, elemental analysis can be employed to determine the geographical origin of food products³ and to control the quality of food and water resources⁴. In addition, it is used for the determination of micro- and macronutrients in soils⁵ and to gain insights into geological processes throughout history by examining the chemical composition of minerals and sediments⁶. Studies have also been made to determine the presence of rare metals in electrical and electronic waste for further metal regeneration⁷, to evaluate the success of drug treatments⁸, and to verify the elemental composition of metal implants⁹.

Inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES) are commonly used techniques for the elemental analysis of various samples¹⁰. They allow simultaneous determination of multiple elements with limits of detection (LOD) and limits of quantification (LOQ) as low as ng/L. In general, ICP-MS has lower LOD values¹¹ and a wider linear concentration range compared to ICP-OES¹². Other techniques to determine elemental composition are microwave-induced plasma optical emission spectrometry¹³ and several variants of atomic absorption spectroscopy (AAS), including flame atomic absorption spectroscopy, electrothermal atomic absorption spectroscopy², cold vapor atomic absorption spectroscopy, and hydride generation atomic absorption spectroscopy¹⁴. Furthermore, elemental determination with low LOD and LOQ is possible with different electroanalytical methods, especially with anodic stripping voltammetry^15,16. Of course, there are other methods to determine the elemental composition of samples, but they are not as frequently employed as the above-mentioned methods.

Direct elemental determination of solid samples is feasible using laser-induced breakdown spectroscopy and X-ray fluorescence¹⁷. However, for elemental determination with ICP-MS, ICP-OES, and AAS it is necessary to convert solid samples into a liquid state. For this purpose, acid digestion is performed using acids and auxiliary reagents (in most cases H₂O₂). Acid digestion is carried out at elevated temperature and pressure, converting the organic part of the sample into gaseous products and converting the metal elements into water-soluble salts, thus dissolving them in the solution¹⁸.

There are two main types of acid digestion, open vessel digestion and closed vessel digestion. Open vessel digestion is cost-effective¹⁴ but has limitations, such as the maximum digestion temperature, which coincides with the boiling temperature of acids at atmospheric pressure. The sample can be heated on hot plates, heating blocks, water baths, sands baths², and by microwaves¹⁹. By heating the sample in that manner, much of the generated heat is lost to the surroundings²⁰, which extends the digestion time¹⁴. Other disadvantages of open vessel digestion include greater chemical consumption, the greater possibility of contamination from the surrounding environment, and possible loss of analytes due to the formation of volatile components and their evaporation from the reaction mixture²¹.

Closed vessel systems are more convenient for the digestion of organic and inorganic samples compared to open vessel systems. Closed vessel systems utilize a variety of energy sources to heat the samples, such as conduction heating and microwaves²². Digestion methods which use microwaves include microwave-induced combustion²³, single reaction chamber systems²⁴, and commonly used microwave-assisted wet acid digestion (MAWD)^25,26. MAWD allows digestion at higher operating temperatures, ranging between 220 °C and 260 °C and maximum pressures up to 200 bar, depending on the instrument's working conditions²⁷.

The efficiency and rate of MAWD depend on several factors, including the chemical composition of the samples, the maximum temperature, the temperature gradient, the pressure in the reaction vessel, the amount of acids added, and the concentration of acids used²⁸. In MAWD, complete acid digestion can be achieved in a few minutes due to the elevated reaction conditions compared to longer-lasting digestions in open vessel systems. Lower volumes and concentrations of acids are required in MAWD, which is in line with current green chemistry guidelines²⁹. In MAWD, a smaller amount of sample compared to open vessel digestion is needed to perform acid digestion, usually up to 500 mg of sample is sufficient^30,31,32. Larger sample quantities may be digested, but they require a larger amount of chemicals.

Since the instrument for MAWD automatically controls the reaction conditions and the person does not come in direct contact with the chemicals during heating, MAWD is safer to operate than open vessel digestions. However, the person should always proceed with caution when adding chemicals to the reaction vessels to prevent them from coming into contact with the body and causing harm. Reaction vessels also need to be opened slowly as the pressure is built up inside them during acid digestion.

Although acid digestion is a useful technique for preparing samples for elemental determination, the person performing it should be aware of its possible limitations. Acid digestion may not be suitable for all samples, especially those with complex matrices and those that are highly reactive or could react explosively. Therefore, sample composition should always be evaluated to select the appropriate chemicals and reaction conditions for complete digestion that dissolves all desired elements in the solution. Other concerns that the user must consider, and address are impurities and loss of analytes at every step of sample preparation. Acid digestion must always be performed according to specific rules or using protocols.

The protocol described below provides instructions for the homogenization of food samples in a laboratory-sized mixer, a procedure for cleaning the mixer's components, properly weighing the sample, adding chemicals, performing acid digestion by MAWD, cleaning the reaction vessels after the digestion is complete, preparing the samples for elemental determination, and performing a quantitative multi-element determination with ICP-MS. By following the instructions given below, one should be able to prepare a sample suitable for elemental determination and perform the measurements of digested samples.

Protocol

1. Sample homogenization

1. Using a clean ceramic knife, manually cut the food samples (broccoli, mushrooms, sausages, and noodles) into smaller pieces to speed up the drying process. Prepare enough samples for a minimum of 6 replicates of the acid digestion (ensure that the minimum mass of the dried samples is 1500 mg).
   NOTE: Increasing the surface area of the sample exposes a larger portion of the sample to the heated surrounding air, increasing the rate of evaporation of the water.
2. Place the sample in a 250 mL glass beaker and dry it at 105 °C to a constant weight using a dryer.
3. Remove the glass beaker with the sample from the dryer and insert it in the desiccator.
4. Allow the sample to cool down to room temperature.
   NOTE: Samples must be weighed at a constant temperature to ensure that the weight accurately reflects the mass. Temperature variations can affect the volume and density of the samples measured.
5. Open the desiccator and transfer the glass beaker with the sample on the analytical balance. Measure the weight of the glass beaker with the sample.
6. After the weighing is completed, put the sample back into the dryer.
   NOTE: If the sample shrank significantly during drying, one could transfer it to a smaller glass beaker using a plastic spatula for more convenient weighing.
7. Repeat the process as described in steps 1.3-1.6 until a constant weight of the sample is achieved.
8. Place the dried heterogeneous sample into the mixer beaker (see Table of Materials), ensuring it does not exceed the maximum volume of the mixer beaker.
9. Insert the mixer beaker into the mixer and close the guard door (Figure 1).
10. Press the start button to activate the blades for grinding and mixing the sample.
11. Perform the grinding until the sample turns into a fine powder or a homogenous paste. To achieve such a product, repeat the grinding process several times.
12. When the sample is homogenized, switch off the mixer, open the guard door, and remove the mixer beaker.
13. Remove the homogenized sample from the mixer beaker and transfer it to a clean 50 mL glass beaker using a clean plastic spatula (Figure 2).
    NOTE: If the sample is too hard and could potentially damage the mixer's components, such as the blades and mixer beaker, it can be homogenized by other means, such as crushing it in mortars. Mixers are usually unsuitable for homogenizing hard materials, frozen samples, or easily flammable samples, which could damage the mixer's components. The use of organic solvents in the mixer is discouraged.
    ​CAUTION: Use safety gear and ensure that the mixer's door is adequately closed as the mixer's blades rotate at high speeds.

2. Mixer cleaning

1. Add ultrapure water (see Table of Materials) to the mark of the empty mixer beaker.
2. Insert the mixer beaker into the mixer and perform the standard mixing procedure.
3. Take the beaker out of the mixer and pour out the wastewater. If necessary, repeat the process with ultrapure water several times until the water remains clean even after mixing.
4. Remove the contaminated blades and diaphragm seal from the mixer and clean them thoroughly with ultrapure water.
   NOTE: Use neutral detergents to improve cleaning efficiency, especially when dealing with samples with a high fat content, as fat easily adheres to the surface of the laboratory inventory.
   CAUTION: Wear appropriate protective equipment, such as gloves, when removing and cleaning the blades to reduce the risk of potential injuries from their sharp edges.
5. Dry the cleaned components in the dryer at 105 °C and reinsert them into the mixer.
   ​NOTE: Ensure that the mixer's components are completely dry before reinstalling them in the mixer, to prevent carry-over of the water to the following sample.

3. Sample weighing

1. Remove the cover lid from the 100 mL trifluoromethoxyl-polytetrafluoroethylene TFM-PTFE reaction vessel³³.
2. Place the open reaction vessel on the analytical balance and ensure that the balance is leveled and zeroed before each measurement (Figure 3).
   NOTE: Weighing must be performed at room temperature. Avoid areas where severe temperature fluctuations and air flow could affect the measured weight. Ensure that the weighing area is clean and free of any contaminants.
3. Transfer the homogenized sample into the reaction vessel using a plastic spatula and weigh 250 mg of the sample. Do not weigh the sample below the minimum weight limit of the analytical balance.
4. Once the weighing is complete, place the cover lid on the reaction vessel to protect the sample from contamination.
   NOTE: Exceeding the weight limit of the digestion procedure can result in incomplete digestion. Handle the sample and reaction vessels with care to avoid any external contamination.

4. Acid addition

1. Pour approximately 40.0 mL of 68 wt% HNO₃ and 5.0 mL of 30 wt% H₂O₂ into separate 50 mL glass beakers, respectively.
   NOTE: Chemicals must be of high purity with trace metal impurities of less than 1.0 µg/L (ppb), ideally in the ng/L (ppt) range. Trace metal impurities affect the accuracy and repeatability of elemental determination.
2. Place the reaction vessels in a fume hood, open the cover lids, and add the below mentioned volumes of 68 wt% HNO₃ and 30 wt% H₂O₂ with 1 mL or 5 mL automatic pipettes, according to the following specifications:
   1. Broccoli, mushrooms, sausages, and noodles; for 250 mg of sample add 5.0 mL 68 wt% HNO₃ and 1.0 mL 30 wt% H₂O₂. Prepare three replicates for every sample.
   2. To determine the accuracy of the method (in terms of recovery, Rec), use the procedure described in step 4.2.1 and add 37.5 µL of 100 mg/L ICP multi-element standard solution (see Table of Materials) into the reaction vessels using a 200 µL automatic pipette. For every sample, prepare three replicates.
      NOTE: The volume of 37.5 µL was selected as it corresponds to an increase of 15.0 µg/L for the spiked solutions of samples compared with the concentration in the non-spiked solutions of samples. Moreover, the increase in concentration for the spiked solution of samples corresponds to the final concentration that is still in the linear concentration range for every analyte measured.
   3. Prepare a sample blank using the same volume of 68 wt% HNO₃ and 30 wt% H₂O₂ as used for the digestion of food samples in step 4.2.1. For a sample blank, do not add the sample to the reaction vessels.
      CAUTION: HNO₃ used for digestion is corrosive and produces fumes. For this reason, acid addition must be performed in a fume hood. Standard laboratory protective equipment must be employed (gloves, safety goggles, and laboratory coat). If there is contact with acid, the affected area should be immediately rinsed under the stream of cold water, and medical help should be sought.
3. Place the cover lid on reaction vessels and allow samples to react with the added 68 wt% HNO₃ and 30 wt% H₂O₂ for 2-3 min.
4. Screw the thread cover on the vessel and tighten the cover lids.
5. Shake the reaction vessel using light hand movements to fully incorporate the samples into chemicals.
   NOTE: Do not leave the specimens on the walls or lids of the reaction vessels, as there is a possibility that they will not be completely digested.

5. Microwave-assisted wet acid digestion

1. Turn on the microwave system (see Table of Materials) for acid digestion by pressing the start button (Figure 4).
2. Open the microwave oven door and remove the rack.
3. Distribute the closed reaction vessels symmetrically in the rack to ensure even irradiation of the samples by microwaves.
4. Insert the rack into the microwave chamber and mount it on a holder (Figure 5).
5. Close the microwave oven door.
6. Set a suitable digestion program on the microwave oven touch screen using a pen shaped tool. Choose an appropriate temperature gradient, the final temperature, and the number of samples to be digested. The recommended digestion program for different food samples is listed below:
   1. Broccoli, mushrooms, sausages, and noodles: 10 min increase to 160 °C, 10 min increase to 200 °C, 15 min at 200 °C, maximum power 900 W.
7. Start the digestion program and monitor the change in reaction conditions on the screen. Stop the digestion process if the temperature does not increase according to the prescribed program.
   NOTE: During digestion, sudden temperature spikes may be seen on the microwave oven screen. They occur when the samples react exothermically with the chemicals. The microwave system will automatically regulate the temperature by adjusting the output power.
8. Wait until microwave-assisted digestion is completed and the temperature of the sample decreases.
9. Open the microwave oven door and remove the rack from the microwave oven chamber. Close the door and switch off the instrument.
10. Remove the reaction vessels from the rack and wait for them to cool down to room temperature.
11. Slowly open the lid covers manually to release the gases formed during acid digestion. Turn the reaction vessels in the direction of the fume hood (Figure 6).
12. Completely remove the cover and rinse the cover and the walls of the reaction vessel with a small amount of ultrapure water.
13. Quantitatively transfer the contents of the reaction vessel into a clean 25 mL glass volumetric flask through a glass funnel by repeated rinsing of the cover and reaction vessel with ultrapure water.
14. Dilute the sample with ultrapure water to the mark of the volumetric flask. Close the volumetric flask with a stopper and mix the content of the volumetric flask.
    NOTE: Further dilution of the digested samples with ultrapure water should be performed as they should contain less than 5% (V/V) of residual acid³⁴ and less than 2 g/L of dissolved elements, also referred to as total dissolved solids³⁵.
15. Take a 20 mL plastic syringe and connect it with a polyamide syringe filter (25 mm diameter, 0.20 µm pore size). Fill the plastic syringe with the diluted sample and filter its content into a 50 mL plastic centrifuge tube by applying pressure. Use a new plastic syringe and syringe filter for every sample to avoid any cross-contamination.
    NOTE: Samples need to be filtered to remove any insoluble materials or solid particles that may remain undigested after MAWD. These particles may interfere with elemental determination measurements by clogging the instrument components. When filtering the samples, ensure to discard the first couple of drops. Use hydrophilic filters (made of polyamide) for aqueous solutions. Hydrophobic filters (PTFE) are not suitable for the filtration of aqueous solutions as they require a higher applied pressure, which could result in membrane rupture³⁶.
16. Close the 50 mL plastic centrifuge tube with a screw cap and put the sample in the refrigerator until the measurements.
    NOTE: Digested samples are stored in the refrigerator at lower temperatures to preserve them and extend their storage time.

6. Reaction vessel cleaning

1. After the digested samples were transferred to 50 mL volumetric flasks, add 5.0 mL of 68 wt% HNO₃ and 5.0 mL of ultrapure water with 5 mL automatic pipettes into the reaction vessels.
2. Close the reaction vessels with the cover lids and insert them into the rack. Transfer the rack to the microwave oven chamber.
3. Apply the following microwave program: 15 min increase to 160 °C, 10 min increase to 180 °C, maximum power 900 W.
4. Monitor reaction conditions during heating. After the heating is completed, let the reaction vessels cool down.
5. Open the microwave oven, remove the reaction vessels from the rack, and slowly open them in the fume hood.
6. Discard the content of the reaction vessels into plastic waste containers.
7. Rinse the reaction vessels with ultrapure water to remove any excess material or chemicals.
8. Dry the reaction vessels in the dryer at 105 °C before the next use.
   ​NOTE: The same microwave procedure (time, power, temperature gradient, and volume of chemicals) used for the acid digestion of samples may be used for cleaning the reaction vessels. Alternatively, the reaction vessels can be cleaned without the microwave system by submerging them in concentrated HNO₃ or HCl for several hours and rinsing them with ultrapure water.

7. Multi-element determination with ICP-MS

1. Take the 50 mL plastic centrifuge tubes containing the digested samples from the refrigerator and allow them to warm to room temperature.
2. Dilute the samples by a factor of 10 to decrease the concentration of acid in the digested sample and to decrease the concentration of the component of the sample matrix. Using an automatic pipette, transfer 2.50 mL of the sample into a 25 mL glass volumetric flask and then fill it up to the mark with ultrapure water.
3. Transfer the diluted samples into the 15 mL plastic tubes and place them into the appropriate positions in the autosampler.
4. Prepare the ICP-MS instrument (see Table of Materials) for the measurements:
   1. Switch on the ventilation and the chiller that supplies the ICP-MS with cooling water and cools its components.
   2. Use the compatible software to ensure that the rinsing solution (1 wt% HNO₃) flows continuously from the autosampler to the ICP-MS without pulsating.
   3. Open Ar (99.999% purity) and He (99.999% purity) gas cylinders to supply the ICP-MS with both gasses. Check the gas flow in the software and adjust it if necessary.
      NOTE: Use collision cell with He gas when spectral interferences are expected due to the formation of polyatomic ions (e.g., ⁴⁰Ar¹⁶O⁺ interfering with ⁵⁶Fe⁺)³⁷.
   4. Start up the plasma and calibrate the instrument using the tuning solution (see Table of Materials).
   5. Once the instrument is calibrated (torch position, gain voltage, lens voltage, mass/resolution, pulse/analog (P/A) calibration, database (DB) calibration, and validation), select the desired measurement method and perform the measurements.
5. When working with unknown samples, perform a semi-quantitative determination to obtain information on what elements are present in the sample and their approximate concentration.
   NOTE: It is advisable to additionally dilute the samples for the semi-quantitative determination as the detectors have a limit of the concentration of elements they can detect at once. Lower sample concentrations can extend the lifetime of the instrument components.
6. After obtaining the data on the approximate concentrations of the elements in the samples, create a method for the quantitative elemental determination in the software. Select the operating conditions of the ICP-MS (Table 1) and select the desired elements (in the present case Cu, Fe, Mn, and Zn). Determine the number and concentrations of solutions of the standard required to create a calibration curve (sometimes referred to as an analytical curve or working curve) (Table 1).
   NOTE: Prepare at least six different concentrations as calibration points for the calibration curve.
7. Prepare solutions of standard for the calibration curve. Using automatic pipettes, pipette the required volume of 100 mg/L multi-element standard solutions into 25 mL glass volumetric flasks, to prepare solutions of standards with the following concentrations: 1.0 µg/L, 2.5 µg/L, 5.0 µg/L, 10.0 µg/L, 20.0 µg/L, 30.0 µg/L, 40.0 µg/L, and 50.0 µg/L. Fill the flasks to the mark with 1 wt% HNO₃. Additionally, prepare a calibration blank using the 1 wt% HNO₃ solution.
8. Transfer the prepared solutions of standard and samples into the 15 mL plastic tubes, place them into the autosampler, and start the instrument following the procedure described in step 7.4.
9. Perform the quantitative measurement of the selected elements using the calibration curve methodology.
10. Once the measurements are completed, turn off the plasma, close the Ar and He gas supplies, switch off the ICP-MS chiller, and turn-off the ventilation system.

Results

Homogenization
All samples were dried to a constant mass with the laboratory dryer to eliminate any moisture. Transferring the sample to a desiccator allowed it to cool to room temperature without binding moisture from the surrounding environment. The food samples were then homogenized using the laboratory mixer to obtain a fine powder. The resulting homogenized particles were uniform in size and evenly distributed, ensuring that subsamples (samples drawn from a larger sample) used for acid digestion...

Discussion

Homogenization
To ensure reproducible results in elemental determination, it is necessary to homogenize samples before acid digestion due to their complex and inhomogeneous structure and composition. Homogenization aims to eliminate constitutional and distributional heterogeneity. Mixing the sample minimizes distributional heterogeneity by evenly redistributing components throughout the sample. Similarly, by bringing the particle size down to a uniform size, constitutional heterogeneity is reduced

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ar gas	Messer	7440-37-1	Ar 5.0 gas (purity 99.999%).
AS-10 Autosampler system	Shimadzu		Autosampler connected to the ICP-MS, containing 68 ports for samples.
Automatic pipettes	Sartorius		200 µL, 1 mL, and 5 mL automatic pipettes.
Balance XSE104	Mettler Toledo, Columbus, Ohio, USA		Analytical balance scale with a maximum weighing mass of 120 g.
Ceramic knife			Ceramic knife used for cutting fresh food samples.
Dessicator			Glass desiccator with lumps of silica gel.
ETHOS LEAN	Milestone, Sorisole, Italy		Microwave system for wet acid digestion in closed 100 mL vessels made of TFM-PTFE.
Fume hood			Laboratory fume hood with adjustable air flow.
Glass beakers RASOTHERM	CarlRoth GmbH + Co.KG		50 mL, 250 mL glass beakers
Glass funnels			Small glass funnels fitting into the neck of volumetric flasks.
He gas	Messer	7440-59-7	He 5.0 gas (purity 99.999%).
Hydrogen peroxide	ThermoFisher Scientific	7722-84-1	Hxdrogen peroxide 100 volumes 30 wt.% solution. Laboratory reagent grade.
ICP multi-element standard solution VIII	Supelco	109492	100 mg/L ICP multi-element standard solution containing 24 elements (Al, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Se, Sr, Te, Tl, Zn) in 2 % dilute nitric acid.
ICPMS 2030	Shimadzu		Inductively coupled plasma mass spectrometry system for multi-element analysis of digested samples.
ICP-MS Tuning Solution A	CarlRoth GmbH + Co.KG		250 mL tuning solution containing 6 elements (Be, Bi, Ce, Co, In, Mn) in 1 % nitric acid.
KIMTECH Purple Nitrile gloves	Kimberly-Clark GmbH		Disposable Purple Nitrile gloves (S, M or L).
Laboratory coat	Any available supplier		/
Mixer B-400	BÜCHI Labortechnik AG, Flawil, Switzerland		Laboratory mixer with ceramic blades.
Nitric acid	ThermoFisher Scientific	7697-37-2	Nitric acid, trace analysis grade, 68 wt%, density 1.42, Primar Plus, For Trace Metal Analysis.
Plastic centrifuge tubes	Isolab		50 mL plastic centrifuge tubes with screw caps, single use.
Plastic syringes Injekt	B. Braun		2 pice, single use 20 mL syringes.
Plastic tubes for autosampler	Shimadzu	046-00147-04	Plastic tubes for autosampler, 15 mL capacity, 16 mm diameter, 100 mm length.
Plastic waste containers			Plastic containers for the removal of chemicals after the cleaning procedure of reaction vessels.
Protective googles			/
Samples (broccoli, sausage, noodles, zucchini, mushrooms)			Fresh samples, which were dried to a constant weight and homogenized during the procedure. The samples were purchased from a local shop.
Spatula			Plastic spatula.
Sterilizator Instrumentaria ST 01/02	Instrumentaria		Dryer with adjustable temperature.
Syringe filters	CHROMAFIL Xtra	729212	Syringe filters with polypropylene housing and polyamide hydrophilic membrane. Membrane diameter 25 mm, membrane pore size 0.2 µm.
Ultrapure water	ELGA Labwater, Veolia Water Technologies.		Ultrapure water with a resistivity of 18.2 MΩcm, obtained with laboratory water purification system.
Volumetric flasks			25 mL glass volumetric flasks.