Name: preparation of food samples using homogenization and microwave-assisted wet acid digestion for multi-element determination with icp-ms
Uploaded: 2023-12-22T12:50:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Author Spotlight: Technologies and Challenges in Elemental Analysis of Food Samples at JoVE.com

The authors acknowledge the financial support of the Slovenian Research Agency (Grant Nos. P2-0414, P2-0118, J1-2470, NK-0001, and J1-4416).

1. Sample homogenization
<ol>
	<li>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.</li>
	<li>Place the sample in a 250 mL glass beaker and dry it at 105 &#176;C to a constant weight using a dryer.</li>
	<li>Remove the glass beaker with the sample from the dryer and insert it in the desiccator.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat the process as described in steps 1.3-1.6 until a constant weight of the sample is achieved.</li>
	<li>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.</li>
	<li>Insert the mixer beaker into the mixer and close the guard door (Figure 1).</li>
	<li>Press the start button to activate the blades for grinding and mixing the sample.</li>
	<li>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.</li>
	<li>When the sample is homogenized, switch off the mixer, open the guard door, and remove the mixer beaker.</li>
	<li>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&#39;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&#39;s components. The use of organic solvents in the mixer is discouraged. 
	&#8203;CAUTION: Use safety gear and ensure that the mixer&#39;s door is adequately closed as the mixer&#39;s blades rotate at high speeds.</li>
</ol>
2. Mixer cleaning
<ol>
	<li>Add ultrapure water (see Table of Materials) to the mark of the empty mixer beaker.</li>
	<li>Insert the mixer beaker into the mixer and perform the standard mixing procedure.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Dry the cleaned components in the dryer at 105 &#176;C and reinsert them into the mixer. 
	&#8203;NOTE: Ensure that the mixer&#39;s components are completely dry before reinstalling them in the mixer, to prevent carry-over of the water to the following sample.</li>
</ol>
3. Sample weighing
<ol>
	<li>Remove the cover lid from the 100 mL trifluoromethoxyl-polytetrafluoroethylene TFM-PTFE reaction vessel33.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Acid addition
<ol>
	<li>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 &#181;g/L (ppb), ideally in the ng/L (ppt) range. Trace metal impurities affect the accuracy and repeatability of elemental determination.</li>
	<li>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:
	<ol>
		<li>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.</li>
		<li>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 &#181;L of 100 mg/L ICP multi-element standard solution (see Table of Materials) into the reaction vessels using a 200 &#181;L automatic pipette. For every sample, prepare three replicates. 
		NOTE: The volume of 37.5 &#181;L was selected as it corresponds to an increase of 15.0 &#181;g/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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Screw the thread cover on the vessel and tighten the cover lids.</li>
	<li>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.</li>
</ol>
5. Microwave-assisted wet acid digestion
<ol>
	<li>Turn on the microwave system (see Table of Materials) for acid digestion by pressing the start button (Figure 4).</li>
	<li>Open the microwave oven door and remove the rack.</li>
	<li>Distribute the closed reaction vessels symmetrically in the rack to ensure even irradiation of the samples by microwaves.</li>
	<li>Insert the rack into the microwave chamber and mount it on a holder (Figure 5).</li>
	<li>Close the microwave oven door.</li>
	<li>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:
	<ol>
		<li>Broccoli, mushrooms, sausages, and noodles: 10 min increase to&#160;160 &#176;C, 10 min increase to&#160;200 &#176;C, 15 min at 200 &#176;C, maximum power 900 W.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Wait until microwave-assisted digestion is completed and the temperature of the sample decreases.</li>
	<li>Open the microwave oven door and remove the rack from the microwave oven chamber. Close the door and switch off the instrument.</li>
	<li>Remove the reaction vessels from the rack and wait for them to cool down to room temperature.</li>
	<li>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).</li>
	<li>Completely remove the cover and rinse the cover and the walls of the reaction vessel with a small amount of ultrapure water.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Take a 20 mL plastic syringe and connect it with a polyamide syringe filter (25 mm diameter, 0.20 &#181;m 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.</li>
	<li>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.</li>
</ol>
6. Reaction vessel cleaning
<ol>
	<li>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.</li>
	<li>Close the reaction vessels with the cover lids and insert them into the rack. Transfer the rack to the microwave oven chamber.</li>
	<li>Apply the following microwave program: 15 min increase to 160 &#176;C, 10 min increase to 180 &#176;C, maximum power 900 W.</li>
	<li>Monitor reaction conditions during heating. After the heating is completed, let the reaction vessels cool down.</li>
	<li>Open the microwave oven, remove the reaction vessels from the rack, and slowly open them in the fume hood.</li>
	<li>Discard the content of the reaction vessels into plastic waste containers.</li>
	<li>Rinse the reaction vessels with ultrapure water to remove any excess material or chemicals.</li>
	<li>Dry the reaction vessels in the dryer at 105 &#176;C before the next use. 
	&#8203;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.</li>
</ol>
7. Multi-element determination with ICP-MS
<ol>
	<li>Take the 50 mL plastic centrifuge tubes containing the digested samples from the refrigerator and allow them to warm to room temperature.</li>
	<li>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.</li>
	<li>Transfer the diluted samples into the 15 mL plastic tubes and place them into the appropriate positions in the autosampler.</li>
	<li>Prepare the ICP-MS instrument (see Table of Materials) for the measurements:
	<ol>
		<li>Switch on the ventilation and the chiller that supplies the ICP-MS with cooling water and cools its components.</li>
		<li>Use the compatible software to ensure that the rinsing solution (1 wt% HNO3) flows continuously from the autosampler to the ICP-MS without pulsating.</li>
		<li>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.</li>
		<li>Start up the plasma and calibrate the instrument using the tuning solution (see Table of Materials).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#181;g/L, 2.5 &#181;g/L, 5.0 &#181;g/L, 10.0 &#181;g/L, 20.0 &#181;g/L, 30.0 &#181;g/L, 40.0 &#181;g/L, and 50.0 &#181;g/L. Fill the flasks to the mark with 1 wt% HNO3. Additionally, prepare a calibration blank using the 1 wt% HNO3 solution.</li>
	<li>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.</li>
	<li>Perform the quantitative measurement of the selected elements using the calibration curve methodology.</li>
	<li>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.</li>
</ol>

Food Sample Homogenization and Digestion for Multi-Element Analysis

The authors have nothing to disclose.

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<...

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 voltammetry15,16. 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 &#176;C and 260 &#176;C and maximum pressures up to 200 bar, depending on the instrument&#39;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 sufficient30,31,32. 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&#39;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.

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

preparation of food samples using homogenization and microwave-assisted wet acid digestion for multi-element determination with icp-ms

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.

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 digesti...

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Author Spotlight: Technologies and Challenges in Elemental Analysis of Food Samples

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&#160;via microwave-assisted wet acid digestion, and multi-element determination performed with inductively coupled plasma mass spectrometry.

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

Sample preparation is crucial for elemental determination, and various techniques are available, one of which involves homogenization followed by acid ...

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2.0K Views.  University of Maribor. 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.

Video: Author Spotlight: Technologies and Challenges in Elemental Analysis of Food Samples

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to the preparation of new organic dyes. Although numerous strategies have been reported to access naphthalene scaffolds, many procedures still present limitations in terms of incorporating functionality, which in turn narrows the range of available substrates. The development of versatile methods for direct access to substituted naphthalenes is therefore highly desirable.
The Diels-Alder (DA) cycloaddition reaction is a powerful and attractive method for the formation of saturated and unsaturated ring systems from readily available starting materials. A new microwave-assisted intramolecular dehydrogenative DA reaction of styrenyl derivatives described herein generates a variety of functionalized cyclopenta[b]naphthalenes that could not be prepared using existing synthetic methods. When compared to conventional heating, microwave irradiation accelerates reaction rates, enhances yields, and limits the formation of undesired byproducts.
The utility of this protocol is further demonstrated by the conversion of a DA cycloadduct into a novel solvatochromic fluorescent dye via a Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. Fluorescence spectroscopy, as an informative and sensitive analytical technique, plays a key role in research fields including environmental science, medicine, pharmacology, and cellular biology. Access to a variety of new organic fluorophores provided by the microwave-assisted dehydrogenative DA reaction allows for further advancement in these fields.

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Environment

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Dose Uptake of Platinum- and Ruthenium-based Compound Exposure in Zebrafish by Inductively Coupled Plasma Mass Spectrometry with Broader Applications

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the toxicokinetics of these compounds have both historical and modern-day relevance. Zebrafish have become an attractive model organism in elucidating pharmaco- and toxicokinetics in environmental exposure and clinical translation studies. Although zebrafish studies have the benefit of being higher-throughput than rodent models, there are several significant constraints to the model.
One such limitation is inherent in the waterborne dosing regimen. Water concentrations from these studies cannot be extrapolated to provide reliable internal dosages. Direct measurements of the metal-based compounds allow for a better correlation with compound-related molecular and biological responses. To overcome this limitation for metals and metal-based compounds, a technique was developed to digest zebrafish larval tissue after exposure and quantify metal concentrations within tissue samples by inductively coupled plasma mass spectrometry (ICPMS).
ICPMS methods were used to determine the metal concentrations of platinum (Pt) from cisplatin and ruthenium (Ru) from several novel Ru-based chemotherapeutics in zebrafish tissue. Additionally, this protocol distinguished concentrations of Pt that were sequestered in the chorion of the larval compared with the zebrafish tissue. These results indicate that this method can be applied to quantitate the metal dose present in larval tissues. Further, this method may be adjusted to identify specific metals or metal-based compounds in a broad range of exposure and dosing studies.

The increased rate of pharmaco- and toxicokinetic analyses of metals and metal-based compounds in zebrafish can be advantageous for environmental and clinical translation studies. The limitation of unknown waterborne exposure uptake was overcome by conducting trace metal analysis on digested zebrafish tissue using inductively coupled plasma mass spectrometry.

Metals and metal-based compounds comprise multifarious pharmaco-active and toxicological xenobiotics. From heavy metal toxicity to chemotherapeutics, the ...

Sample Preparation for Analytical Characterization

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Sample preparation is the way in which&#160;a sample is treated to prepare&#160;for analysis. Careful sample preparation is critical in analytical chemistry to accurately generate either a standard or unknown sample for a chemical measurement. Errors in analytical chemistry methods are categorized as random or systematic. Random errors are errors due to change and are often due to noise in instrument. Systematic errors are due to investigator or instrumental bias, which introduces an offset in the measured value. Errors in sample preparation are systematic errors, which will propagate&#160;through analysis, causing&#160;uncertainty or inaccuracies through improper calibration curves. Systematic errors can be eliminated through correct sample preparation and proper use of the instrument. Poor sample preparation can also sometimes cause harm to the instrument.

Source: Laboratory of Dr. B. Jill Venton - University of Virginia
Sample preparation is the way in which&#160;a sample is treated to prepare&#160;for ...

Education

JoVE Science Education

Analytical Chemistry

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.

Mass Spectrometer: Principle, Operation, Applications

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the ...

Sample Preparation for Analysis: Advanced Techniques

Accurate analysis of complex samples often requires advanced preparation techniques to achieve reliable and reproducible results. Samples containing inorganic or organic materials can be challenging to dissolve or decompose effectively. Standard sample preparation methods include acid digestion, fusion, dry ashing, and wet digestion.
Acid digestion with strong acids is commonly used to dissolve inorganic materials that are insoluble (do not dissolve) in water. This method can be useful for analyzing soil or mineral samples. During acid digestion, the insoluble solid sample is finely ground or crushed to increase its surface area, which allows for a more effective reaction with the acids. The typically used acids include hydrochloric acid, nitric acid, sulfuric acid, and aqua regia &#8211; a mixture of nitric and hydrochloric acid. Each acid has specific advantages for different sample types:
<ol style="list-style-type: circle;">
	<li>Hydrochloric acid (HCl) is often used to dissolve chlorides, oxides, and carbonates and is also effective on materials such as metals and ores.</li>
	<li>Nitric acid (HNO&#8323;) is excellent for breaking down organic matter and oxidizing metals, making it a common choice for biological tissues or metal analysis.</li>
	<li>Sulfuric acid (H&#8322;SO&#8324;) is used when a strong dehydrating agent is needed, for instance, in dissolving more resistant samples like sulfides or metals.</li>
	<li>Aqua regia, a mixture of nitric and hydrochloric acids, is particularly effective for dissolving noble metals like gold and platinum, which are resistant to individual acids.</li>
</ol>
The sample is treated with the chosen high-strength acid in a suitable container, where it is broken down into soluble forms or ions. Heat may also be applied to speed up the digestion process. After digestion, the resulting mixture diluted with water is suitable for further analysis.
The fusion technique is an alternative method to dissolve inorganic materials that cannot be broken down to a soluble form by acid digestion. Here, the weighted sample is mixed with a flux material in a specific ratio, typically about 1 to 10 or 20. The sample-flux mixture is placed in a platinum crucible that is resistant to high temperatures and chemical reactions. This crucible is heated in a high-temperature furnace to obtain a molten mixture. After heating, the molten mixture is allowed to cool till it yields a new material soluble in water or dilute acid.
Several examples of flux usage in sample preparation for dissolving solid and liquid compounds are:
<ol style="list-style-type: circle;">
	<li>Na2CO3 and Na2O2 as Fluxes for Iron and Chromium Alloys: Sodium carbonate (Na2CO3) is a commonly used flux, but sodium peroxide (Na2O2) is employed due to its stronger oxidizing ability in certain cases. This combination is particularly useful for dissolving iron and chromium alloys in a nickel crucible.</li>
	<li>K2S2O7 for Refractory Oxides: When the target is refractory oxides (but not silicates), potassium disulfate (K2S2O7) serves as an effective flux. It can be prepared by heating potassium hydrogen sulfate (KHSO4) to decompose any foaming byproducts. K2S2O7 is effective in dissolving oxides and silicates with the application of heat.</li>
	<li>Li2B4O7 and Li2SO4 Mixture for Rapid Dissolution: A mixture of lithium tetraborate (Li2B4O7) and lithium sulfate (Li2SO4) in a 2:1 ratio by weight is highlighted for its rapid ability to dissolve refractory silicates and oxides.</li>
</ol>
Dry ashing and wet digestion methods are popularly used to decompose organic materials. In dry ashing, the organic sample is heated in a muffle furnace at a high temperature. The heat causes the combustion of the organic material in the presence of atmospheric oxygen, leaving behind an inorganic residue, or &#39;ash.&#39; This ash can then be dissolved in an appropriate solvent for further analysis. Wet digestion involves the use of acids to break down the organic material.

Accurate analysis of complex samples often requires advanced preparation techniques to achieve reliable and reproducible results. Samples containing ...