Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate

Summary

This protocol details a facile, one-pot synthesis of manganese oxide (MnO) nanoparticles by thermal decomposition of manganese(II) acetylacetonate in the presence of oleylamine and dibenzyl ether. MnO nanoparticles have been utilized in diverse applications including magnetic resonance imaging, biosensing, catalysis, batteries, and waste water treatment.

Abstract

For biomedical applications, metal oxide nanoparticles such as iron oxide and manganese oxide (MnO), have been used as biosensors and contrast agents in magnetic resonance imaging (MRI). While iron oxide nanoparticles provide constant negative contrast on MRI over typical experimental timeframes, MnO generates switchable positive contrast on MRI through dissolution of MnO to Mn²⁺ at low pH within cell endosomes to ‘turn ON’ MRI contrast. This protocol describes a one-pot synthesis of MnO nanoparticles formed by thermal decomposition of manganese(II) acetylacetonate in oleylamine and dibenzyl ether. Although running the synthesis of MnO nanoparticles is simple, the initial experimental setup can be difficult to reproduce if detailed instructions are not provided. Thus, the glassware and tubing assembly is first thoroughly described to allow other investigators to easily reproduce the setup. The synthesis method incorporates a temperature controller to achieve automated and precise manipulation of the desired temperature profile, which will impact resulting nanoparticle size and chemistry. The thermal decomposition protocol can be readily adapted to generate other metal oxide nanoparticles (e.g., iron oxide) and to include alternative organic solvents and stabilizers (e.g., oleic acid). In addition, the ratio of organic solvent to stabilizer can be changed to further impact nanoparticle properties, which is shown herein. Synthesized MnO nanoparticles are characterized for morphology, size, bulk composition, and surface composition through transmission electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy, respectively. The MnO nanoparticles synthesized by this method will be hydrophobic and must be further manipulated through ligand exchange, polymeric encapsulation, or lipid capping to incorporate hydrophilic groups for interaction with biological fluids and tissues.

Introduction

Metal oxide nanoparticles possess magnetic, electric, and catalytic properties, which have been applied in bioimaging^1,2,3, sensor technologies^4,5, catalysis^6,7,8, energy storage⁹, and water purification¹⁰. Within the biomedical field, iron oxide nanoparticles and manganese oxide (MnO) nanoparticles have proven utility as contrast agents in magnetic resonance imaging (MRI)^1,2. Iron oxide nanoparticles produce robust negative contrast on T₂* MRI and are powerful enough to visualize single labeled cells in vivo^11,12,13; however, the negative MRI signal cannot be modulated and remains “ON” throughout the duration of typical experiments. Due to endogenous iron present in the liver, bone marrow, blood and spleen, the negative contrast generated from iron oxide nanoparticles may be difficult to interpret. MnO nanoparticles, on the other hand, are responsive to a drop in pH. MRI signal for MnO nanoparticles can transition from “OFF” to “ON” once the nanoparticles are internalized inside the low pH endosomes and lysosomes of the target cell such as a cancer cell^{14,15,16,17,18,19}. The positive contrast on T₁ MRI produced from the dissolution of MnO to Mn²⁺ at low pH is unmistakable and can improve cancer detection specificity by only lighting up at the target site within a malignant tumor. Control over nanoparticle size, morphology and composition is crucial to achieve maximum MRI signal from MnO nanoparticles. Herein, we describe how to synthesize and characterize MnO nanoparticles using the thermal decomposition method and note different strategies for fine-tuning nanoparticle properties by altering variables in the synthesis process. This protocol can be easily modified to produce other magnetic nanoparticles such as iron oxide nanoparticles.

MnO nanoparticles have been produced by a variety of techniques including thermal decomposition^{20,21,22,23,24,25}, hydro/solvothermal^26,27,28,29, exfoliation^{30,31,32,33,34}, permanganates reduction^35,36,37,38, and adsorption-oxidation^39,40,41,42. Thermal decomposition is the most commonly used technique which involves dissolving manganese precursors, organic solvents, and stabilizing agents at high temperatures (180 – 360 °C) under the presence of an inert gaseous atmosphere to form MnO nanoparticles⁴³. Out of all of these techniques, thermal decomposition is the superior method to generate a variety of MnO nanocrystals of pure phase (MnO, Mn₃O₄ and Mn₂O₃) with a narrow size distribution. Its versatility is highlighted through the ability to tightly control nanoparticle size, morphology and composition by altering reaction time^44,45,46, temperature^44,47,48,49, types/ratios of reactants^{20,45,47,48,50} and inert gas^47,48,50 used. The main limitations of this method are the requirement for high temperatures, the oxygen-free atmosphere, and the hydrophobic coating of the synthesized nanoparticles, which requires further modification with polymers, lipids or other ligands to increase solubility for biological applications^14,51,52,53.

Besides thermal decomposition, the hydro/solvothermal method is the only other technique that can produce a variety of MnO phases including MnO, Mn₃O₄, and MnO₂; all other strategies only form MnO₂ products. During hydro/solvothermal synthesis, precursors such as Mn(II) stearate^54,55 and Mn(II) acetate²⁷ are heated to between 120-200 °C over several hours to achieve nanoparticles with a narrow size distribution; however, specialized reaction vessels are required and reactions are performed at high pressures. In contrast, the exfoliation strategy involves treatment of a layered or bulk material to promote dissociation into 2D single layers. Its main advantage is in producing MnO₂ nanosheets, but the synthesis process is long requiring several days and the resulting size of the sheets is difficult to control. Alternatively, permanganates such as KMnO₄ can react with reducing agents such as oleic acid^56,57, graphene oxide⁵⁸ or poly(allylamine hydrochloride)⁵⁹ to create MnO₂ nanoparticles. Use of KMnO₄ facilitates nanoparticle formation at room temperature over a few minutes to hours within aqueous conditions⁴³. Unfortunately, the rapid synthesis and nanoparticle growth makes it challenging to finely control resulting nanoparticle size. MnO₂ nanoparticles can also be synthesized using adsorption-oxidation whereby Mn²⁺ ions are adsorbed and oxidized to MnO₂ by oxygen under basic conditions. This method will produce small MnO₂ nanoparticles with a narrow size distribution at room temperature over several hours in aqueous media; however the requirement for adsorption of Mn²⁺ ions and alkali conditions limits its widespread application⁴³.

Of the MnO nanoparticle synthesis methods discussed, thermal decomposition is the most versatile to generate different monodisperse pure phase nanocrystals with control over nanoparticle size, shape and composition without requiring specialized synthesis vessels. In this manuscript, we describe how to synthesize MnO nanoparticles by thermal decomposition at 280 °C using manganese(II) acetylacetonate (Mn(II) ACAC) as the source of Mn²⁺ ions, oleylamine (OA) as the reducing agent and stabilizer, and dibenzyl ether (DE) as the solvent under a nitrogen atmosphere. The glassware and tubing setup for nanoparticle synthesis is explained in detail. One advantage of the technique is the inclusion of a temperature controller, thermocouple probe, and heating mantle to enable precise control over the heating rate, peak temperature, and reaction times at each temperature to fine-tune nanoparticle size and composition. Herein, we show how nanoparticle size can also be manipulated by changing the ratio of OA to DE. Additionally, we demonstrate how to prepare nanoparticle samples and measure nanoparticle size, bulk composition and surface composition using transmission electron microscopy (TEM), x-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR), respectively. Further guidance is included on how to analyze the collected images and spectra from each instrument. To generate uniformly shaped MnO nanoparticles, a stabilizer and adequate nitrogen flow must be present; XRD and TEM results are shown for undesired products formed in the absence of OA and under low nitrogen flow. In the Discussion section, we highlight crucial steps in the protocol, metrics to determine successful nanoparticle synthesis, further variation of the decomposition protocol to modify nanoparticle properties (size, morphology and composition), troubleshooting and limitations of the method, and applications of MnO nanoparticles as contrast agents for biomedical imaging.

Protocol

1. Glassware and tubing assembly – to be performed only the first time

NOTE: Figure 1 shows the experimental setup for MnO nanoparticle synthesis with numbered tubing connections. Figure S1 shows the same setup with the main glassware components labeled. If there is a mismatch between the chemical resistant tubing and the glass connection size, cover the glass connection first with a short piece of smaller tubing before adding the chemical resistant tubing to make the connections snug.

1. Secure the air-free nitrogen tank to the wall close to a chemical fume hood using approved strap restraints. Add the appropriate nitrogen regulator to the tank.
   CAUTION: Gas cylinders must be properly secured since they can be very dangerous if tipped over.
2. Fill the gas drying column with desiccant. Attach chemical resistant tubing from the air-free nitrogen regulator to the bottom inlet of the gas drying column (#1 in Figure 1).
3. Secure the glass manifold containing at least 2 outlet stopcocks to the top of the fume hood using two metal claw clamps. Attach chemical resistant tubing from the outlet of the gas drying column (#2 in Figure 1) to the inlet of the manifold (#3 in Figure 1).
4. Place and secure 3 mineral oil bubblers in the fume hood using metal claw clamps according to Figure 1. Put two bubblers to the left and one bubbler to the right.
5. Fill the leftmost bubbler (by #9 in Figure 1) with the smallest amount of silicone oil (~1 inch of oil from the bottom of the bubbler). Fill the middle bubbler (by #7,8 in Figure 1) with a medium amount of silicone oil (~1.5 inches of oil from the bottom of the bubbler). Fill the rightmost bubbler (by #11 in Figure 1) with the largest amount of silicone oil (~2 inches of oil from the bottom of the bubbler).
   NOTE: The relative amount of silicone oil between the mineral bubblers is very important to achieve appropriate flow of the air-free nitrogen gas through the system. Do not add too much oil (over ~2.5 inches), as the oil will bubble during the reaction and can exit the bubblers if overfilled.
6. Connect the outlet on the right stopcock of the manifold (#4 in Figure 1) to the threaded end of a glass elbow adapter (#5 in Figure 1) using chemical resistant tubing.
7. Attach the threaded end of another glass elbow adapter (#6 in Figure 1) to the inlet of the middle bubbler (#7 in Figure 1) using chemical resistant tubing. Connect the outlet of the middle bubbler (#8 in Figure 1) to the inlet of the leftmost bubbler (#9 in Figure 1) using chemical resistant tubing.
8. Connect the outlet on the left stopcock of the manifold (#10 in Figure 1) to the inlet of the rightmost bubbler (#11 in Figure 1).
9. Leave the preliminary setup in the fume hood if space accommodates. Secure the two glass elbow adapters with tubing attached (#5,6 in Figure 1) to the metal latticework in the fume hood when the experiment is not running.

2. Equipment and glassware setup – to be performed during every experiment

CAUTION: All steps involving solvents require the use of a chemical fume hood as well as proper personal protective equipment (PPE) including safety glasses, lab coat and gloves. The nanoparticle fabrication setup should be assembled in the fume hood.

1. Place the stir plate in the fume hood and put the heating mantle on top of the stir plate.
   NOTE: The heating mantle must be able to withstand temperatures above 300 °C.
2. Put the 4 neck 500 mL round bottom flask onto the heating mantle and secure the middle neck with a metal claw clamp. Add a magnetic stir bar to the round bottom flask. Place the glass funnel in the middle neck of the round bottom flask.
3. Check the manifold: make sure the safety stopcock (#10 in Figure 1) and input stopcock (#4 in Figure 1) are open.
   CAUTION: The safety stopcock needs to be open at all times to assure no pressure is built up in the system. If the stopcock is closed, an explosion can occur.
4. Weigh 1.51 g of manganese(II) acetylacetonate (Mn(II) ACAC) and place inside the round bottom flask using the glass funnel.
5. Add 20 mL of oleylamine and 40 mL of dibenzyl ether to the round bottom flask using a glass pipette and the glass funnel. Remove the funnel and clean it with hexane.
   CAUTION: The experiment can be scaled up (e.g., 2 times), but it is recommended to be conservative when using any larger quantities of reactants. Larger amounts of reactants could cause the reaction to become less stable, and therefore dangerous.
6. Attach the condenser to the left neck of the round bottom flask and secure the condenser with a metal claw clamp. Add the glass elbow adapter (#6 in Figure 1) on top of the condenser.
   NOTE: The adapter should be connected with chemical resistant tubing to the middle mineral oil bubbler (#7 in Figure 1).
7. Connect water compatible tubing from the water outlet spout in the fume hood (#12 in Figure 1) to the inlet of the condenser (#13 in Figure 1). Also use water compatible tubing to connect the outlet of the condenser (#14 in Figure 1) to the drain in the fume hood (#15 in Figure 1). Secure the tubing to the condenser connections (#13,14 in Figure 1) with interlocked worm gear metal hose clamps.
8. Add the rotovap trap to the right neck of the round bottom flask. Place the glass elbow adapter (#5 in Figure 1) on top of the rotovap trap.
   NOTE: The adapter should be connected with chemical resistant tubing to the right stopcock manifold outlet (#4 in Figure 1).
9. Attach the rubber stopper to the middle neck of the round bottom flask and fold it over so the sides cover the neck of the flask. Add the plastic conical joint clips (4 green clips in Figure 1) to secure the following glassware neck connections: elbow adapter and rotovap trap, rotovap trap and round bottom flask, round bottom flask and condenser, and condenser and elbow adapter.
10. Place the temperature probe into the smallest neck in the round bottom flask, tightening and securing the probe with the neck cap and the o-ring. Seal the connection with paraffin plastic film.
    NOTE: Make sure the temperature probe is immersed within the fluid mixture, but does not touch the bottom of the glass. If the probe is in contact with the glass surface, the temperature measured will be inaccurate compared to the true fluid temperature, which will cause the temperature controller to provide an incorrect amount of heat to the reaction.
11. Connect the temperature probe to the input of the temperature controller. Connect the heating mantle to the output of the temperature controller.
12. Turn on the stir plate and start stirring vigorously.
13. Open the air-free nitrogen tank and slowly begin flowing nitrogen into the system (this will remove the air). Adjust the nitrogen flow using the regulator until a steady slow stream of bubbles form in the middle mineral oil bubbler (#7 in Figure 1).
14. Turn on the cold water in the fume hood (#12 in Figure 1) to the condenser and check that no water leaks from the tubing.
15. Put the sash of the fume hood down before the reaction begins.

3. Nanoparticle synthesis

1. Turn on the temperature controller (power and heating supply) to start the reaction. Observe and record the color of the reaction mixture in each stage. The reaction will begin as a dark brown color in stages 1 to 3 and will turn green during stage 4.
   NOTE: Each temperature controller will work differently. Make sure to use the correct manual and program.
2. Stage 1: Observe the temperature controller display to confirm the temperature increases from room temperature to 60 °C over 30 min.
3. Stage 2: Ensure that the temperature controller stabilizes at 60 °C for 1 min as it prepares for a faster heating rate in stage 3.
4. Stage 3: Check the temperature controller display as the temperature rises to 280 °C at 10 °C per minute over 22 min. Make sure the water flow through the condenser is sufficient, as the mixture will start to evaporate during this stage.
5. Stage 4: Confirm the temperature controller displays a constant reaction temperature of 280 °C for 30 min. Observe the reaction color change to a green tone, which indicates MnO formation. Once the reaction reaches 280 °C, turn off the nitrogen tank and close the right stopcock for the inlet of the reaction on the manifold (#4 in Figure 1).
   CAUTION: Keep the safety stopcock (#10 in Figure 1) open.
6. Stage 5: Check the temperature controller display to ensure that the heating stops automatically. Keep the temperature probe inside (do not open the round bottom flask) and wait until the temperature reaches room temperature to proceed with nanoparticle collection.
   CAUTION: The flask will be extremely hot. Heat resistant gloves should be worn to remove the heating mantle if a faster cooling rate is desired.
   NOTE: The protocol can be paused here.

4. Nanoparticle collection

1. Turn off the temperature controller, the stir plate and the cold water. Remove the water compatible tubing from the condenser, water faucet in the fume hood and the drain. Remove all the plastic conical joint clips from glassware connections.
2. Remove the glass elbow adapters from the rotovap trap (#5 in Figure 1) and the condenser (#6 in Figure 1). Secure the elbow adapters to the metal latticework in the hood to use for a future experiment.
3. Detach the condenser and rotovap trap from the round bottom flask and rinse the insides of the condenser and rotovap trap with hexane.
4. Remove the rubber stopper and temperature probe, and clean with 70% ethanol.
5. Pour the MnO nanoparticle solution from the round bottom flask into a clean 500 mL beaker. Use hexane (~5 mL) to rinse the round bottom flask and add the hexane with residual MnO nanoparticles into the 500 mL beaker.
   NOTE: Hexane will resuspend the MnO nanoparticles while 200 proof ethanol will act as the precipitant agent.
6. Note the current volume of the MnO nanoparticle mixture. Add 200 proof ethanol to the MnO nanoparticle mixture using a volume ratio of 2:1 (e.g., add 150 mL of ethanol if the nanoparticle mixture is 75 mL).
7. Pour the nanoparticle mixture equally into four centrifuge tubes, around 3/4 full. Screw on the appropriate caps. Check to make sure the fluid levels are balanced.
   NOTE: Any extra nanoparticle mixture will be added to the tubes on the next round of centrifugation.
8. Centrifuge nanoparticles for 10 min at 17,400 x g at 10 °C.
   NOTE: Longer centrifugation times and/or higher centrifugation speeds can be used to increase collection of smaller nanoparticle fractions, but nanoparticle aggregation can be increased.
9. Discard the supernatant into a waste beaker, being careful not to disturb the pellet. If needed, use a transfer pipette to collect the supernatant.
   NOTE: It is normal for the early rounds of centrifugation to produce a brown colored supernatant. The supernatant should be brown and clear, but not cloudy. Any cloudiness indicates that the nanoparticles are still present in the supernatant. If the supernatant is cloudy, centrifuge the tubes again before discarding the supernatant; centrifuging again will reduce loss of the synthesized nanoparticles, but can cause more agglomeration.
10. Add 5 mL of hexane and any extra nanoparticle solution left to each centrifuge tube containing the MnO nanoparticle pellets. Resuspend the nanoparticles using a bath sonicator and/or vortex. Continue until the solution becomes cloudy and the pellet disappears, which indicates successful nanoparticle resuspension.
11. Add more 200 proof ethanol to the centrifuge tubes until 3/4 full.
12. Repeat steps 4.8-4.10. Then, combine the resuspended nanoparticles from four centrifuge tubes to two centrifuge tubes. Next, repeat step 4.11.
13. Repeat steps 4.8-4.10 once more, which will make a total of three washes with hexane and 200 proof ethanol. Do not add any 200 proof ethanol to the centrifuge tubes.
14. Combine and transfer the MnO nanoparticles resuspended in hexane into a preweighed 20 mL glass scintillation vial. Leave the lid of the vial off to allow the hexane to evaporate overnight in the fume hood.
15. The next day, transfer the uncovered glass scintillation vial containing the nanoparticles into a vacuum oven. Keep the lid for the vial in a safe place outside the oven. Dry out the nanoparticles at 100 °C for 24 hours.
16. Once nanoparticles are dried, use a spatula to break up the powder inside the vial. Weigh the vial containing dried MnO nanoparticles and subtract the known weight of the glass scintillation vial to determine the nanoparticle yield.
    CAUTION: Dried nanoparticles can easily become airborne and should be handled by personnel using a particle respirator such as N95 or P100.
17. Store nanoparticles at room temperature inside the glass scintillation vial with the lid on. Wrap the lid with paraffin plastic film.

5. Nanoparticle size and surface morphology (TEM)

1. Pulverize the MnO nanoparticles into a thin powder using a mortar and pestle.
2. Add 5 mg of MnO nanoparticles to a 15 mL conical centrifuge tube. Add 10 mL of 200 proof ethanol.
   NOTE: 200 proof ethanol evaporates quickly to obtain a more homogeneous spread of nanoparticles on the TEM grid. Another solvent could have better nanoparticle suspension, but would take longer to evaporate, and due to surface tension, the nanoparticles would accumulate on the borders of the TEM grids.
3. Bath sonicate the nanoparticle mixture for 5 min or until full resuspension of the nanoparticles.
4. Immediately upon resuspension, add three 5 µL drops of the nanoparticle mixture onto a 300 mesh copper grid support film of carbon type-B. Let air dry.
   1. Use reverse tweezers for easier sample preparation. Position the grid on the tweezers with the darker side up before adding the drops containing nanoparticles.
      NOTE: The grids are fragile, so be careful not to bend and damage the grids for better imaging. Once dry, grids should be kept inside commercially available TEM grid storage boxes for protection.
5. Assess nanoparticle shape and size using transmission electron microscopy (TEM). Apply typical parameters for TEM including a beam strength of 200 kV, a spot size of 1, and a magnification of 300x.
6. Collect images on areas of the grid where enough nanoparticles (10 - 30 nanoparticles) are evenly distributed. Avoid areas containing nanoparticle aggregations, as accurate sizing cannot be made if nanoparticles are not visibly separated.
   1. Image areas from different grid squares to assure an even distribution. For an optimal size distribution, take between 25 - 30 images from each sample to obtain a sufficient sample size.

6. Quantitative analysis of nanoparticle diameter

1. To analyze TEM images with ImageJ, first open one of the images by clicking File | Open. Select the desired image and click Open.
2. To calibrate the distance measurement in ImageJ from pixels to nanometers, first click the straight-line tool. Hold the Shift key and trace the length of the scale bar. Then, click Analyze | Set Scale.
3. In the Set Scale pop-up window, type the true scale bar measurement into the Known distance box (e.g., type 50 if the scale bar is 50 nm). Change the unit of length to the corresponding units (e.g., type nm for nanometers). Check the Global box to keep the scale consistent in all the images, and click OK.
4. After setting the scale, use the straight-line tool to trace the diameter of a nanoparticle. Then click Analyze | Measure or click Ctrl+M keys.
5. Look for a results pop-up window to appear with different information about the measurement. Confirm that the Length column is present, as it will provide the diameter of the nanoparticles with the units specified during step 6.3.
6. Repeat step 6.4 until all the nanoparticles in the image are sized. To move to the next image, either click File | Open Next, or Ctrl+Shift+O keys.
7. After all nanoparticles are sized in all images, go to the Results window and click File | Save As. Rename the results file and click Save. View and analyze all nanoparticle diameters in a spreadsheet program after importing the results file.

7. Nanoparticle bulk composition (XRD)

1. If not done during step 5.1, pulverize the MnO nanoparticles into a thin powder using a mortar and pestle. Place the fine nanoparticle powder into the sample holder using a spatula. Follow the sample loading procedure specified for the X-ray diffraction (XRD) machine to be used.
2. Determine bulk composition of MnO nanoparticles using XRD. Collect XRD spectra over a 2θ range from 10° to 110° to view peaks of MnO (30° to 90°) and Mn₃O₄ (15° to 90°).
   NOTE: Other setting parameters recommended for XRD are a step size of 0.05 s, a beam mask of 10 mm, and a scan step time of 64.77 s.
3. Save the generated .XRD file and open it in the XRD analysis program.

8. Analysis of XRD spectra

1. In the XRD analysis program, identify all the main peaks in the sample’s measured XRD spectrum by clicking on the IdeAll button in the software.
2. To save the data, select File on the toolbar, followed by Save as… to save the data as an ASC file that can be opened with a spreadsheet program.
3. Use the program to pattern match the XRD database of known compounds to find the best composition match to the sample. To narrow the search, specify anticipated compounds (e.g., manganese and oxygen).
   1. To pattern match the spectrum, select Analysis | Search & Match. In the pop-up window, select Chemistry and click the desired chemical elements to restrict the program search based on the sample.
   2. Once all elements are chosen, select Search. Wait for a list of chemical compositions matching the XRD spectrum to appear.
      NOTE: The program will provide the likelihood that known XRD spectra correspond to the sample’s composition. If two or more compositions are chosen, the program would give the composition percentage of each of them (e.g., MnO versus Mn₃O₄).
4. If desired, remove the background from the XRD spectrum by clicking the Fit Background button (). Then, click Background in the pop-up window, followed by Subtract. Confirm that the spectrum appears starting with 0 on the y-axis.
   1. Save the data again without the background as shown in step 8.2.
5. When plotting the XRD spectrum, show the characteristic peaks of each matched compound (e.g., MnO and Mn₃O₄).
   1. To obtain the list of the characteristic peaks for matched compounds from the database, first right click on the pattern match spectrum, and then select Show Pattern. Wait for a pop-up window to appear with all the peak information corresponding to the selected pattern.
   2. Select, copy and paste the desired information from that compound and plot the characteristic peaks with the measured XRD spectrum in a spreadsheet program.

9. Nanoparticle surface composition (FTIR)

1. Add dry MnO nanoparticle powder to the sample holder for Fourier-transform infrared spectroscopy (FTIR) analysis.
2. Evaluate nanoparticle surface chemistry using FTIR. Collect FTIR spectra between a 4000 and 400 cm^-1 wavelength range with a resolution of 4 cm^-1.
3. Clean the FTIR sample holder and add liquid oleylamine. Repeat Step 9.2.

10. Analysis of FTIR spectra

1. In the FTIR analysis program, remove the background from the collected FTIR spectrum by selecting Transforms in the drop-down menu, followed by Baseline Correct. Select Linear as the correction type.
2. Use the left mouse click to select the baseline points on the original spectrum. Once finished, save the spectrum under another name by selecting Add or replace the old spectrum by selecting Replace.
   NOTE: Background correction can enhance the prevalence of weaker FTIR peaks of interest.
3. To export the FTIR spectrum, first select the specific spectrum from the list. Then, click File on the toolbar, followed by Export spectrum.
4. Choose csv file format from the Save As window and click Save. Open and graph the csv file using a spreadsheet program.
5. Compare acquired MnO nanoparticle with oleylamine FTIR spectra as detailed in the Representative Results section to evaluate nanoparticle capping with oleylamine.

Results

To confirm successful synthesis, MnO nanoparticles should be assayed for size and morphology (TEM), bulk composition (XRD), and surface composition (FTIR). Figure 2 shows representative TEM images of MnO nanoparticles synthesized using decreasing ratios of oleylamine (OA, the stabilizer) to dibenzyl ether (DE, the organic solvent): 60:0, 50:10, 40:20, 30:30, 20:40, 10:50. Ideal TEM images consist of individual nanoparticles (shown as dark rounded octagons in Figure 2

Discussion

The protocol herein describes a facile, one-pot synthesis of MnO nanoparticles using Mn(II) ACAC, DE, and OA. Mn(II) ACAC is utilized as the starting material to provide a source of Mn²⁺ for MnO nanoparticle formation. The starting material can be easily substituted to enable production of other metal oxide nanoparticles. For example, when iron(III) ACAC is applied, Fe₃O₄ nanoparticles can be generated using the same nanoparticle synthesis equipment and protocol described

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals and Gases
Benzyl ether (DE)	Acros Organics	AC14840-0010	Concentration: 99%, 1 L
Drierite	W. A. Hammond Drierite Co. LTD	23001	Drierite 8 mesh, 1 lb
Ethanol	Decon Laboratories	2701	200 proof, 4 x 3.7 L
Hexane	Macron Fine Chemicals	5189-08	Concentration:  ≥98.5%, 4 L
Hydrochloric acid	VWR	BDH3030-2.5LPC	Concentration: 36.5 - 38.0 % ACS, 2.5 L
Manganese (II) acetyl acetonate (Mn(II)ACAC)	Sigma Aldrich	245763-100G	100 g
Nitrogen gas tank	Airgas	NI R300	Research 5.7 grade nitrogen, size 300 cylinder
Nitrogen regulator	Airgas	Y11244D580-AG	Single stage brass 0-100 psi analytical cylinder regulator CGA-580 with needle outlet
Oleylamine (OA)	Sigma Aldrich	O7805-500G	Concentration: 70%, technical grade, 500 g
Silicone oil	Beantown Chemical	221590-100G	100 g
Equipment
Centrifuge	Beckman-Coulter	Avanti J-E	JA-20 fixed-angle aluminum rotor, 8 x 50 mL, 48,400 x g
Hemisphere mantle	Ace Glass Inc.	12035-17	115 V, 270 W, 500 mL, temperature up to 450 °C
Hot plate stirrer	VWR	97042-642	120 V, 1000 W, 8.3 A, ceramic top
Temperature controller	Yokogawa Electric Corporation	UP351
Temperature probe	Omega	KMQXL-040G-12	Immersion probe, temperature up to 1335 °C
Vacuum oven	Fisher Scientific	282A	120 V, 1800 W, temperature up to 280 °C
Vortex mixer	Fisher Scientific	02-215-365	120 V, 50/60 Hz, 150 W
Water bath sonicator	Fisher Scientific	FS30H	Ultrasonic power 130 W, 3.7 L tank
Tools and Materials
Dumont tweezer	Electron Microscopy Sciences	72703D	Style 5/45, Dumoxel, 109 mm, for picking up TEM grids
Dumont reverse tweezer	Ted Pella	5748	Style N2a, 118 mm, NM-SS, self-closing, holding TEM grids in place for sample preparation
Mortar and pestle	Amazon	BS0007	BIPEE agate mortar and pestle, 70 X 60 X 15 mm labware
Nalgene™ Oak Ridge tubes	ThermoFisher Scientific	3139-0050	Polypropylene copolymer, 50,000 x g, 50 mL, pack of 10
Scintillation vials	Fisher Scientific	03-337-4	20 mL vials with white caps, case of 500
TEM grids	Ted Pella	01813-F	Carbon Type-B, 300 mesh, copper, pack of 50
Glassware Setup
4-neck round bottom flask	Chemglass Life Sciences	CG-1534-01	24/40 joint, 500 mL, #7 chem thread for thermometers
6-port vacuum manifold	Chemglass Life Sciences	CG-4430-02	480 nm, 6 ports, 4 mm PTFE stopcocks
Adapter	Chemglass Life Sciences	CG-1014-01	24/40 inner joint, 90°
Condenser	Chemglass Life Sciences	CG-1216-03	24/40 joint, 365 mm, 250 mm jacket length
Drierite 26800 drying column	Cole-Parmer	EW-07193-00	200 L/hr, 90 psi
Funnel	Chemglass Life Sciences	CG-1720-L-02	24/40 joint, 100 powder funnel, 195 mm OAL
Interlocked worm gear hose clamp	Grainger	16P292	1/2" wide stainless steel clamp, 3/8" to 7/8" diameter, to secure condenser tubing, 10 pack
Keck clips	Kemtech America Inc	CS002440	24/40 joint
Metal claw clamp	Fisher Scientific	05-769-7Q	22cm, three-prong extension clamps
Metal claw clamp holder	Fisher Scientific	05-754Q	Clamp regular holder
Mineral oil bubbler	Kemtech America Inc	B257040	185 mm
Rotovap trap	Chemglass Life Sciences	CG-1319-02	24/40 joints, 100 mL, self washing rotary evaporator
Rubber stopper	Chemglass Life Sciences	CG-3022-98	24/40 joints, red rubber
Tubing for air/water	McMaster-Carr	6516T21	Clear Tygon PVC for air/water, B-44-3, 1/4" ID, 1/16" wall, 25 ft
Tubing for air/water	McMaster-Carr	6516T26	Clear Tygon PVC for air/water, B-44-3, 3/8" ID, 1/16" wall, 25 ft
Tubing for chemicals	McMaster-Carr	5155T34	Clear Tygon PVC for chemicals, E-3603, 3/8" ID, 1/16" wall, 50 ft
Analysis Programs
XRD analysis program	Malvern Panalytical	N/A	X'Pert HighScore Plus
FTIR analysis program	Varian, Inc.	N/A	Varian Resolutions Pro