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JoVE Journal Bioengineering

Bioengineering

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

Published: June 18, 2020 doi: 10.3791/61572
Automatic Translation
1, 1
1Department of Chemical and Biomedical Engineering, West Virginia University

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 bioimaging1,2,3, sensor technologies4,5, catalysis6,7,8, 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 vivo11,12,13; 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 cell14,15,16,17,18,19. 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 decomposition20,21,22,23,24,25, hydro/solvothermal26,27,28,29, exfoliation30,31,32,33,34, permanganates reduction35,36,37,38, and adsorption-oxidation39,40,41,42. 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 time44,45,46, temperature44,47,48,49, types/ratios of reactants20,45,47,48,50 and inert gas47,48,50 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 applications14,51,52,53.

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) stearate54,55 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 acid56,57, 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.

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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 (Equation 1). 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.

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Representative 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), with minimal overlap. It is crucial to achieve adequate separation of nanoparticles for accurate manual sizing of the nanoparticle diameters using the line trace tool in ImageJ.

Figure 3 shows suboptimal TEM sample preparation. If a high concentration of MnO nanoparticles are suspended in ethanol or too many drops of nanoparticle suspension are added to the TEM grid, each image will consist of large agglomerations of nanoparticles (Figure 3A,B). Due to the substantial overlap of nanoparticles, the limits of each nanoparticle diameter cannot be distinguished, which prevents accurate measurement. If a low nanoparticle concentration is prepared in ethanol, nanoparticles could be well separated, but distributed sparsely on the TEM grid (Figure 3C,D). When only one or two nanoparticles appear in each TEM image, more images need to be taken to gain a large enough sample size and the full size distribution may not be precisely captured. The TEM preparation protocol described herein aims to produce TEM images with approximately 10-30 nanoparticles per image (more nanoparticles can be accommodated per image if the diameter is small).

TEM can be used to evaluate changes in nanoparticle size with a variation in synthesis parameters. Figure 4 shows the average diameters of MnO nanoparticles synthesized with decreasing ratios of OA:DE. Diameters for each synthesis condition were quantified from 75 to 90 TEM images, with a total of 900 to 1100 MnO nanoparticles analyzed per condition. To ensure reproducibility, 3 batches of nanoparticles were synthesized for each OA:DE ratio. Overall, a decrease in the ratio of OA:DE yielded smaller MnO nanoparticles with less variation in size; the only exception occurred when OA alone was used during synthesis, which produced similar sized nanoparticles to the 30:30 ratio. Histograms showing the full size distribution of all MnO nanoparticle groups are displayed in Figure S2.

After confirming nanoparticle size and morphology with TEM, the bulk nanoparticle composition can be tested using XRD. Through measuring the angle and intensity of the X-ray beam diffracted by the sample, XRD can be used to determine crystal structure and phase of the nanoparticles. Figure 5A-F shows the raw collected XRD spectra for each synthesized MnO nanoparticle sample with decreasing ratios of OA:DE. The XRD peaks obtained on sample spectra are matched to XRD peaks from known compounds such as MnO and Mn3O4 through the XRD analysis program database. The standard peaks for MnO appear at 35°, 40°, 58°, 70°, 73°, and 87°, which are shown in Figure 5G. When comparing the nanoparticle XRD spectra with known MnO, it is evident that all nanoparticle spectra possess the 5 highest peaks of MnO, indicating successful synthesis of MnO nanoparticles. XRD can also be utilized to estimate nanoparticle size using the Scherrer equation; wider peaks on XRD indicate smaller nanoparticle diameters. For example, Figure 5F with the widest XRD peaks is associated with the smallest nanoparticles as shown by TEM (18.6 ± 5.5 nm).

Figure 6 shows XRD spectra of two undesired products in MnO nanoparticle synthesis. To encourage the formation of the MnO phase at high temperatures (280 oC), nitrogen is used during nanoparticle synthesis to purge air out of the system. If inadequate nitrogen flow is applied, a mixed phase composition of Mn3O4 (51%) and MnO (49%) is produced (Figure 6A). Through comparison with the standard peaks of Mn3O4 (Figure 6C) and MnO (Figure 6D), low nitrogen flow produces XRD spectra with the 8 highest peaks for Mn3O4 and the 5 highest peaks for MnO. TEM of nanoparticles synthesized under low nitrogen flow revealed a mixed population of large nanoparticles surrounded by smaller nanoparticles (Figure 6E). Nitrogen flow can be monitored through the nitrogen regulator reading and the rate of bubbling through the mineral oil bubbler. Another critical parameter in MnO nanoparticle synthesis is the inclusion of a stabilizer. In an attempt to produce even smaller MnO nanoparticles than the 10:50 OA:DE ratio, pure DE was used without any OA. A very small amount of an unknown powder was synthesized in the absence of stabilizer. As shown in Figure 6B, the XRD spectra for the 0:60 OA:DE ratio was noisy and contained the 3 highest peaks of Mn3O4. From analysis in the XRD program database, the compound had a chemical composition of 67% Mn3O4 and 33% MnO. As supported by the wide peaks in the XRD spectra, the TEM confirmed that very small nanoparticles were synthesized in the absence of stabilizer (Figure 6F). Nanoparticles also appeared irregularly shaped and agglomerated. Additionally, only a 33% yield was obtained without any stabilizer, meaning that a small amount of product was synthesized. Therefore, high nitrogen flow and inclusion of a stabilizer such as OA or oleic acid is necessary for synthesis of MnO nanoparticles.

To complement bulk nanoparticle composition with XRD, surface composition can be evaluated using FTIR. Figure 7 shows the FTIR spectra of MnO nanoparticles after background correction. All spectra show the symmetric and asymmetric CH2 peaks (2850-2854 and 2918-2926 cm-1, marked by asterisks) associated with oleyl groups60, in addition to the NH2 bending vibration peaks (1593 cm-1 and 3300 cm-1, marked by squares) associated with amine groups61. Since MnO nanoparticles share the same peaks for oleyl groups and amine groups present in the FTIR spectra of OA (Figure S3), it can be concluded that the nanoparticles are coated with a surface layer of OA. Furthermore, all nanoparticle FTIR spectra contain Mn-O and Mn-O-Mn bond vibrations around 600 cm-1 (marked by triangles), which confirm the composition found through XRD62.

Figure 1
Figure 1: Nitrogen and water flow through the MnO nanoparticle synthesis setup.
Tubing connections are labeled 1-15. Air-free nitrogen enters (1) and exits (2) the drying column and is fed into the inlet of the manifold (3). During the reaction, nitrogen purges air from the system by entering the right stopcock on the manifold (4). Nitrogen flows from the stopcock to the glass elbow adapter (5), rotovap trap, round bottom flask, condenser, glass elbow adapter (6) and through a series of two mineral oil bubblers (7-9). In the manifold, excess nitrogen not flowing through the reaction will leave the system through the left stopcock (10), which is connected to the mineral oil bubbler with the largest amount of silicone oil (11). Stopcock #10 is to always remain open. Water will flow from the faucet (12) through the condenser inlet (13) and outlet (14) and into the fume hood drain (15). The tubing is secured to the condenser with metal clamps. All tubing should be chemical resistant tubing except for the water compatible tubing used for the condenser. The main glassware and equipment are labeled in Figure S1. Please click here to view a larger version of this figure.

Figure 2
Figure 2: TEM images of MnO nanoparticles synthesized with decreasing ratios of OA:DE.
The following ratios were used: (A) 60:0, (B) 50:10, (C) 40:20, (D) 30:30, (E) 20:40, (F) 10:50. MnO nanoparticles appear as separate, rounded octagons with minimal overlap to allow for clear delineation of nanoparticle borders. The reactant ratio was observed to affect overall nanoparticle size, with 50:10 synthesizing the largest nanoparticles and 10:50 producing the smallest nanoparticles. Scale bars are 50 nm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Suboptimal TEM images resulting from incorrect TEM grid preparation.
(A,B) If the nanoparticle suspension is too concentrated or if excess drops of nanoparticle suspension get loaded onto the TEM grid, nanoparticles will aggregate into large masses with substantial overlap. Individual nanoparticles cannot be observed in most areas of the grid. (C,D) Alternatively, a low nanoparticle concentration could result in TEM grids populated with a scarce amount of nanoparticles. Individual nanoparticles are spread far apart, but require more images to capture the sample’s population size distribution. Scale bars are 50 nm. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Average MnO nanoparticle diameters measured from TEM images.
In general, a lower amount of stabilizer (OA) with a higher amount of organic solvent (DE) resulted in smaller, more uniform MnO nanoparticles. A total of 900 to 1100 nanoparticle diameters were calculated on TEM images using the line trace tool in ImageJ for each group. Error bars show standard deviation. Please click here to view a larger version of this figure.

Figure 5
Figure 5: XRD spectra of MnO nanoparticles synthesized with decreasing ratios of OA:DE.
The following ratios were used: (A) 60:0, (B) 50:10, (C) 40:20, (D) 30:30, (E) 20:40, (F) 10:50. (G) The standard diffraction peaks for MnO are shown from the XRD analysis program database. All nanoparticles produced exhibit the 5 highest intensity XRD peaks for MnO, indicating successful synthesis of MnO nanoparticles. Please click here to view a larger version of this figure.

Figure 6
Figure 6: XRD spectra and TEM images of undesired nanoparticles.
XRD spectra are shown for MnO nanoparticle synthesis using (A) low nitrogen flow and (B) a 0:60 ratio of OA:DE (no stabilizer is present). The standard diffraction peaks for (C) Mn3O4 and (D) MnO are displayed from the XRD analysis program database. Through comparison with standard spectra, inadequate nitrogen flow (A) created nanoparticles with a mixture of Mn3O4 (51%) and MnO (49%). In the absence of oleylamine (B), a broader XRD spectrum is obtained, which matches the 3 highest peaks of Mn3O4. Based on the analysis performed by the XRD program database, these synthesized nanoparticles are 67% Mn3O4 and 33% MnO. TEM images of (E) nanoparticles synthesized with low nitrogen flow show large nanoparticles surrounded by smaller ones. TEM images of (F) nanoparticles synthesized with a 0:60 ratio of OA:DE display very small aggregated nanoparticles with irregular shape. Scale bars are 50 nm. Please click here to view a larger version of this figure.

Figure 7
Figure 7: FTIR spectra of MnO nanoparticles synthesized with decreasing ratios of OA:DE.
The following ratios were used: (A) 60:0, (B) 50:10, (C) 40:20, (D) 30:30, (E) 20:40, (F) 10:50. Asterisks and squares correspond to oleyl groups and amine groups, respectively, while triangles indicate the vibration of Mn-O and Mn-O-Mn bonds. The boxed insets highlight the two distinct peaks of oleyl groups. FTIR spectra indicate that MnO nanoparticles are coated with oleylamine, as confirmed through comparison with the oleylamine only FTIR spectrum in Figure S3. Please click here to view a larger version of this figure.

Figure S1: Major glassware and equipment of the MnO nanoparticle synthesis setup. The manifold is secured to the metal lattice by metal claw clamps and disperses nitrogen into the reaction. Mn(II) ACAC, dibenzyl ether, oleylamine and a stir bar are added to the round bottom flask with four necks. The right neck of the flask is attached to the rotovap trap and an elbow adapter, while the left neck is attached to a condenser and an elbow adapter. The middle neck of the round bottom flask is covered with a rubber stopper. The temperature probe is inserted into the smallest opening of the round bottom flask, and is surrounded by an o-ring and paraffin plastic film to form an air-tight seal. The round bottom flask sits on top of a heating mantle and a stir plate to vigorously stir the reaction while heating. The temperature probe and heating mantle are connected to the temperature controller to provide real-time automated regulation of the temperature profile. The round bottom flask and condenser are secured to the metal lattice with metal claw clamps. There are three mineral oil bubblers, two on the left and one on the right, filled with increasing amounts of silicone oil from the left bubbler to right bubbler in the image. The bubblers are also attached to the metal lattice with claw clamps. Green plastic conical joint clips are attached to secure glassware connections before the reaction begins. The tubing connections are detailed in Figure 1. Please click here to download this figure.

Figure S2: Histograms showing distribution of MnO nanoparticle size for decreasing ratios of OA:DE. The following ratios were used: (A) 60:0, (B) 50:10, (C) 40:20, (D) 30:30, (E) 20:40, (F) 10:50. Overall as the ratio approaches 10:50, the nanoparticle size distribution shifts to the left (indicating smaller diameters) and becomes more compact (indicating more uniform nanoparticle size). The average diameter for each distribution is shown in Figure 4. Please click here to download this figure.

Figure S3: FTIR spectrum of oleylamine. Asterisks and squares represent the oleyl groups and amine groups of oleylamine, respectively. Please click here to download this figure.

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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 described63. DE serves as an ideal organic solvent for thermal decomposition reactions, as it has a high boiling point of 295-298 °C. OA is a commonly used inexpensive stabilizer/mild reducing agent, which aids in capping and coordinating metal oxide nanoparticle nucleation and growth61,63. Similar to DE, OA has a high boiling point of 350 °C to withstand the high temperatures of thermal decomposition. The following two observations can be used as evidence of successful generation of MnO nanoparticles during synthesis: 1) the appearance of a green hue to the reaction mixture during thermal decomposition at 280 °C and 2) the formation of a dark brown large pellet on the bottom of the centrifuge tubes following centrifugation in hexane and ethanol. Resulting nanoparticles should be further characterized by TEM, XRD and FTIR to evaluate size/morphology, bulk composition and surface composition, respectively.

During nanoparticle synthesis, several variables must be noted and controlled to ensure production of uniform nanoparticles with the MnO crystalline phase. First, the ratio of all starting materials should remain the same, as we have shown that decreasing ratios of OA to DE decrease nanoparticle size (Figure 4). Second, the reaction should be vigorously stirred to enable adequate dispersion of nucleating nanoparticles, uniform heating, and reduction of size variation. Third, as temperature plays a large role in controlling metal oxide nanoparticle size47,48,50 and phase composition47,48,50, it is critical to properly immerse the temperature probe tip into the reaction mixture while not contacting the glass of the round bottom flask that will read an inaccurate temperature. Fourth, the flow of nitrogen should be high enough to purge all air from the reaction to encourage formation of the MnO crystalline phase over Mn3O4. As shown in Figure 6A, low nitrogen flow will result in nanoparticles with a mixed MnO/Mn3O4 composition. Correct filling of the mineral oil bubblers with increasing amounts of silicone oil from the left bubbler (1 inch of oil) to the middle bubbler (1.5 inches of oil) to the right bubbler (2 inches of oil) will set the resistance for nitrogen flow to be lowest through the reaction (#4 in Figure 1). The bubbling rate of the middle mineral oil bubbler (by #7,8 in Figure 1) can be used to measure the rate of nitrogen flowing through the reaction. Finally, a stabilizer such as OA must be added to the reaction mixture to coordinate nanoparticle nucleation and growth. As shown in Figure 6B, DE without OA created a small amount of product, mostly of a Mn3O4 (67%) composition. This product was also observed to have an irregular shape with aggregated nanoparticles by TEM, which did not occur when OA was present in the reaction (Figure 6F).

Several variables of the thermal decomposition reaction can be modified to optimize nanoparticle size, morphology, and composition including the type of inert gas47,48,50, peak reaction temperature44,47,48,49, total reaction time44,45,46, and types/ratios of initial chemical compounds utilized in the reaction20,45,47,48,50. Salazar-Alvarez et al.50 and Seo et al.48 have shown that argon flow during thermal decomposition of Mn(II) forms Mn3O4 at lower peak reaction temperatures ranging from 150 °C to 200 °C. When using nitrogen or air, Nolis et al.47 achieved similar results for Mn(III) ACAC decomposition where Mn3O4 nanoparticles were produced at lower temperatures (150 oC or 200 oC) and MnO nanoparticles were generated only at higher temperatures (250 °C and 300 °C)47. Higher peak reaction temperatures and longer times held at the peak reaction temperature, also known as the aging time, have also been associated with an increase in nanoparticle size44,45,46,47,48,49. Furthermore, the heating rate of the reaction can impact nanoparticle size. Schladt et al.44 found that increasing the heating rate from 1.5 °C/min up to 90 oC/min dropped nanoparticle size from 18.9 nm to 6.5 nm, respectively. Finally, different chemicals can be added as reducing agents and stabilizers in manganese thermal decomposition reactions; however, OA20,47,48,50 and oleic acid20,45 are most commonly used. The ratio of OA to oleic acid has been proven to affect the chemistry and shape of synthesized MnO nanoparticles. According to Zhang et al.20, OA only resulted in the formation of Mn3O4 nanoparticles, a combination of OA and oleic acid led to a mixture of Mn3O4 and MnO nanoparticles, and oleic acid only produced MnO nanoparticles. Interestingly, experience shows that MnO nanoparticles can be fabricated with OA only, and that oleic acid is not necessary to promote formation of the MnO crystalline phase. Furthermore, the use of OA by itself fabricated spherical nanoparticles, while oleic acid alone generated star shaped nanoparticles20,64. Clearly, there is much flexibility in altering synthesis parameters to impact resulting physical and chemical properties of MnO nanoparticles.

Despite the detailed protocol, instances may arise that require troubleshooting. The following paragraph details some common issues and solutions. During the reaction, if the temperature seems to stabilize around 100 °C, some water may have leaked into the heating mantle. Visibly inspect the surrounding area for water leakage from the condenser. Do not directly touch the mantle or round bottom flask without heat resistant gloves, as they will be very hot. If water is observed, immediately turn off the temperature controller, unplug the heating mantle, and let it dry overnight. To prevent future leakages, use an interlocked worm gear hose clamp to secure the water tubing to the condenser. In the case that the desired product is MnO, but only Mn3O4 is produced, it is important to check the nitrogen flow during the reaction. The middle bubbler should have a constant stream of bubbles (see the video for correct bubbling rate), while the right bubbler should only have one or two bubbles forming in it. Incorrect nitrogen flow can occur if the differential silicone oil levels in each mineral oil bubbler are not maintained. Check the oil levels before every experiment and fill up the bubblers according to step 1.5 if needed. During nanoparticle collection, the protocol specifies to pour out the supernatant without disturbing the nanoparticle pellet. The best way to discard the supernatant is to pour it out with one fast continuous motion rather than a slow one. However, if the pellet gets easily detached from the centrifuge tube, the use of a transfer pipette is recommended to remove the supernatant. During nanoparticle collection and TEM grid preparation, bath sonication is a key step. If the nanoparticles are not resuspending correctly, move the tube around the water bath sonicator until an area is located where the sonication can be felt by the hand holding the tube. The nanoparticle pellet can also be visibly seen disintegrating under strong bath sonication if the tube is in the correct spot. After nanoparticle resuspension, it is important that the TEM grid is suspended in the air with reverse tweezers rather than placed onto a wipe or directly onto an absorbent bench surface. The wipe or absorbent bench surface will wick the nanoparticle suspension off of the TEM grid before drying, resulting in insufficient nanoparticle deposition on the grid for imaging.

Although the thermal decomposition reaction is fairly simple and straightforward to follow to synthesize MnO nanoparticles, there are some limitations associated with the method. While it is possible to control the physical and chemical properties of nanoparticles to some extent, some variables such as temperature and aging time impact both nanoparticle size and phase composition simultaneously. Therefore, it is difficult to always have precise independent control of nanoparticle properties using this method. In addition, scaling up of the nanoparticle synthesis by tripling or quadrupling the amounts of starting materials can cause the reaction to become unstable and violent. Larger batch size is also associated with a decreased yield. Furthermore, despite storage of MnO nanoparticles inside capped scintillation vials wrapped in paraffin plastic film, we have seen oxidation of the nanoparticle surface to Mn3O4 as evaluated by X-ray photoelectron spectroscopy. Finally, the MnO nanoparticles generated by this technique will be hydrophobic and capped with OA (Figure 7). Further surface modification to transition nanoparticles to a hydrophilic state will need to be applied to enable nanoparticle suspension in aqueous media. Several methods have been established to promote the dispersion of nanoparticles in biological solutions including nanoparticle encapsulation inside of polymers14, coating of the nanoparticle surface with lipids52, or ligand exchange to substitute the OA on the nanoparticle surface with hydrophilic ligands such as poly(acrylic acid)20. To achieve encapsulation of MnO nanoparticles within poly(lactic-co-glycolic acid) (PLGA) polymer, follow McCall and Sirianni’s detailed JoVE protocol65; MnO nanoparticles can be added directly to the PLGA polymer solution as described for hydrophobic drugs in step 8 of the Nanoparticle Preparation section. MnO nanocrystal distribution inside of PLGA nanoparticles can be assessed using TEM and loading of Mn inside the PLGA polymer can be determined by thermogravimetric analysis as shown in Bennewitz et al.14.

Although MnO nanoparticles can be utilized for a wide variety of applications due to their magnetic, electronic and catalytic properties, we are interested in applying MnO nanoparticles as switchable, T1 MRI contrast agents. Previously, our group and others have shown that intact MnO nanoparticles have negligible T1 MRI contrast (MRI signal is “OFF”) at physiological pH 7.4 mimicking the blood14,15,16,17,18,19. However, MnO dissolves to create substantial Mn2+ ions at low pH 5 mimicking cellular endosomes; released Mn2+ will coordinate with surrounding water molecules to turn “ON” MRI signal at low pH14,15,16,17,18,19. MnO nanoparticles can be localized to different cells of interest, such as cancer cells, through addition of targeting peptides or antibodies to the nanoparticle surface51,66. Here, we describe the synthesis of MnO nanoparticles with an average diameter ranging from 18.6 nm to 38.8 nm. Control of nanoparticle size can be useful for improving MRI contrast agent effectiveness. Specifically, it is anticipated that larger nanoparticles will have more surface area for attachment of targeting ligands to enhance nanoparticle accumulation at the site of interest such as tumors. However, overall nanoparticle size with added surface groups should be limited to 50-100 nm to maximize tumor accumulation67,68. Smaller nanoparticles, on the other hand, have a higher surface area-to-volume ratio to facilitate faster release of Mn2+ under acidic environments and should allow for enhanced nanoparticle packing volumes inside of polymeric delivery systems. Synthesis of MnO over Mn3O4 should also improve MRI contrast, as MnO has been shown to dissolve faster than Mn3O4 in concentrated acidic solutions to generate more Mn2+ ions69. In summary, we have described a thermal decomposition protocol for fabrication of MnO nanoparticles that is relatively straightforward and customizable to allow for optimizing nanoparticle design for future use in applications such as smart MRI contrast agents, biosensors, catalysts, batteries and water purification.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

This work was supported by WVU Chemical and Biomedical Engineering Department startup funds (M.F.B.). The authors would like to thank Dr. Marcela Redigolo for guidance on grid preparation and image capture of nanoparticles with TEM, Dr. Qiang Wang for support on evaluating XRD and FTIR spectra, Dr. John Zondlo and Hunter Snoderly for programming and integrating the temperature controller into the nanoparticle synthesis protocol, James Hall for his assistance in assembly of the nanoparticle synthesis setup, Alexander Pueschel and Jenna Vito for aiding in quantification of MnO nanoparticle diameters from TEM images, and the WVU Shared Research Facility for use of the TEM, XRD, and FTIR.

Materials

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

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Manganese Oxide Nanoparticle Synthesis Thermal Decomposition Manganese(II) Acetylacetonate Metal Oxide Nanoparticles Particle Size Control Shape Control Chemical Composition Control One Pot Synthesis Metal Precursor Organic Solvent Stabilizer Celia Martinez De La Torre Graduate Research Assistant Round Bottom Flask Heating Mantle Magnetic Stir Bar Glass Funnel Allylamine Di-benzyl Ether Condenser Metal Claw Clamp Rotovap Trap
Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate
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Martinez de la Torre, C., Bennewitz, … More

Martinez de la Torre, C., Bennewitz, M. F. Manganese Oxide Nanoparticle Synthesis by Thermal Decomposition of Manganese(II) Acetylacetonate. J. Vis. Exp. (160), e61572, doi:10.3791/61572 (2020).

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