Jun 20, 2024

Public workspaceMicrowave synthesis of low molecular weigth deacylated chitosan V.3

Version 1 is forked from Microwave Synthesis of Lanthanum-Doped Carbon Dots
DOI
dx.doi.org/10.17504/protocols.io.x54v9d6w4g3e/v3
Microwave synthesis of low molecular weigth deacylated chitosan
  • 1Department of Plant and Environmental Sciences, Clemson University, USA;
  • 2Department of Agricultural Science, Clemson University, USA;
  • 3Global Alliance for Rapid Diagnostics, Michigan State University, USA;
  • 4Agricultural Sciences Department, Clemson University, USA
Maria J Torres
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9d6w4g3e/v3
Protocol Citation: Maria J Torres, Eric S McLamore, Geisianny AM Moreira 2024. Microwave synthesis of low molecular weigth deacylated chitosan. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9d6w4g3e/v3Version created by Maria J Torres
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2023
Last Modified: June 20, 2024
Protocol Integer ID: 102152
Keywords: nanoparticle, microwave, synthesis, chitosan
Funders Acknowledgement:
National Science Foundation
Grant ID: CBET-2019435
Abstract
This protocol describes the synthesis of low molecular weight deacylated chitosan using microwave irradiation. The process requires approximately 2.5 hours (including baseline analysis).

An Anton-Parr microwave reactor (Monowave 50p) was used throughout this protocol. ADA-compliant procedures are used throughout the protocol (including building lab space/design and analysis of synthesized particles).
Guidelines
This protocol describes the synthesis of chitosan nanobrushes using microwave irradiation. The complete process requires approximately 2.5 hours (see diagram for overview of steps).

A link to the Monowave Resource Manual with additional introductory information on the instrument is here (link).
Figure 1. Process flow for the synthesis of nano brushes in a microwave reactor. The steps are separated into sections: preparation procedures (blue), particle synthesis, including the setup of the settings in the equipment (red), stabilization and storage of the solution (green), data collection (grey), and cleanup of the working station and dispose of waste (black).
Figure 1. Process flow for the synthesis of nano brushes in a microwave reactor. The steps are separated into sections: preparation procedures (blue), particle synthesis, including the setup of the settings in the equipment (red), stabilization and storage of the solution (green), data collection (grey), and cleanup of the working station and dispose of waste (black).

Materials
MATERIALS

HARDWARE

SOFTWARE
Safety warnings
Attention
SAFETY

General
  • Lab coat, gloves, and closed-toed shoes are mandatory.
  • Safety issues specific to Monowave 50p.
  • The maximum pressure for the glass vial is 20 bar.
  • The maximum volume in the glass vial at any time is 6 mL.

Eye protection
  • Goggles or eye protection is required when handling acidic solutions outside a chemical hood (i.e., transport in the lab).

Skin
  • Immediately rinse under water and wash with soap for at least 5 min.

Fumes/aerosols
  • Any open containers should be processed/handled under a chemical hood.
  • All acidic solutions should be covered with parafilm when outside the chemical hood.
  • Aerosolization of particles (nano or colloidal) and subsequent health effects are unknown, and samples should be treated as hazardous and capped.

Heat and Flammable materials
  • The Monowave heats to temperatures as high as 250°C, and the instrument undersurface may be very hot. Do not touch the undercarriage at any time.
  • The instrument panel and handle for the cover are safe to touch when the reactor has cooled to 50°C (default instrument safety standard).

Disposal
  • Vials of carbon particles should be discarded in the waste container and not disposed of in the sink.

ADA COMPLIANCE
The following guidance is summarized by Perry and Baum 1 where relevant to this protocol.

1) General building codes for laboratory
  • Minimum 2 Exits for labs ≥500 sf (150 sm) net area.
  • Minimum 2 Exits for labs using chemical fume hoods or glove box
  • Minimum 2 Exits for labs using flammable and combustible liquids, gases, cryogenics, dust and solids.
  • Minimum 2 Exits for labs using oxidizers, unstable reactives, water reactives, organic peroxides, highly toxics, corrosives.

2) Egress for wheelchair 360° Turn is 1.5 m (5 ft) clearance. Wheelchair clearance must be provided for:
  • Both sides of Exit and Entry doors to
  • Emergency Eyewash & Safety Shower
  • In front of wall benches, sinks, equipment
  • In front of chemical fume hoods
  • At chalk/marker board
  • Between benches
  • Aisles that lead to Primary Exits, back to front
  • Aisles that allow passage side to side in lab

3) Standard accommodations for use of chemical hood or other exhaust air containment systems
  • knee space obstructions
  • adjustable work surface height
  • accessible receptacles and alarm control

Common equipment
  • Where visual inspection is utilized, alternative technologies should be listed as optional (colorimeters, spectro-radiometers, etc.)

References
1. Perry, J. & Baum, J. Assessing the Laboratory Environment. in Accessibility in the Laboratory vol. 1272 3–25 (American Chemical Society, 2018).

Before start
  • Wear appropriate safety PPE throughout (lab coat, gloves, eyewear).
  • An electronic or physical lab notebook may be used throughout.
  • See the experimental plan guide for tips on planning your work.
SECTION 1) Materials and solution preparation
SECTION 1) Materials and solution preparation
58m
Prepare Solution (1% deacylated chitosan -0.05M Acetic Acid)
  • In a glass container or sealed plastic weight Amount2 g of Chitosan (CHI) 85% deacetylated and solvated in Amount98 mL of 0.1M of acetic acid, mix and look carefully to ensure no precipitates. Use the magnetic stirrer base to provide a mix of the solution.
  • Add Amount49 mL of the previous solution and mix with Amount49 mL of DI/nano-pure water to obtain 1% deacylated CHI - 0.05M acetic acid solution. Use the magnetic stirrer base to provide a mix of the solution.
  • Shake/stir well and look carefully to ensure there are no precipitates. Use the magnetic stirrer base to provide a mix of the solution.
  • Seal the vial immediately (contamination is a common problem).

Note
Shelf life solution: The same solution can continue to be used for about one week of storage.


42m
Prepare reaction vials

  • Check the vial cap and Teflon liner to make sure neither is damageddamage to the cap and both are clean (Fig 1A-B)

Figure 1. Reaction vials for monowave. A) Check vial cap and B) Teflon liner. Both the cap and liner should be free of damage and clean.
Figure 1. Reaction vials for monowave. A) Check vial cap and B) Teflon liner. Both the cap and liner should be free of damage and clean.
Note
Critical step: If the Teflon liner is damaged, or if the reaction vial is capped, dispose of it, and replace it with a new cap/liner. If damage to the cap is not observed, solutions will likely be "overcooked", see Figure 5A.

10m
Add solution to the reaction vial.

  • Pipette Amount8000 µL of 1% deacylated chitosan -0.05M acetic acid solution to a glass reactor vial.
  • Add a nano stir bar to the reactor vial.
  • Place Teflon liner in cap.
  • Seal the reaction vial with cap.
  • Invert the vial three times to mix solutions.
  • Place in a test tube rack.

Note
Critical step: Be careful not to lose the nano stir bars. They are the most frequently misplaced item in this protocol.

6m
SECTION 2) Particle synthesis
SECTION 2) Particle synthesis
36m
Set up microwave reactor.

  • Ensure the Anton-Paar reactor is in the chemical hood and plugged into the power outlet (Fig 2A).
  • If not placed in the chemical hood, the institutional safety committee must approve the exhaust hose. setup (using either a snorkel exhaust and/or a valid tube/hose system).
  • Open the lid and place the glass reactor in the Anton-Paar reactor chamber (Fig 2B).
  • Close the chamber lid and seal it shut with the handle.

Figure 2. Microwave reactor (Anton-Paar Monowave) for hydrolysis of deacylated chitosan. A) Ensure the reactor is in the chemical hood and plugged into the power outlet. B) Open the lid to show the lining is not damaged or dirty.
Figure 2. Microwave reactor (Anton-Paar Monowave) for hydrolysis of deacylated chitosan. A) Ensure the reactor is in the chemical hood and plugged into the power outlet. B) Open the lid to show the lining is not damaged or dirty.
  • Before starting the Monowave, go to the third bottom option on the screen to ensure the settings are correct and have not been changed. Then, press "step type" and "ramp to temp" and click "✓".

Note
Critical step: When running for the first time, method must be created.

Create a method with the following settings:
  • Target cooling temperature: 50°C
  • Stir speed: 600 rpm
  • Maximum set temperature: 170 °C
  • Time to ramp: 5 minutes
  • Hold time: 1 minute.

Figure 3. Photos of Monowave 50 reactor touch screen used for setup. A) Select the method. B) Choose “Run by Method.” C) Enter a file name to save data.
Figure 3. Photos of Monowave 50 reactor touch screen used for setup. A) Select the method. B) Choose “Run by Method.” C) Enter a file name to save data.

  • Press “Run by Methods” section (Fig 3) by pressing the Menu button.
  • Check the settings above (see note) and ensure “Run by Methods” is correct in the instrument (Fig 3).
  • Press Menu
  • Press the "Methods" required to complete button required to complete
  • Choose the method, then "Done."
  • Click back.
  • Choose Run Name and name in the text box as your preference.
  • Click "START".

Note
The time required to complete the synthesis of the chitosan nanobrushes is approximately 10 minutes.

26m
SECTION 3) Reactor data collection
SECTION 3) Reactor data collection
5m
Download reactor temp/pressure profile

  • Insert USB to port on right side of Monowave 50 p.
  • Press “Details”.
  • Press “Export”.
  • Choose the Export option, and press “OK” when done.
  • Remove USB.
5m
SECTION 4) Stabilization and analysis (post cook)
SECTION 4) Stabilization and analysis (post cook)
35m
Qualitative analysis (color)

  • The first step of qualitative analysis is visual inspection of the solution color.
  • Analysis of polychromatic visible (VIS) color is an important quality control mechanism in particle synthesis (Fig 4)

Figure 4. Visual inspection is an important quality control mechanism in particle synthesis. If visual inspection is impossible, a colorimeter or other device may be used to visually inspect the samples (see note below for an example of a mobile phone app). Image courtesy of Shutterstock standard license (no. 514067857)
Figure 4. Visual inspection is an important quality control mechanism in particle synthesis. If visual inspection is impossible, a colorimeter or other device may be used to visually inspect the samples (see note below for an example of a mobile phone app). Image courtesy of Shutterstock standard license (no. 514067857)
Note
If preferred, a cell phone app may be used to detect the color of the sample.

For example, Color Name AR is a useful tool that is available for both iPhone and Android (as well as tablets) (https://apps.apple.com/us/app/color-name-ar/id906955675)

  • Observe the glass vials under white light (valuable lab lighting)
  • Representative photos of "as prepared" particle solutions are shown in Fig 5.

Figure 5. Representative photos of lower molecular weight deacylated chitosan solutions. A) Sample with a light color before using the microwave reactor to reduce molecular weight. B) Sample that is the optimum color for this protocol (based on extensive spectroscopy and material analysis). This represents the characteristic color after low molecular weight processes assisted my microwave.
Figure 5. Representative photos of lower molecular weight deacylated chitosan solutions. A) Sample with a light color before using the microwave reactor to reduce molecular weight. B) Sample that is the optimum color for this protocol (based on extensive spectroscopy and material analysis). This represents the characteristic color after low molecular weight processes assisted my microwave.

  • In addition to visual inspection, UV-VIS may be used to analyze the samples.
  • Color determination to evaluate the quality of the sample after the use of microwave to reduce the molecular weight of deacylated chitosan is shown in Fig. 6
Figure 6. Analysis of solution color using an iPhone app (Color Name AR). A) Sample of lower molecular weigth deacylated chitosan. B) associated color identification using Color Name AR (identified as Russet; RGB 196, 194, 150).
Figure 6. Analysis of solution color using an iPhone app (Color Name AR). A) Sample of lower molecular weigth deacylated chitosan. B) associated color identification using Color Name AR (identified as Russet; RGB 196, 194, 150).

5m
Qualitative analysis (fluorescence emission)
  • Using either low light conditions or a prepared lightbox for analysis (e.g., black styrofoam), position a 395 nm UV pen perpendicular to the glass storage vial,
  • Turn on the pen and inspect for an emission beam.
  • Fig 7A shows an example of fluorescence for a sample following the protocol described here.
  • This rapid analysis is helpful for quality control immediately after synthesizing particles but should be supported by subsequent quantitative analysis (shown in the step below).

Figure 7. Reference comparison for UV pen fluorescence check of carbon dots. A) Photo of low molecular weight deacylated chitosan in a lightbox under 395nm excitation using a UV pen. B) associated color identification using Color Name AR (150,222,209) in the RGB color space. C) EE Toolkit was used to analyze the emission signal. Based on visual observation, an emission peak of approximately 488 nm was detected using EE Toolkit (https://ee-toolkit.com/).
Figure 7. Reference comparison for UV pen fluorescence check of carbon dots. A) Photo of low molecular weight deacylated chitosan in a lightbox under 395nm excitation using a UV pen. B) associated color identification using Color Name AR (150,222,209) in the RGB color space. C) EE Toolkit was used to analyze the emission signal. Based on visual observation, an emission peak of approximately 488 nm was detected using EE Toolkit (https://ee-toolkit.com/).


10m
SECTION 5) Cleanup
SECTION 5) Cleanup
10m
Clean up space and dispose of waste.

  • Turn off Monowave and unplug it from the waste area. It has a dedicated chemical disposal container for the chitosan outlet.
  • Dispose of used chemicals according to the lab safety plan.

Note
Note: The waste area has a dedicated chemical disposal container for chitosan residues.

  • Wash all glass reactors with mild detergent and warm water.
  • Carefully remove the Teflon lining from the cap and wash with mild detergent and warm water.
If the cap or the Teflon lining is damaged, it should be discarded according to the lab safety plan.

Note
Note: Clean up the chemical hood and process pipette tips or other materials used for handling HCl before discarding them according to the safety plan (otherwise, they contain acid residue.

10m
SECTION 6) Data management
SECTION 6) Data management
5m
Data management (reactor data only).

  • File naming: For saving all versions of this protocol, use the following file structure:
Method_(insert date here).ai

  • File storage: Store all methods in the Desktop folder.

  • Backup files: At least once per year, ensure the folder is backed up on the lab's external hard drive.
5m