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).
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.
In a glass container or sealed plastic weight 2g of Chitosan (CHI) 85% deacetylated and solvated in 98mLof 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 49mLof the previous solution and mix with 49mLof 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.
2.
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.
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 .
2.
2.
3.
Add solution to the reaction vial .
Pipette 8000µL 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.
SECTION 2) Particle synthesis
4.
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.
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°CStir speed: 600 rpmMaximum set temperature: 170 °CTime to ramp: 5 minutesHold 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.
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.
SECTION 3) Reactor data collection
5.
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.
SECTION 4) Stabilization and analysis (post cook)
6.
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)
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) (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.
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. 6Figure 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).
7.
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/).
SECTION 5) Cleanup
8.
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.
SECTION 6) Data management
9.
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.
