Paraformaldehyde, Formaldehyde, and Formalin: Understanding the Key Differences
When I first encountered the need to fix my cells, I struggled to understand the distinctions between the various terms (and, by extension, the substances) involved.
FORMALDEHYDE
Formaldehyde can form covalent bonds with various chemical groups containing a reactive hydrogen atom: R-H + HCHO → R-CH2OH
It is typically used as a fixative in a solution at approximately 4% concentration, with a pH range of 6.9 to 7.4, and at room temperature or 4°C under atmospheric pressure.
The primary reaction of formaldehyde in these conditions involves interactions with aromatic rings and amino groups (NH2).
Once bound to a protein, formaldehyde can react with the reactive hydrogen of another molecule, forming intermolecular bridges.
Although formaldehyde reacts relatively slowly with biological structures, it penetrates tissues deeply.
Formaldehyde preserves most lipids (making them insoluble in solvents), partially preserves glycogen, and causes tissue hardening.
FORMALIN
Formaldehyde is a gas at normal temperature and pressure and is commercially available as formol or formalin. A saturated formalin solution typically contains around 40% formaldehyde by volume (37% by mass). For instance, a 10% (vol/vol) formalin solution is made by mixing 10 ml of commercial formalin with 90 ml of water or other solvents, resulting in a 4% formaldehyde solution.
Formalin is prone to oxidation, so commercial solutions are often stabilized with 10-12% methanol to prevent oxidation and polymerization. However, the presence of methanol may negatively impact some experiments. Formalin is typically used in buffered solutions with added calcium chloride to enhance the stability of lipid structures in samples.
PARAFORMALDEHYDE
Paraformaldehyde is a polymer of formaldehyde and is commercially available as a white powder. It is commonly used to prepare formaldehyde solutions for cell cultures as it can be stored at room temperature without stabilizers, providing a pure formaldehyde solution. Paraformaldehyde is preferred in electron microscopy and immunohistochemistry (IHC) because formalin contains methanol, which can interfere with these applications.
Formaldehyde, Formalin, Paraformaldehyde And Glutaraldehyde: What They Are And What They Do
Preparing a Paraformaldehyde Solution: Protocol
Depolymerization of paraformaldehyde in solution can be achieved by using either acidic or basic catalysts.
In summary, the solution is heated, and NaOH (sodium hydroxide) is added to facilitate the solubilization of paraformaldehyde. The initially cloudy solution clears as formaldehyde forms.
Formaldehyde (derived from paraformaldehyde) is produced for cultured cells using PBS or other physiological saline solutions.
Reagents required
- PBS
- Paraformaldehyde powder
- NaOH solution 1N
- HCl 1 N
Required material
- A beaker and a magnetic bead
- Hot plate with magnetic stirrer
- A thermometer
- Chemical hood
- A laboratory balance.
Procedure for the preparation of a paraformaldehyde solution
To prepare 100 mL of a 4% formaldehyde solution, follow these steps:
- Prepare the PBS solution: Add approximately 80 mL of 1X PBS into a glass beaker. Insert a magnetic stirrer and place the beaker on a stir plate inside a vented hood.
- Heat the solution: Begin stirring and heat the PBS solution to approximately 60°C. Avoid boiling, as this can release dangerous formaldehyde vapors.
- Add paraformaldehyde: Weigh 4 g of paraformaldehyde (ensure to follow proper safety protocols, as it is toxic) and add it to the heated PBS. The solution will appear milky at first, as the paraformaldehyde dissolves.
- Adjust the pH: Gradually add 1 N NaOH dropwise using a pipette to increase the pH until the solution becomes clear.
- Cool and adjust pH: Allow the solution to cool down. Once cooled, check the pH (it will likely be basic) and adjust it to 6.9 by adding small amounts of 1 N HCl.
- Adjust the volume: Bring the final volume of the solution to 1 L by adding 1X PBS.
- Filter the solution: To remove undissolved particles, filter the solution through Whatman No. 1 filter paper or a 0.45 μm membrane filter.
This formaldehyde solution can then be used for further experiments.
The solution can be aliquoted and stored at -20°C for up to a month. Avoid repeated freeze-thaw cycles to maintain the integrity of the solution.
Formaldehyde can easily be photo-oxidized in sunlight to carbon dioxide under atmospheric conditions.(A qualitative exploratory study on the effects of formalin on mortuary attendants – PMC (nih.gov).