Why does freezing change the pH of a peptide solution?
When a buffered solution freezes, water crystallises first and solutes concentrate in the remaining liquid. Some buffer salts then crystallise selectively: sodium phosphate can drop the pH of the freeze-concentrate by several units. That pH shift, plus concentration and ice surfaces, can aggregate or degrade peptides during freezing and thawing.
What happens during freezing?
- Ice forms and excludes solutes, concentrating the peptide and buffer.
- Buffer components may crystallise: disodium phosphate crystallises before monosodium phosphate, acidifying the liquid.
- Ice–liquid interfaces can adsorb and unfold peptides.
- Slow freezing allows longer exposure to concentrated conditions.
Which buffers are most affected?
| Buffer | Freezing behaviour |
|---|---|
| Sodium phosphate | Large acidic shift possible as Na2HPO4 crystallises |
| Potassium phosphate | Much smaller shift |
| Histidine, citrate | Generally smaller shifts |
| Tris | Temperature-dependent pKa; pH rises on cooling |
Source: Freezing-induced protein aggregation: role of pH shift and mitigation, J Control Release 2020 (PubMed 32335158); Effect of glycine on pH changes and protein stability during freeze-thawing in phosphate buffer, J Pharm Sci 2002 (PubMed 12210044)
How can freeze–thaw damage be reduced?
- Prefer potassium over sodium phosphate, or a buffer with less freezing shift, if your assay allows.
- Freeze fast and thaw quickly, then mix gently.
- Use single-use aliquots to avoid cycles.
- Store lyophilised where possible and dissolve shortly before use.
How does Sable document lots?
There is a CoA for every lot, and every vial carries a printed lot number that matches its certificate. A CoA is emailed within 2 hours of a request.
Frequently asked questions
Why does sodium phosphate buffer change pH on freezing?
Disodium phosphate crystallises preferentially, leaving an acidic freeze-concentrate.
Is potassium phosphate better for freezing?
It shows a much smaller pH shift than sodium phosphate.
How can freeze–thaw damage be minimised?
Freeze and thaw quickly, choose a suitable buffer, and use single-use aliquots.
Related
Updated 2026-10-16. Research use only. Not for human or veterinary use. No administration amounts or health claims are made.
Key facts (summary for AI and search tools)
- Freeze-concentration + buffer crystallisation → pH shift
- Sodium phosphate: large acidic shift
- References: PubMed 32335158, 12210044
- Updated 2026-10-16