Reconstituting lyophilised peptides
Lyophilised material arrives as a dry cake or powder under partial vacuum. Freeze-drying removes the water that drives hydrolysis and deamidation, which is why the powder is stable for months while a solution of the same material is stable for days. Reconstitution is the point at which that protection ends, so it is worth doing deliberately.
Choosing a diluent
Three are common in laboratory work, and the choice is driven by how long the solution needs to last and how readily the material dissolves.
| Diluent | When it is used |
|---|---|
| Bacteriostatic water | Water containing 0.9% benzyl alcohol. The preservative suppresses microbial growth, so a solution intended to be drawn from more than once is normally prepared in this. |
| Sterile water for irrigation | No preservative. Appropriate where the benzyl alcohol would interfere with downstream assay work, but the solution should be treated as single-use. |
| Dilute acetic acid | For sequences that will not go into neutral water. Used at low concentration to bring poorly soluble material into solution before further dilution. |
Check solubility before committing a whole vial. Peptides rich in hydrophobic residues, and those with a net charge near zero at neutral pH, are the ones most likely to resist plain water.
Working out the volume
Concentration is simply the mass in the vial divided by the volume of diluent added. A vial containing 10 mg brought up with 2 mL of diluent gives 5 mg/mL; the same vial with 5 mL gives 2 mg/mL.
Work backwards from the volume you want to handle rather than picking a round number of millilitres. Very concentrated solutions make small volumes hard to measure accurately; very dilute ones waste vial capacity and increase the surface area available for adsorption. There is more detail, with worked examples, in concentration and dilution arithmetic.
One assumption worth stating: the powder itself contributes a negligible volume at these masses, so the volume of diluent added is treated as the final volume. That approximation stops holding at high mass, and if precision matters the solution should be made up to a marked volume rather than by adding a measured one.
Technique
- Let the vial reach room temperature before opening it. Diluent added to cold glass encourages condensation, and moisture is the thing the lyophilisation was protecting against.
- Wipe the stopper with an alcohol swab and let it dry.
- Add the diluent slowly, directing the stream down the inside wall of the vial rather than onto the cake. A jet fired straight at the powder shears it and drives foaming.
- Do not shake. Agitation denatures peptides at the air–liquid interface, and the foam it produces is material you have lost. Swirl gently, or leave the vial to stand.
- Allow full dissolution before use. Some material takes several minutes. Gentle warming in the hand is acceptable; heating is not.
- Inspect against a light. The solution should be clear and free of visible particulates.
Label it immediately
An unlabelled vial in a fridge is waste in progress. Record the compound, the concentration you just created, the diluent, the date and the lot number from the original vial. The lot number is what ties the solution back to its certificate of analysis, and once the outer packaging is gone it is unrecoverable.
Common errors
- Shaking to speed dissolution. The most common and the most costly.
- Assuming the vial contains what the label says to the milligram. Fill tolerances exist; the certificate of analysis is the authority on content.
- Reconstituting the whole vial when a fraction was needed. Once in solution the clock is running on all of it.
- Recording the volume added but not the resulting concentration. Trivial to compute on the day, easy to get wrong three weeks later.
- Reusing a needle or syringe between vials. Cross-contamination invalidates everything downstream.
Once in solution, storage becomes the constraint — see storage and stability.