What you are starting with
Lyophilised peptide is a freeze-dried cake or powder, often barely visible as a film at the bottom of the vial. A vial that looks empty usually is not; a few milligrams of material spread across the base of a glass vial is easy to miss and easy to lose by handling the vial carelessly before opening it.
Two properties matter before anything is added. The material is hygroscopic, so it draws moisture from the air the moment it is exposed. And it is under partial vacuum in many cases, which affects how solvent enters the vial.
Let the vial reach room temperature before opening it. Opening a cold vial causes condensation inside, which introduces exactly the water that lyophilisation was performed to remove.
Choosing a solvent
| Solvent | Properties | Typical use |
|---|---|---|
| Bacteriostatic water | Sterile water with a preservative, conventionally 0.9% benzyl alcohol | The standard choice where a vial will be entered more than once. The preservative inhibits microbial growth between entries. |
| Sterile water | No preservative | Single-entry work. Offers no protection once the vial has been opened. |
| Acetic acid, dilute | Lowers pH | Peptides that resist dissolving in neutral water, typically those with a basic isoelectric point. |
Solubility is a property of the specific sequence, not of peptides in general. Where a compound is known to be poorly soluble in water, that is usually stated on the supplier documentation, and the documentation is worth reading before opening anything.
Procedure
- Bring the vial to room temperature while sealed. Do not rush this with heat.
- Wipe both septa, the peptide vial and the solvent vial, with 70% isopropyl alcohol and allow them to dry.
- Draw the intended volume of solvent.
- Introduce the solvent slowly, letting it run down the inside wall of the vial rather than jetting directly onto the powder. Peptides are shear-sensitive and a direct stream can damage them.
- Leave the vial to stand. Most material dissolves on its own within a few minutes.
- If undissolved material remains, roll the vial gently between the palms or swirl it. Do not shake. Shaking generates foam, and the air-liquid interface in foam denatures peptides.
- Inspect against a light. The solution should be clear. Cloudiness, visible particulates or a persistent film indicate the material has not gone fully into solution or has been compromised.
- Label immediately with compound, concentration, date and source lot number.
Concentration arithmetic
Concentration is mass divided by volume. The arithmetic is trivial; the errors come from units and from assumptions about mass.
The calculation
Mass of peptide in the vial, divided by volume of solvent added, gives concentration.
10 mg into 2 mL = 5 mg/mL
10 mg into 1 mL = 10 mg/mL
5 mg into 2 mL = 2.5 mg/mL
The quantity of peptide does not change with the volume of solvent. Only the concentration does.
Where this goes wrong
- Mixing units. Milligrams against micrograms, or millilitres against the unit graduations on a syringe barrel. A U-100 syringe is graduated in insulin units, not millilitres, and 100 units correspond to 1 mL.
- Assuming label mass is peptide mass. A vial labelled 10 mg contains 10 mg of material, which includes counterion and residual water. Where a net peptide content figure is supplied, that is the figure the calculation should use.
- Reconstituting to a round number rather than a useful one. Choosing a volume that produces an awkward concentration guarantees arithmetic at every subsequent step.
Record the concentration on the vial at the moment of preparation. Reconstructing it later from memory is where most laboratory arithmetic errors actually originate.