Reconstitution arithmetic is simple, but it is easy to get wrong under time pressure, and the cost of getting it wrong is a stock solution at the wrong concentration for the life of the vial. This is the arithmetic only — it is not guidance on use.
The one equation
concentration (mg/mL) = vial mass (mg) ÷ diluent volume (mL)
A 10 mg vial reconstituted with 2 mL of bacteriostatic water gives 5 mg/mL. The same vial with 1 mL gives 10 mg/mL. Nothing else affects it: the lyophilised powder contributes negligible volume.
Working in micrograms
Most laboratory work is easier in µg/mL. The conversion is 1 mg/mL = 1000 µg/mL. A 5 mg vial in 5 mL is 1 mg/mL, or 1000 µg/mL, which means 0.1 mL contains 100 µg.
Serial dilution
To take a stock down to a working concentration, use C₁V₁ = C₂V₂. To make 10 mL at 50 µg/mL from a 1000 µg/mL stock:
V₁ = (50 × 10) ÷ 1000 = 0.5 mL of stock, brought to 10 mL total.
Note “brought to”, not “plus”. Adding 10 mL of diluent to 0.5 mL of stock gives 10.5 mL and a concentration that is 5% off.
The two mistakes
Injecting the diluent into the powder
Directing a stream of water straight onto a lyophilised cake causes foaming and can denature the material at the air–liquid interface. Angle the needle so the diluent runs down the inside wall of the vial, then let it dissolve without agitation. Swirl gently if needed. Do not shake and do not vortex.
Assuming the label mass is peptide mass
A lyophilised vial contains peptide plus residual water plus counter-ions. If the COA states a peptide content of, say, 82%, then a 10 mg vial holds about 8.2 mg of peptide. Where your work depends on peptide mass rather than powder mass, calculate from the COA figure, not the label.
Planning volume before you start
Choose the diluent volume for the concentration you actually need, because you cannot undo it. A general approach: decide the smallest volume you will need to measure accurately, then pick a concentration where that volume falls comfortably within your pipette's range. Measuring 0.02 mL accurately is harder than measuring 0.2 mL.
Storage after reconstitution
A reconstituted solution is far less stable than the lyophilised powder. Store at 2–8 °C, protect from light, and aliquot if the vial will be accessed repeatedly — each freeze–thaw cycle and each needle entry costs you material and adds contamination risk.
Aegis Bio supplies bacteriostatic water in 3 mL and 10 mL vials for reconstitution, and every compound page carries a reconstitution reference table showing resulting concentration at common diluent volumes.