HPLC dilution and injection calculator

Three calculations that decide whether an injection is informative: how much stock goes into the flask, how much peptide reaches the column, and what the dilution series actually contains. Arithmetic only — no protocol, no method recommendation.

The arithmetic

• Dilution: C₁V₁ = C₂V₂, so stock volume = (target concentration × final volume) ÷ stock concentration. Diluent volume = final volume − stock volume.
• Injection mass: concentration in µg/mL equals ng/µL, so mass on column (ng) = concentration (µg/mL) × injection volume (µL).
• Moles: pmol = ng × 1000 ÷ molecular weight in g/mol.
• Serial dilution: each tube is the previous concentration divided by the dilution factor; with a fixed volume per tube, the volume transferred at each step is tube volume ÷ dilution factor.

Worked with the defaults: 2 mg/mL = 2000 µg/mL; 100 × 10 ÷ 2000 gives 0.5 mL (500 µL) of stock in a 10 mL flask, a 20-fold dilution. At 100 µg/mL and a 10 µL injection, 1,000 ng reaches the column. A 1,000 µg/mL top standard diluted 2-fold over six points gives 1000, 500, 250, 125, 62.5 and 31.25 µg/mL, transferring 0.5 mL into 0.5 mL of diluent at each step.

What the calculator does not know

• Whether the stock concentration is real. A nominal concentration computed from fill weight ignores peptide content, counter-ion and water. The inputs must come from a measured or corrected value — see net peptide content.
• Column capacity and void volume. 1,000 ng on a 2.1 mm column is a very different load from 1,000 ng on a 4.6 mm column. The number is a mass, not a suitability judgement.
• Sample solvent strength. Injecting in a solvent stronger than the mobile phase distorts early peaks regardless of how accurately the dilution was made.
• Adsorption losses. At low concentrations, a meaningful fraction of peptide can bind to the flask, the vial or the needle — see adsorption to labware.
• Detector linearity. The top standard must sit inside the validated range of the method, not merely inside the detector's nominal maximum.
• Volumetric glassware accuracy. A 10 mL flask and a 100 µL pipette have tolerances; at low volumes they dominate the error budget.

Practical notes on making the dilution

• Bring the stock and the diluent to the same temperature before pipetting; thermal expansion of a small volume is not negligible relative to a 100 µL transfer.
• Rinse the pipette tip with the stock before transferring, especially for a solution containing organic solvent.
• Add the stock into the diluent, not the reverse, and mix by inversion rather than vortexing if the peptide is prone to aggregation.
• Prepare the working solution fresh where the method allows it; a dilute solution has more surface area per unit mass and less stability than the stock.
• Record the actual values — stock lot, measured concentration, pipette volumes, diluent composition — so that a later result can be reconstructed.

Related reading & products

• Reading an HPLC chromatogram — peak shape, integration and area percent
• Method validation — why the range and LOQ matter before the injection
• Measuring concentration — verifying the stock before diluting it
• Adsorption to labware — the low-concentration loss
• Reconstitution calculator — vial to stock solution
• All tools
• All knowledge guides