Peptide Dilution Calculator

This calculator solves C₁V₁ = C₂V₂ for one dilution step. Enter the stock concentration, the working concentration and the final volume, and it returns the volume of stock to pipette, the volume of diluent to add and the dilution factor. Both concentrations are read in the unit selected (mg/mL, µg/mL, mM, µM or nM), so convert a mass-based stock to molar units first with the molarity calculator if the assay is designed in µM.

A dilution spreads the same amount of peptide through a larger volume, so the amount taken from the stock equals the amount in the final solution. Everything the calculator reports follows from that single conservation statement.

The formula

C₁ × V₁ = C₂ × V₂

C₁ is the stock concentration, V₁ the stock volume taken, C₂ the working concentration and V₂ the final volume. Rearranged:

V₂ is entered in millilitres; V₁ is returned in both mL and µL. Treating V₂ − V₁ as the diluent volume assumes volumes add, which holds closely for dilute aqueous solutions.

Worked example: a ten-fold series from a Semax stock

A 5 mg vial of Semax (Met-Glu-His-Phe-Pro-Gly-Pro, 813.92 g/mol) reconstituted in 2.0 mL gives 2.5 mg/mL, which is 3,071.6 µM. An in-vitro concentration–response experiment needing 10 µM down to 10 nM in 1 mL volumes runs like this, with every concentration entered in µM:

The last row is arithmetically correct and practically useless: no air-displacement pipette delivers 3 nL. The calculator returns whatever the equation gives, so check each V₁ against the working range of the pipette that will deliver it. Relative error is largest near the bottom of a pipette's range, and in a serial series each step inherits the error of the one before, which is why the early transfers deserve the most care.

Units and conventions

• One unit for both concentrations. The unit menu labels C₁ and C₂ but converts nothing. A mg/mL stock and a µM target must be brought to the same unit first; the molarity shortcut is mM = mg/mL ÷ MW × 1,000, so for a 1,000 g/mol peptide 1 mg/mL is 1 mM.
• Factor notation. "1 : 10" here means one volume of stock in ten volumes of final solution, not one part stock plus ten parts diluent.
• Prefixes move in steps of 1,000. A slipped mM/µM or µM/nM prefix is a thousand-fold error; an unexpected factor in the result is the quickest way to spot one.
• Vehicle carry-over. V₁ ÷ V₂ is the fraction of the final solution that came from the stock. A 1 : 1,000 dilution of a stock made in 100% DMSO leaves 0.1% DMSO; diluted serially in plain buffer, the solvent falls along with the peptide, so vehicle controls have to match each step. The solubility troubleshooting guide covers when a co-solvent is needed at all.

Correcting C₁ for net peptide content

The calculator trusts C₁. A stock calculated from the fill weight assumes the powder is entirely peptide, but lyophilised peptides also carry counter-ions and water, so the net peptide content (NPC) is below 100%. Correct the stock before entering it:

Corrected C₁ = nominal C₁ × NPC

At an NPC of 80%, the Semax stock above is 2,457.2 µM, and the 100 µM intermediate needs 40.7 µL rather than 32.6 µL. Because dilution factors are ratios, an uncorrected series stays internally consistent, but every concentration on it is labelled 25% higher than it really is, and so is any EC₅₀ read from the curve. For APL vials, request the batch documentation when an exact molar figure matters.

Where the arithmetic stops

C₁V₁ = C₂V₂ assumes no peptide is lost between tubes. In dilute solutions that assumption fails. The decapeptide cetrorelix adsorbed to vial surfaces up to a plateau of about 0.4 µg/cm², enough to make HPLC responses poor and variable at 0.2–4 µg/mL ^[1]. Recovery of eight radiolabelled endocrine peptides differed widely between glass and plastic tubes, and 1% bovine serum albumin improved it ^[2]. Surface capacity is roughly fixed per area, so the fraction lost rises as concentration falls: 1 mL of 10 nM Semax holds only about 8 ng of peptide. Make the lowest concentrations last, in low-binding tubes and, where the assay allows, in buffer with carrier protein. Keep the concentrated stock as frozen aliquots, prepared with the reconstitution calculator and the methods in the reconstitution guide.