Dosage Calculation Principles for In Vitro Research
Understanding concentration, dilution, and measurement accuracy.
Concentration errors are silent killers of experimental reproducibility. An off-by-two error in a dilution series produces data that looks plausible but is entirely wrong. This guide covers the fundamentals every researcher should know.
Key Units and Conversions
Peptide concentrations are expressed in multiple units depending on context. Knowing how to convert between them is foundational:
- mg/mL (mass concentration): most common for stock solutions
- µg/mL (micrograms per mL): common for working solutions
- nM / µM / mM (molar concentration): used when biological activity is molar-dependent
- To convert: molarity (µM) = (mg/mL × 1000) / molecular weight (Da)
The C1V1 = C2V2 Formula
The dilution formula is the most frequently used calculation in research bench work. C1 is your starting concentration, V1 is the volume you need to take from it, C2 is your target concentration, and V2 is the final total volume.
Example: You have a 1 mg/mL stock and need 200 µL at 100 µg/mL. (1000 µg/mL)(V1) = (100 µg/mL)(200 µL) → V1 = 20 µL of stock, make up to 200 µL total with diluent.
Always double-check dilution calculations by working backwards: confirm that C2 × V2 = C1 × V1 before pipetting.
Serial Dilutions
For dose-response experiments, serial dilutions are more accurate than individual dilutions for each concentration. Each step uses the previous concentration as the source, maintaining equal dilution factor across the series.
A 1:3 serial dilution from 1000 nM produces: 1000 → 333 → 111 → 37 → 12.3 → 4.1 nM. To set this up, take 33 µL from each well and add to 67 µL of diluent in the next well (100 µL final volume).
Accuracy vs. Precision in Pipetting
Inaccurate pipetting compounds across a dilution series. A 5% error at each of three dilution steps produces a cumulative 15%+ error in final concentration. Best practices:
- Calibrate pipettes every 6 months — or verify with gravimetric analysis
- Pre-wet tips: aspirate and dispense once before your actual transfer
- Pipette slowly to prevent bubble formation
- Use the correct pipette range (avoid pipetting 2 µL with a P200)
- Avoid aspirating to the very bottom of a vial — this introduces air
Accounting for Purity in Calculations
Peptide purity (listed on the COA as % purity by HPLC) directly affects actual active compound concentration. If you weigh out 1 mg of a peptide with 95% purity, you have 0.95 mg of active compound. Adjust stock concentrations accordingly: effective concentration = (nominal concentration × purity%) / 100.