For micropipette calibration, weigh dispensed water at set volumes, apply density correction, and adjust until results meet ISO 8655 limits.
Good pipetting lives and dies on calibration. This guide walks you through a gravimetric check that any trained lab worker can run with a balance, pure water, and time. You’ll set volumes, take repeat measurements, convert mass to volume, and compare your numbers with the standard tolerance band. If the device drifts, you’ll tweak, retest, and log the data so audits go smoothly.
Calibrating Micropipettes Step-By-Step (Gravimetric)
Below is a clear path from setup to sign-off. The method follows widely accepted gravimetric practice and aligns with tolerance criteria from the ISO family for piston-operated devices. The flow works for air-displacement and positive-displacement models alike.
Plan Your Test Points
Test three settings per channel: the top of the usable range (100%), a mid point (50%), and the low end (10% or the lowest settable value, whichever is higher). Take at least ten replicates at each setting. That sample size gives a stable coefficient of variation (CV) and a clean mean for the systematic error check.
ISO Tolerance Snapshot (Read Before You Start)
Your results will be judged against percentage limits. Use the table below as a quick reference when reviewing accuracy (systematic error) and repeatability (random error). Values reflect single-channel devices at the 100% setting.
| Volume Band (µL) | Max Systematic Error (±%) | Max Random Error (% CV) |
|---|---|---|
| 5 | 2.5 | 1.5 |
| >5–10 | 2.0 | 1.0 |
| >10–20 | 2.0 | 0.80 |
| >20–100 | 1.4 | 0.60 |
| >100–1,000 | 1.2 | 0.40 |
What You Need
- Analytical balance with 0.01 mg or 0.1 mg readability (match to volume range).
- Type-I or Type-II water, temperature-equilibrated near 20–21 °C.
- Evaporation trap or covered weighing vessel sized to your volumes.
- Clean tips matched to the model; fresh box for the run.
- Thermometer (0.1 °C), barometer, and hygrometer for ambient readings.
- Anti-vibration table, draft shield, and lint-free tissues.
- Calibration log sheet or LIMS template.
For background on converting mass to volume with pure water and why temperature control matters, see this NIST gravimetric procedure. For tolerance limits and test structure, refer to the current ISO 8655 part on pipettes.
Setup For Reliable Weighing
Stabilize The Room
Balance readings hate drafts and heat spikes. Close doors, steady the HVAC, and let the balance warm up per the manual. Keep the weigh cell clean and level. Aim for steady temperature in the low twenties and mid-range relative humidity. Record all three ambient numbers; they go in the report.
Condition The Device
Mount a new tip and pre-wet at least three times at the first test setting. Pre-wetting saturates the plastic and cuts tip-to-tip drift. Keep your hand off the barrel while weighing; body heat leads to slow creep in delivered volume. Hold the device upright during aspiration and keep immersion depth consistent across replicates.
Prepare Water And Vessels
Equilibrate water and the weighing cup to the same temperature. Use a tight-fitting lid or an evaporation trap for low volumes. Tare each empty cup with its lid. If droplets splash, stop and re-tare; outside moisture ruins a run.
Run The Weighing Series
- Pick the first setting (100%, then 50%, then low). Set blow-out and speed as you normally run the instrument.
- On each replicate: aspirate smoothly, touch off on the vessel wall, dispense, pause a beat, then push the second stop.
- Close the lid and weigh at once. Log mass to the balance’s last digit.
- Repeat until you have ten masses at the same setting. Swap tips every two to five dispenses for small volumes to limit carryover.
- Move to the next setting and repeat the full series.
Convert Mass To Volume And Score Results
From Grams To Microliters
Convert mass to volume with a density factor at your measured water temperature. A simple lab-grade approach uses:
Volume (µL) = Mass (mg) ÷ ρ (mg/µL)
At 20 °C, ρ for pure water is near 0.9982 mg/µL. If you run slightly warmer or cooler, update ρ using a trusted table or software. The reference above explains density use in gravimetry and why steady temperature keeps the math tight.
Calculate Accuracy And Precision
- Mean delivered volume at each setting: average of the ten converted volumes.
- Systematic error (%) = 100 × (mean − set volume) ÷ set volume.
- Random error (% CV) = 100 × (standard deviation ÷ mean).
Compare both numbers to the limits for your volume band and setting. Most labs use the same acceptance band for the 50% point and a wider band at the low point; if your SOP mirrors ISO, you’ll also check the 50% and 10% rows in the full table from the standard.
Worked Micro Example
Say the set point is 10 µL and your ten masses average 9.91 mg at 20 °C. Using ρ = 0.9982 mg/µL, the mean is 9.93 µL. The systematic error is −0.7%. If your SD is 0.05 µL, the CV is 0.5%. Both sit inside the limits for that band in the snapshot table, so the channel passes at this setting.
Adjust And Recheck
If accuracy is out at the top setting and the CV looks tight, adjust the device. Most models offer a dial under the cap or a set screw near the display. Make a small change, re-wet the tip, and repeat the 100% series. Once the top setting clears, check the lower points. Repeat adjustments in small steps; big jumps often overshoot.
When Precision Suffers
High CV with a mean near target points to technique or a hardware fault. Swap tips, slow the plunge, and control immersion depth. If CV stays high, inspect the seal, piston grease, and spring. A quick service with new seals and lube often restores repeatability.
Technique Notes That Save Runs
- Hand heat: Hold near the hook, not the barrel. Use a pipette stand between series.
- Immersion: Keep depth shallow and constant; deep dips trap extra liquid.
- Angle: Aspirate upright, then touch off at a slight angle on the vessel wall.
- Speed: Even, unhurried strokes. Snap moves trap air bubbles.
- Tip fit: Seat tips firmly with a slight twist to seal micro leaks.
Troubleshooting Patterns And Fixes
Use this map to hunt down drift, bias, or noisy replicates. Most issues track back to just a few habits or wear points.
| Symptom | Likely Cause | Quick Fix |
|---|---|---|
| Mean low at all settings | Under-delivery from mis-adjustment or leaks | Adjust at 100%, replace seal, retest |
| High CV, mean near target | Technique drift, poor tip seal, drafts | Standardize strokes, reseat tips, close shields |
| Low setting fails only | Evaporation loss, timing lag, wrong tip | Use trap, weigh fast, switch to low-retention tip |
| High at 100%, low at 10% | Non-linear adjustment | Center the mid point, set again in smaller steps |
| Air bubbles in tip | Fast aspiration or deep immersion | Slow draw, shallow depth, pre-wet |
Positive-Displacement Notes
These models move a plunger inside the tip. They shine with viscous or volatile liquids. Calibration uses the same gravimetric flow, but you’ll run the matching capillaries and plungers. Follow the device’s cleaning and re-use limits, and log which tip type you used on each run so the record matches the setup.
Pass/Fail Rules And Recordkeeping
For each setting, both the mean error and the CV must land inside the allowed band. Log the raw masses, converted volumes, temperature, pressure, humidity, tip lot, and any adjustment you made. Save the balance ID and the date of its last check. Your report should state the water temperature used for density conversion and the exact number of replicates per point.
How Often To Calibrate
Match the interval to risk: daily high-stakes assays get tighter cycles than general use. Many core labs run monthly checks for heavy use, quarterly for light use, and ad-hoc after repairs or drops. Keep a simple calendar that flags devices past due. A short monthly verification at one setting between full checks adds extra confidence.
Air-Displacement Sources Of Error To Watch
Several factors nudge results off target: barometric swings, temperature gaps between water and device, tip wetting, timing during blow-out, and wiping the tip on the vessel wall. The tolerance pages in the standard also list technique fixes, from pre-wetting to consistent handling depth. If your lab SOP calls for a wetting protocol, stick to it across every replicate in a series.
Service Or Replace?
If adjustment cannot pull the mean inside limits and parts show wear, send the unit for service. A pro service adds new seals, lube, leak checks, and a traceable report. If the body is cracked or the plunger is scarred, replacement is often cheaper than chasing drift across runs.
One-Page Checklist
- Warm up balance; level and clean.
- Record room temp, pressure, humidity.
- Use matched tips; pre-wet three times.
- Weigh covered vessels; re-tare when needed.
- Run 10 replicates at 100%, 50%, and low.
- Convert with water density at measured temp.
- Check % error and % CV against the band for your volume.
- Adjust in small steps; recheck top, mid, then low.
- Log raw data, settings, tip lot, and any changes.
Wrap-Up: Reliable Pipetting Starts Here
This process gives you numbers you can trust. A steady setup, clean strokes, and clear math will keep daily pipetting on track. With a short monthly verification and a full run on your schedule, you’ll catch drift early and keep reports clean for audits and method runs alike.