💉 Micropipette Verification
PKKK/200/044 — Verifikasi Mikropipet (ISO 8655-2:2022)
Gravimetric verification procedure for single and multichannel micropipettes. Ensures volumetric accuracy and precision per ISO 8655.
◈ Key Parameters
3 Mo
Frequency
10
Measurements
20±3°C
Room Temp
45-80%
Rel. Humidity
◈ Test Volumes
- 📏 Nominal volume — maximum range of the micropipette
- 📏 50% of nominal volume
- 📏 10% of nominal volume or lower limit (whichever is larger)
- 📏 Any additional volumes specified by the laboratory
◈ 1. Balance Resolution Requirements (ISO 8655-6)
The analytical balance used must meet the minimum resolution requirements strictly based on the nominal volume of the micropipette under test.
| Selected Volume ($V$) | Resolution (mg) | Repeatability & Linearity (mg) | Standard Uncertainty (mg) |
|---|---|---|---|
| $10 \, \mu\text{L} \le V \le 100 \, \mu\text{L}$ | 0.01 | 0.025 | 0.05 |
| $100 \, \mu\text{L} < V \le 1000 \, \mu\text{L}$ | 0.1 | 0.2 | 0.4 |
| $1 \, \text{mL} < V \le 10 \, \text{mL}$ | 1.0 | 2.0 | 4.0 |
◈ 2. Immersion Depth & Wait Times
| Volume Range ($\mu\text{L}$) | Immersion Depth (mm) | Wait Time (seconds) |
|---|---|---|
| $1$ to $2$ | 1 | 1 |
| $> 1$ to $100$ | 2 to 3 | 1 |
| $> 100$ to $1000$ | 2 to 4 | 1 |
| $> 1000$ to $20000$ | 3 to 6 | 3 |
Pre-wet tip 3-5 times before measurement
Aspirate vertically
Dispense at 30°–45° against wall
◈ 3. Gravimetric Procedure
- Record room conditions from calibrated thermohygrometer. Must be $T = 20 \pm 3^{\circ}\text{C}$ (max variation $\pm 0.5^{\circ}\text{C}$), $RH = 45–80\%$.
- Record deionized water temperature ($T_w$). It must be within $\pm 0.5^{\circ}\text{C}$ of room temperature.
- Determine Z-factor corresponding to the water temperature (assumes air pressure approx. $1013 \, \text{hPa}$).
- Select appropriate balance (Tare a suitable receiving vessel with a small amount of water to humidify the vessel).
- Attach a new tip and pre-wet the tip by aspirating and dispensing the nominal volume multiple times to saturate the dead air volume.
- Hold micropipette vertically. Immerse tip to correct depth. Aspirate steadily. Wait the required time (1–3s).
- Withdraw tip and dispense into vessel at a 30°–45° angle against the inner wall. Drag the tip upwards slightly along the wall to remove any remaining droplet.
- Record the mass ($M$). Tare the balance.
- Repeat for a total of 10 consecutive measurements ($M_1$ to $M_{10}$).
◈ 4. Mass Loss (Evaporation Correction)
For test volumes $< 50 \, \mu\text{L}$, evaporation during the weighing cycle significantly affects accuracy. A Mass Loss ($ML$) correction must be applied.
- Start a timer at the beginning of the 10 weighings. Record the total time $T_{cycle}$ (in seconds) taken to complete all 10 weighings.
- After the 10th reading ($M_{10}$) is recorded, leave the vessel on the balance and wait for exactly $T_{cycle}$ seconds.
- Record the new balance reading ($M_{11}$).
- Calculate Total Mass Loss during the cycle: $\Delta M = M_{10} - M_{11}$.
- Calculate average Mass Loss per measurement: $ML = (M_{10} - M_{11}) / 10$.
- Add $ML$ to each recorded mass ($M_i$) before applying the Z-factor.
◈ 5. Final Calculations
Corrected Mass ($M_i^*$) = $M_i + ML$
Volume ($V_i$) = $M_i^* \times Z$
Mean Volume ($\overline{V}$) = $\frac{1}{10} \sum_{i=1}^{10} V_i$
Accuracy ($A$) = $100 \times \frac{\overline{V} - V_s}{V_s}$ %
CV = $100 \times \frac{S_r}{\overline{V}}$ %
Volume ($V_i$) = $M_i^* \times Z$
Mean Volume ($\overline{V}$) = $\frac{1}{10} \sum_{i=1}^{10} V_i$
Accuracy ($A$) = $100 \times \frac{\overline{V} - V_s}{V_s}$ %
CV = $100 \times \frac{S_r}{\overline{V}}$ %
- $V_s$: Selected Test Volume
- $S_r$: Standard Deviation of the 10 calculated volumes ($V_i$)
- PASS Criterion: $A_{found} \le A_{max}$ AND $CV_{found} \le CV_{max}$
◈ Max Permissible Errors — Single Channel
| Nominal (µL) | Volume % | Accuracy A (%) | CV (%) |
|---|---|---|---|
| 1–3 | 100 | 2.5 | 2.0 |
| 1–3 | 50 | 5.0 | 4.0 |
| >3–5 | 100 | 2.5 | 1.5 |
| >5–10 | 100 | 1.2 | 0.8 |
| >10–50 | 100 | 1.0 | 0.5 |
| >50–5000 | 100 | 0.80 | 0.30 |
| >50–5000 | 50 | 1.6 | 0.60 |
| >50–5000 | 10 | 8.0 | 3.0 |
| >5000–20000 | 100 | 0.60 | 0.30 |
◈ Max Permissible Errors — Multichannel
| Nominal (µL) | Volume % | Accuracy A (%) | CV (%) |
|---|---|---|---|
| 2 | 100 | 8.0 | 8.0 |
| >2–5 | 100 | 5.0 | 3.0 |
| >5–10 | 100 | 2.4 | 1.6 |
| >10–20 | 100 | 2.0 | 1.0 |
| >20–50 | 100 | 2.0 | 0.80 |
| >50–2000 | 100 | 1.6 | 0.60 |
◈ Z-Factor Table (Selected)
| Temp (°C) | Z (mL/g) | Temp (°C) | Z (mL/g) |
|---|---|---|---|
| 15 | 1.0020 | 23 | 1.0035 |
| 17 | 1.0023 | 24 | 1.0038 |
| 18 | 1.0025 | 25 | 1.0040 |
| 19 | 1.0027 | 26 | 1.0043 |
| 20 | 1.0029 | 27 | 1.0045 |
| 21 | 1.0031 | 28 | 1.0048 |
| 22 | 1.0033 | 30 | 1.0054 |
◈ Corrective Action
- ⚠️ If FAIL: Switch to Analyst 2 → repeat the verification
- ❌ If Analyst 2 also fails → micropipette is considered defective, remove from service
- ✅ If Analyst 2 passes → send to Analyst 3 for confirmation
- ✅ If Analyst 3 passes → verification is confirmed PASS
- ❌ If Analyst 3 fails → micropipette is considered defective
◈ Forms & References
- 📋 PKKK/044A — Single Channel Verification Form
- 📋 PKKK/044B — Single Channel (Alternate) Form
- 📋 PKKK/044C — Multichannel Verification Form
- 📋 PKKK/200/044 — Quality Procedure: Verifikasi Mikropipet
- 📖 ISO 8655-2:2022 — Piston-operated volumetric apparatus — Part 2: Pipettes
- 📖 ISO 8655-6:2022 — Part 6: Gravimetric reference measurement