Standard Operating Procedure (SOP) and Protocol for Operation and Calibration of Micropipette (fixed volume or variable volume micropipettes).
Operation and Calibration Procedure of Micropipette
1.0 Purpose:
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- To lay down the procedure for Operation and calibration of the Micropipette.
2.0 Scope:
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- This Standard Operating Procedure is applicable at microbiology section of Quality Control department for operation and calibration of micropipette.
3.0 References & Annexures :
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- References:
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- In House
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- Annexures:
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- Micropipette calibration record (Annexure- 1)
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- Micropipette calibration record for Fixed Volume (Annexure- 2)
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- Calibration Status Label (Annexure- 3)
4.0 Responsibilities – Micropipette Calibration and Operation:
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- Microbiologist / Executive – Quality control shall be responsible for the operation and calibration of the Micropipette.
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- Quality Control (QC) Head shall ensure proper control and compliance of the SOP for Operation and calibration of Micropipette.
5.0 Abbreviations and Definition of Terms :
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- Abbreviations :
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- CC : Change Control
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- CV : Coefficient Value
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- IPA : Iso Propyl Alcohol
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- NMT : Not more than
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- RSD : Relative standard deviation
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- UV : Ultra Violet
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- v/v : Volume by Volume
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- µL : Micro litter
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Definition of Terms :
- Accuracy (systematic error):
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- Accuracy is the difference between the dispensed volume and the selected volume of a pipette.
A = V -V0 A = accuracy
V = mean volume
V0 = nominal volume
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- Accuracy can be expressed as a relative value: A% = 100% x A / V0
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- Precision (random error):
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- Precision refers to the repeatability of the pipetting. It is expressed as standard deviation (s) or coefficient of variation (cv)
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- Conversion factor (Z):
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- The factor Z is for converting the weight of the water to volume at test temperature and pressure.
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- A typical value is 1.0041µl/mg at 25°C and 1067 mbar.
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- See the conversion Values for Z (µg/mg), as a function of temperature and atmospheric pressure, for purified water
Temp. | 800 mbar | 853 mbar | 907 mbar | 960 mbar | 1013 mbar | 1067 mbar |
19.0 °C | 1.0024 | 1.0025 | 1.0025 | 1.0026 | 1.0027 | 1.0027 |
19.5 °C | 1.0025 | 1.0026 | 1.0026 | 1.0027 | 1.0028 | 1.0028 |
20.0 °C | 1.0026 | 1.0027 | 1.0027 | 1.0028 | 1.0029 | 1.0029 |
20.5 °C | 1.0027 | 1.0028 | 1.0028 | 1.0029 | 1.003 | 1.003 |
21.0 °C | 1.0028 | 1.0029 | 1.003 | 1.003 | 1.0031 | 1.0031 |
21.5 °C | 1.003 | 1.003 | 1.0031 | 1.0031 | 1.0032 | 1.0032 |
22.0 °C | 1.0031 | 1.0031 | 1.0032 | 1.0032 | 1.0033 | 1.0033 |
22.5 °C | 1.0032 | 1.0032 | 1.0033 | 1.0033 | 1.0034 | 1.0035 |
23.0 °C | 1.0033 | 1.0033 | 1.0034 | 1.0035 | 1.0035 | 1.0036 |
23.5 °C | 1.0034 | 1.0035 | 1.0035 | 1.0036 | 1.0036 | 1.0037 |
24.0 °C | 1.0035 | 1.0036 | 1.0036 | 1.0037 | 1.0038 | 1.0038 |
24.5 °C | 1.0037 | 1.0037 | 1.0038 | 1.0038 | 1.0039 | 1.0039 |
25.0 °C | 1.0038 | 1.0038 | 1.0039 | 1.0039 | 1.004 | 1.0041 |
25.5 °C | 1.0039 | 1.004 | 1.004 | 1.0041 | 1.0041 | 1.0042 |
26.0 °C | 1.004 | 1.0041 | 1.0042 | 1.0042 | 1.0043 | 1.0043 |
6.0 Procedure for Operation and Calibration of Micropipette:
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Precaution during handling of Micropipette:
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- The tip should fit tightly on to the tip cone.
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- Clean the tip cone, if it gets clogged and the pipette does not pick up.
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- Do not pipette liquid without tip
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- Do not put down pipette with filled tips
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- If small bubbles collect in tips, the tip must be changed.
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- Always control the push button movements with the thumb for consistency.
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Operating procedure of Micropipette :
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- Forward Pipetting :
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- Press the operating button up to the first stop
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- Dip the tip attached to the pipette in to the solution to a depth of about 1 cm, and slowly release the operating button.
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- Wait for a while then withdraw it form liquid reservoir.
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- Dispense the liquid into the receiving vessel by gently pressing the operation button to the first stop.
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- After a second, press the operating button to the second stop.
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- This will empty the tip completely. Remove the pipette from the vessel.
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- Release the operating button to the ready position.
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- Reverse Pipetting :
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- Press the operating button to the second stop.
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- Dip the tip attached to the pipette in to the solution to a depth of about 1 cm, and slowly release the operating button.
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- This action will fill the tip with a volume that is larger than the set volume.
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- Wait 1-2 seconds and withdraw the tip from the liquid reservoir.
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- Dispense the liquid into the receiving vessel by pressing the operation button gently and steadily to the first stop.
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- This volume is equal to the set volume.
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- Hold the button in this position.
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- Some liquid will remain in the tip, which should not to be dispensed.
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- The liquid remaining in the tip can be pipetted back into the original solution or disposed together with the tip.
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- Release the operating button to the ready position
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- Reverse Pipetting technique is recommended for viscous solution, solution having tendency to foam.
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Calibration Procedure of Micropipette:
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- Take 100 mL of purified water in a clean beaker.
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- Place a beaker on balance and tare the weight.
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- Set the volume of micro pipette to required volume.
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- Fix the tip of desired volume into the micro pipette probe.
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- Take purified water and adjust it’s temperature to 25±1 ºC.
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- Pipette out the purified water using the micropipette and pour the volume to the Weighing bottle/beaker.
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- Remove the typographically error merge.
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- Wait till get a stable display on the screen and record the weight, repeat the same for 10 times.
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- Perform the calibration by using different set volumes of 100 µl, 500 µl and 1000 µl for 1000 µl pipette (Refer Table-1).
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- Calibration by using different set volumes of 10 µl, 50 µl and 100 µl for 100 µl pipette (Refer Table-2)
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- Calibration by using different set volumes of 500 µl, 2500 µl and 5000 µl for 5000 µl pipette (Refer Table-3)
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- Calculate the volume of purified water delivered by the Micro Pipette by each time by using the formula.
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Quantity of Purified water (in ml) =
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(Weight of Purified Water/D)
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- Where, D is the density of Purified Water at 25 ºC (i.e. 0.99602 g/cm3) or 0.9982 g/cm3 at 20 ºC
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- Conversion factor (Z) = 1.0040 µl/mg
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The percent variation can be calculate as formula given below
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% Variation =(100 x Actual weight – Observed weight)/D
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- Record the observation in the Annexure- 1.
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- Label on the Micropipette as per Annexure-3.
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- If any deviation observed during calibration of Micropipette, the analyst shall be inform to supervisor for necessary corrective action.
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- Frequency: Quarterly
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Acceptance Criteria
Table-1
Set Volume | Error | Tolerance Limit | |
Lower Limits | Upper Limits | ||
100 µL | ± 0.03 % | 99.9 µL | 100.1 µL |
500 µL | ± 0.03 % | 499.5 µL | 500.5 µL |
1000 µL | ± 0.03 % | 999.0 µL | 1001.0 µL |
RSD: NMT 1.0 % |
Table-2
Set Volume | Error | Tolerance Limit | |
Lower Limits | Upper Limits | ||
10 µL | ± 0.03 % | 09.99 µL | 10.01 µL |
50 µL | ± 0.03 % | 49.95 µL | 50.05 µL |
100 µL | ± 0.03 % | 99.9 µL | 100.1 µL |
RSD: NMT 1.0 % |
Table-3
Set Volume | Error | Tolerance Limit | |
Lower Limits | Upper Limits | ||
500 µL | ± 0.03 % | 499.5 µL | 500.5 µL |
2500 µL | ± 0.03 % | 2497.5 µL | 2502.5 µL |
5000 µL | ± 0.03 % | 4995.0 µL | 5005.0 µL |
RSD: NMT 1.0 % |
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Calculate the pipette volume as per following formula given below
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Volume = (Weight of Purified Water) / D
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Calibration of Fixed Volume Micropipettes
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- Attach a disposable tip and pre-rinse the tip.
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- Set the desired or lowest volume at which the pipette will be used.
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- Make 10 measurements of purified water, recording the weights.
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- Adjust for environmental factors e.g., Temperature NMT 25 ºC
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- Calculate the Accuracy and Precision (% CV) as per below formula-
Accuracy (%Error) = Mean – Set Volume x 100
Set Volume
Precision (% CV) =Standard Deviation x 100
Mean
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Performance Specification of Fixed Volume Pipettes
Volume Range | Accuracy Within ±% | At 100% of Pipette Volume Precision Within ±% |
100 µl | 0.80 | 0.20 |
50 µl | 0.60 | 0.30 |
Annexure- 1 – Micropipette calibration record
Micropipette Identification No. | Make | ||
Date of Calibration | Frequency | Quarterly | |
Next Due On | Balance Identification No. | ||
Balance Calibration On | Calibration due On |
Set Volume | ||||||
S. No. | Weight
(mg) |
Wt. of water x Z factor (1.004 µl/mg) | Weight
(mg) |
Wt. of water x Z factor (1.004 µl/mg) | Weight
(mg) |
Weight of water x Z factor (1.004 µl/mg) |
1. | ||||||
2. | ||||||
3….10 |
Average
SD RSD |
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Remarks: Micropipette Calibration Found OK/Not OK | ||||||
The RSD should be NMT 1.0% |
Annexure- 2 – Micropipette calibration record for Fixed Volume
Make | Model | ||
ID No. | Frequency | Quarterly | |
Serial No. | Liquid Type | ||
Balance Make | Balance ID | ||
Room Temperature | 25 ºC | Density of Liquid | 0.99602 |
Date of Calibration | Next Due Date | ||
Set Volume | Calibrated By |
TEST DATA
S.N. | Weight of water in mg | Actual volume dispensed
(Weight of water x Z factor (1.004 µl/mg) |
TEST RESULTS
Average Volume :
Standard Deviation :
(I) Accuracy (%Error) = [{(Mean – Set Volume) * 100}/Set Volume]
Accuracy (%Error) Spec. | |
Accuracy (%Error) Actual | |
Pass/Fail |
(II) Precision (% CV) = (Standard Deviation *100) / Mean
Accuracy (%Error) Spec. | |
Accuracy (%Error) Actual | |
Pass/Fail |
Remarks: Micropipette Calibration Found OK/Not OK.
Calibration Status Label (Annexure- 3)
CALIBRATION STATUS LABEL |
ID No.: |
Date of Calibration: |
Next Due Date: |
Calibrated By: |
Format No.: |
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