Micropipette Calibration and Operation Procedure

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:

    • To lay down the procedure for Operation and calibration of the Micropipette.

2.0   Scope:

    • This Standard Operating Procedure is applicable at microbiology section of Quality Control department for operation and calibration of micropipette.

3.0   References & Annexures :

    • References:
    • In House
    • Annexures:
    • Micropipette calibration record (Annexure- 1)
    • Micropipette calibration record for Fixed Volume (Annexure- 2)
    • Calibration Status Label (Annexure- 3)

4.0   Responsibilities – Micropipette Calibration and Operation:

    • Microbiologist / Executive – Quality control shall be responsible for the operation and calibration of the Micropipette.
    • 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 :

    • Abbreviations :
    • CV :   Coefficient Value
    • IPA  :   Iso Propyl Alcohol
    • NMT :   Not more thanMicropipette
    • RSD :   Relative standard deviation
    • UV :   Ultra Violet
    • v/v :   Volume by Volume 
    • µL :    Micro litter
    • Definition of Terms :

    • Accuracy (systematic error):
    • 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

    • Accuracy can be expressed as a relative value: A% = 100% x A / V0
    • Precision (random error):
    • Precision refers to the repeatability of the pipetting. It is expressed as standard deviation (s) or coefficient of variation (cv)
    • Conversion factor (Z):
    • The factor Z is for converting the weight of the water to volume at test temperature and pressure.
    • A typical value is 1.0041µl/mg at 25°C and 1067 mbar.
    • 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:

    • Precaution during handling of Micropipette:

    • The tip should fit tightly on to the tip cone.
    • Clean the tip cone, if it gets clogged and the pipette does not pick up.
    • Do not pipette liquid without tip
    • Do not put down pipette with filled tips
    • If small bubbles collect in tips, the tip must be changed.
    • Always control the push button movements with the thumb for consistency.
    • Operating procedure of Micropipette :

    • Forward Pipetting :
    • Press the operating button up to the first stop
    • Dip the tip attached to the pipette in to the solution to a depth of about 1 cm, and slowly release the operating button.
    • Wait for a while then withdraw it form liquid reservoir.
    • Dispense the liquid into the receiving vessel by gently pressing the operation button to the first stop.
    • After a second, press the operating button to the second stop.
    • This will empty the tip completely. Remove the pipette from the vessel.
    • Release the operating button to the ready position.
    • Reverse Pipetting :
    • Press the operating button to the second stop.
    • Dip the tip attached to the pipette in to the solution to a depth of about 1 cm, and slowly release the operating button.
    • This action will fill the tip with a volume that is larger than the set volume.
    • Wait 1-2 seconds and withdraw the tip from the liquid reservoir.
    • Dispense the liquid into the receiving vessel by pressing the operation button gently and steadily to the first stop.
    • This volume is equal to the set volume.
    • Hold the button in this position.
    • Some liquid will remain in the tip, which should not to be dispensed.
    • The liquid remaining in the tip can be pipetted back into the original solution or disposed together with the tip.
    • Release the operating button to the ready position
    • Reverse Pipetting technique is recommended for viscous solution, solution having tendency to foam.
  • Calibration Procedure of Micropipette:

    • Take 100 mL of purified water in a clean beaker.
    • Place a beaker on balance and tare the weight.
    • Set the volume of micro pipette to required volume.
    • Fix the tip of desired volume into the micro pipette probe.
    • Take purified water and adjust it’s temperature to 25±1 ºC.
    • Pipette out the purified water using the micropipette and pour the volume to the Weighing bottle/beaker.
    • Remove the typographically error merge.
    • Wait till get a stable display on the screen and record the weight, repeat the same for 10 times.
    • Perform the calibration by using different set volumes of 100 µl, 500 µl and 1000 µl for 1000 µl pipette (Refer Table-1).
    • Calibration by using different set volumes of 10 µl, 50 µl and 100 µl for 100 µl pipette (Refer Table-2)
    • Calibration by using different set volumes of 500 µl, 2500 µl and 5000 µl for 5000 µl pipette (Refer Table-3)
    • Calculate the volume of purified water delivered by the Micro Pipette by each time by using the formula.
    • Quantity of Purified water (in ml) =

(Weight of Purified Water/D)

    • 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
    • Conversion factor (Z) = 1.0040 µl/mg
    • The percent variation can be calculate as formula given below

% Variation  =(100 x Actual weight – Observed weight)/D

    • Record the observation in the Annexure- 1.
    • Label on the Micropipette as per Annexure-3.
    • If any deviation observed during calibration of Micropipette, the analyst shall be inform to supervisor for necessary corrective action.
    • Frequency: Quarterly
  • 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 %
    • Calculate the pipette volume as per following formula given below

Volume = (Weight of Purified Water) / D

  • Calibration of Fixed Volume Micropipettes

    • Attach a disposable tip and pre-rinse the tip.
    • Set the desired or lowest volume at which the pipette will be used.
    • Make 10 measurements of purified water, recording the weights.
    • Adjust for environmental factors e.g., Temperature NMT 25 ºC
    • 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

  • 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

 
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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Janki Singh is experienced in Pharmaceuticals, author and founder of Pharma Beginners, an ultimate pharmaceutical blogging platform. Email: [email protected]

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