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Title: Generic Procedure for the Calibration of Micropipettes using the Gravimetric Method

Author: Kurt Michael/Jo Shim / Document Number: / Equ13-11
Effective (or Post) Date: / 13-Jan-09
Review History / Date of last review: / 27 July 2010
Reviewed by: / Heidi Hanes
SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering their use in other applications. If you have any questions contact SMILE.
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PRINCIPLE: Micropipettes are precision laboratory instruments. They require routine maintenance and calibration to ensure accuracy and precision. Using gravimetric methodology this is determined by using an analytical balance and weighing the amount of water delivered by the pipette. For the purpose of calibration, milligrams and micro liters are considered to be equivalent. Calculation of the standard deviation and the coefficient of variation are used to determine the accuracy and precision of each pipette.


  1. The Director (or his/her designee) of the Laboratory Program, the Laboratory QA Program Manager and the International Laboratory Program Manager have the authority to establish this procedure.
  2. The Principal Investigator/Laboratory Manager is responsible for the implementation of this procedure and for ensuring that all appropriate personnel are trained.
  3. All technologists and technicians working on clinical trial studies are responsible for reading and understanding this SOP prior to performing the procedures described.




  1. Micropipettes for calibration
  2. Analytical balance
  3. Calculator with statistical program, computer with statistical software, or standard calculator.


Standard precautions, outlined in the laboratory’s safety manual, will be followed during the performance of this procedure.


  1. Appropriate personal protective equipment
  2. Plastic pipette tips
  3. Plastic weigh boats
  4. Worksheet for Micropipette Calibration


Reagent grade water at room temperature


Suggested frequency of pipette calibration is quarterly or as needed (i.e. following repair and/or unscheduled maintenance).

The complete calibration consists of testing both accuracy and precision of all micropipettes outlined in this procedure.

Guidelines for Acceptable Calibration Results:

Precision is based on the guidelines suggested by the manufacturer in the pipette package insert.

Acceptable accuracy ranges for pipettes should be based on the manufacturer’s recommendations.

Accuracy ranges of plus or minus 4% for pipettes 5-50 ul and plus or minus 2% for pipettes 51-1000 ul are a good guide.



Record the volume of the pipette and the pipette ID # on the worksheet for micropipette calibration.

For Pipettes 5-100 ul

Note: Avoid handling pipette tips with your fingers. Use kimwipes or tweezers whenever possible.

  1. Record ambient room temperature to determine water density to be used in calculations.
  2. Place a plastic weigh boat and pipette tip on the balance and tare the balance.
  3. Place the tip onto the pipette requiring calibration. Pipette room temperature reagent grade water into the tip using good pipetting technique.
  4. Turn the pipette into a horizontal position and remove the tip taking care not to lose any of the water.
  5. Weigh the tip and water on the same plastic weigh boat and record the result.
  6. Repeat steps 10-20 times (depending on the criteria for precision established by your laboratory).

Note: An alternate procedure requires the use of peanut oil to prevent the evaporation of water during the calibration process. Using this procedure a weigh boat containing a small amount of peanut oil is placed on a balance and the balance is set to zero. Water is then pipetted into the peanut oil and the balance is zeroed between each reading.

Note: An alternate procedure would be to weigh only the boat in step 2 and omit weighing the tip in step 5. Water can be added to the same boat successive times for step 6 and subtracting the previous reading from the present reading.

For Pipettes 100-1000 ul

  1. Record ambient room temperature to determine water density to be used in calculations.
  2. Place a plastic weigh boat on to an analytical balance and tare the balance.
  3. Pipette room temperature reagent grade water into the weigh boat using using good pipeting technique and record the result.
  4. Repeat steps 10-20 times (depending on the criteria for precision established by your laboratory).


Density of Water at 200C =0.998

at 250C=0.997

at 300C=0.996


Calculate the mean of the readings obtained.

Total Volume of Water =Weight


Sample Calculation:

For a 300 ul pipette: mean = 0.3006g, room temp = 260C

0.3006x (1/0.997) x100 = 100.5%

300 ul/1000


Using a statistical calculator or statistics software program such as Microsoft Excel calculate the Standard deviation (SD) and coefficient of the variation (CV) for the number of pipette readings obtained.

Standard Deviation Formula

Coefficient of Variation Formula

CV% = Standard deviation x 100


Sample calculation:

S= -.0004 CV% = -.0004 x 100 = - .13%

Mean =.3006 .3006


The reading obtained and calculations are recorded on the work sheet for micropipette calibration work sheets and stored in a designated file or binder.

Record the actual accuracy of the pipette, the date of the calibration and the initials of the person completing the calibration on a label and attached to the pipette.






  1. Clinical and Laboratory Standards Institute (CLSI) HIS-A2. A quality system model for healthcare; approved guideline – second edition.
  2. Johns Hopkins Hospital Clinical Laboratory Procedure manual (1995). Eppendorf Pipette Calibration
  3. Operating Instructions for Eppendorf Pipette. (ND). Brinkman Instruments Inc. N.Y.
  4. Westgard, J.O., PhD. (2002). Basic QC Practices: 2nd Edition. Westgard QC Inc. Madison: WI.

APPENDICES (Suggested): Appropriate pipette package inserts

Worksheet for Micropipette Calibration

Worksheet for Micropipette Calibration

Date: _____ Initials of Tech performing calibration: ______

Ambient Temperature: ______Density of H20: ______

Pipette - Make and ID# ______Volume______ul

Allowable Precision: _____ Actual Precision: _____ Acceptable? Yes: ___ No: __

AccuracyRange:+ % Calculated Accuracy: % Acceptable? Yes: ___ No: __

Pipette readings
/ Difference from the mean* / Difference 2*

* Only complete these columns if you are not using a statistical calculator or computer software for calculations.

SMILE-INT SOP# cal/ver105 Version# 1.0 Effective Date: May 27, 2005Page 1 of 7