PIPETTE CALIBRATION

PIPETTE CALIBRATION
Pipette Calibration is a fundamental part of Good Laboratory Practice (GLP) and must be considered a vital part of any laboratory regime where precise volumes of fluid need transferring or diluting. Pipettes are a ubiquitous tool in laboratories and come in many shape and sizes, from many different manufacturers, but all perform the same function. This function is the transfer, through aspirate and dispense actions of precise amounts of fluid.

To look at all types of pipettes would entail many hundreds of thousands of words, so we have covered a number of the pipette types, the actions involved, the mechanics or electronics of standard units and the physics of pipette calibration.

There are many suppliers of manual pipettes and the vast majority are of the same design, with a thumb plunger action which depresses a plunger and a spring mechanism to return the piston.

There are some however that have slightly different designs such as those made by Brand or the Ovation pipette from Vista labs, that have unusual designs.



Inaccuracy & Imprecision: Prior to discussing pipette calibration it is important to understand accuracy and precision or to be more precise inaccuracy and imprecision.  Inaccuracy can be expressed as the deviation of the mean of a number of sample replicates from a set point volume and is expressed in either absolute units such as microliters, or relative units such as percent.  Imprecision is expressed as the standard deviation (STD) of the number sample replicates and is expressed as the coefficient of variation (CV) of samples volume replicates.  ISO standards recommend 10 readings.

Innaccuracy & Imprecision

 

This concept can be much more easily understood through use of an image. The first image shows the average is accurate (not precise), the second is precise (not accurate) and the third, both accurate and precise..

 

Nominal Volume: This is the greatest possible user-selectable volume which is specified by the manufacturer. For maintaining accuracy and precision even through repeated pipetting regimes, pipettes should be calibrated at periodic intervals.
 

PIPETTE FUNCTIONALITY
All of the air-displacement manual pipettes (90% of the pipette market) use the same principles for measuring and transferring accurate amounts of fluid. They operate by use of a piston mechanism which is activated by either the thumb or hand and which subsequently generates a vacuum by vertical travel of the metal or ceramic piston within an airtight shaft.  As the piston moves up, driven by the release of a plunger, a vacuum is created in the space left by the piston. This vacuum is then replaced by air. When a disposable tip is attached to the tip cone and immersed in liquid, the liquid is then drawn into the tip and subsequently dispensed by depression of the plunger.

 

Factors Affecting a Pipettes Performance

A pipette’s design and use is fundamental to the accuracy of a pipette and its continued precision.


 

 

 

  1. An operator should be trained in the correct use of a pipette and occasionally compared against standards to ensure continued good practise.

     

  2. Most users will be aware (if made conscious of the fact) that both air temperature and relative humidity can affect a pipettes performance as can the temperature of the sample (freshly thawed serum for example).

     

  3. In early pipettes heat transference from operator's hands caused small changes in the pipetting volumes. The heat was absorbed through the handle of the instrument and transferred to the components inside, the interaction between which caused these discrepancies. More modern pipettes have air spaces and less thermo conductive materials and the “heat Effect” is considered minimal.  If this is a concern however the best technique for maximum accuracy is to employ multiple pipettes and rotate them often in use.


     
  4. One of the most common causes of inaccuracy and mistakes is operator fatigue. Even the best trained and experienced operator will see a decrease in accuracy and precision as time increases.  Pipette calibration companies restrict the number of pipettes a technician can calibrate in a single day for this reason. In the last 20 years this effect has also become recognised by Health & Safety due to increasing numbers of Work Related Upper Limb Disorders (WURLD) often known as Repetitive Strain Injury an example of which is Carpal Tunnel Syndrome. Many pipettes have now reduced thumb forces needed to use a pipette and have decreased the weight of pipettes to ease a users discomfort when pipetting for long periods. Another solution is choosing an electronic pipette which significantly reduces hand fatigue. Once the operating button is touched the pipette always operates always the same way producing user independent accuracy and precision.


     
  5. Component integrity is critical to the performance of a pipette. With any mechanical device where several components interact in movement there is friction, “wear and tear”, fractures, weakness and loss of tolerance. This is no different in a pipette where the objective is to create a precise vacuum through use of a piston, mechanical travel, springs, and rubber and/or polymer seals.  Add to this the possible corrosive effects of fluid, poor pipette handling, damage to external components, volume adjustment problems and you can see the reasons for checking performance and calibration.


     

Maintaining Accuracy & Precision

For maintaining accuracy and precision even through repeated pipetting regimes, pipettes should be calibrated at periodic intervals. The interval at which a pipette needs calibrating depends on several factors:

 

  • The skill and training of the operators and the care with which the pipettes are used.

     
  • How intensively the pipettes are used.

     
  • The type of liquid dispensed by the pipettes.  Volatile liquids or corrosive liquids may emit vapours or come into contact with metal pistons, springs, seals and o-rings.

     
  • The accuracy and precision required by the pipette.  Pipette applications that require superior accuracy also demand more frequent calibration.

     
  • There is not absolute for calibration intervals but each institute or organisation should produce their own risk analysis/calibration requirement document. A pharmaceutic
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