8/13/2019 Pipettes Calibration Facts

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Pipettes Calibration Facts

On the surface, the process of calibrating a pipette appears to

be a simple procedure; however several important factors

can greatly influence the accuracy of a pipette. Among those

factors are: the calibration technique, balance resolution,

how the water density is determined , the

calibration/refurbishing procedure , and the parts replaced .

Calibration Technique

There are numerous ways to calibrate a pipette including

measuring of radio nuclide, color change reactions, air

pressure/vacuum testing, and gravimetric.

Gravimetric - In gravimetric calibration you are measuring the

weight of water a pipette dispenses at a given dial setting.

This weight must then be converted into a volume by using

the physical property of water density.

Volume ( ul ) = Weight of water dispensed ( mg ) / density of

water (mg/ml)

Gravimetric calibration is one of the most accurate ways to

calibrate a pipette and is the accepted standard for the

calibration of pipettes.

Radio nuclideThe principle behind radio nuclide calibration

is that if a fluid containing a known amount of radioactivity

per unit volume is used one can determine the volume of a

pipette by the equation:

Volume ( ul ) = ( Disintegrations per minute (dpm ) / counter

efficiency for the isotope ) / dpm/ml in the standard solution

Although this technique can be highly accurate the obvious

drawbacks of using radioactivity make it an impractical

calibration technique.

Color change reactionsIn color change calibrations, a

chemical reaction producing color is used to calibrate a

pipette. Using a test tube or microtiter plate, a known and

equal amount of one reactant is added to each well, then the

pipette to be calibrated is used to dispense the second

reactant into the well. Since chemical reactions are

dependant upon the concentration of each reactant, the

more reactant that is pipetted into the well the more intense

the color that will develop in that well. The color intensity is

then converted into a volume by using a standard curve of

color intensity versus volume of reactant.

Methods based on color change are secondary calibration

techniques, ie. you must transform data using a standard

curve, and as such can be inaccurate, inconsistent and highly

susceptible to error.

Air pressure/vacuumThis technique is not suited to

calibrate pipettes, in fact the technique is not quantitative

and only serves as a qualitative test for the condition of the

seal inside the pipette.

Any service using this technique is not calibrating your

pipette, they are merely testing if the pipette seal is worn or

not!

Since gravimetric calibration is the most widely used

standard, lets look into factors that can affect the accuracy of

the calibration.

Balance

BalanceTypically when calibrating a pipette you should use

a balance that has at least a four times higher resolution than

the quantity you are testing against. As an example if a P20 is

being calibrated at 2 ul where the accuracy tolerance is +/-

.012 ul, the balance used should have a resolution of at least

.03 mg. When a 4 to 1 resolution is not possible (either

because the technology does not exist or conditions prevent

measure to that level resolution), the resolution ratio should

be stated on any calibration document.

A company calibrating your pipettes should be using a 5 place

balance for pipettes whose maximum volume is 10 ul or

greater and a 6 or 7 place balance for pipettes less than 10 ul.

Water Density

Water density is dependant upon temperature and

barometric pressure. Because we are using the water density

to convert the weight of water dispensed by a pipette into

the volume of water dispensed by a pipette, it is extremely

important that the water density is determined properly and

accurately. Most services use air temperature instead ofwater temperature to determine the water density, which

will produce an incorrect value for the water density. The

reason for this is that typically the water being used to test

your pipette is in an open container, be it a beaker or trough.

As such, the water in the vessel is being cooled by the

evaporation of water into the air from the surface of the

water. This evaporative cooling typically produces water

temperatures that are 1 or more degrees centigrade cooler

than the air temperature.

A calibration service should measure the temperature of the

water being used to calibrate your pipette and the

barometric pressure at the instant that pipette is being

calibrated.

Calibration / Refurbishing procedures

Your pipette should always be calibrated to manufacturers

specifications and over the entire range it was designed to

operate. A Gilson P200 should be calibrated at its extremes of

50 and 200ul, and at the midpoint 100ul. Many services do

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not calibrate either to manufacturers specifications or over

the entire range the pipette was designed to operate.

A calibration service should always calibrate your pipettes to

manufacturers specifications and over its entire operational

range (minimum, maximum and midpoint design volumes)

unless the user specifies otherwise.

Typically the piston inside a pipette is made of highly polished

steel or ceramic. In either case, abrasives should never beused to clean these surfaces. Some companies use either

abrasives or steel wool to clean pistons both of which will

damage the piston of the pipette.

A calibration service should only use non-abrasive cleaning

methods to refurbish the internal components of your

pipette.

Misconception About Parts

Replacing parts on your pipette will not effect the pipettes

calibration. This statement is patently false; no two parts

produced will be identical, there will always be a variation in

a parts dimension. It is therefore extremely likely that

changing a part such as a seal, piston or shaft will alter the

calibration of your pipette.

A pipettes calibration should always be checked when a part

is replaced.

Pipettes should only be serviced if they are not working

correctly. Typically a pipette can appear to be functioning

properly and may in fact be in calibration even though the

piston may be contaminated with salts and other laboratory

chemicals.

As a rule a pipette should be serviced at least once a year to

minimize or prevent damage to internal components such as

pistons and springs.

http://www.pipettecal.com/facts.html

Most manufacturers stamp TC or TD near the top of the pipet

to alert the user as to the type of pipet. Like other TC-

designated labware, a TC pipet holds or contains a particular

volume but does not dispense that exact vol- ume,

sa bishop po yan meaning ng Tc

page 12

http://www.austincc.edu/mlt/ser/LaboratoryGlassware.ppt

Calibrating a Volumetric Pipet

Obtain some distilled water. Record the temperature of the

water.

Weigh a beaker and record the mass of the beaker. Then,

transfer some water to the beaker using the pipet.

Determine the mass of the water that was transferred.

Look up the density of water at the temperature recorded

above (a CRC Handbook of Chemistry and Physics will contain

an appropriate table) and use the relationship between the

density and the mass to determine the actual volume of

water that was delivered.

Repeat several times and calculate the average volume

delivered and the standard deviation.

Record the calibrated volume of the pipet in your notebook.

Discuss calibrated volume of the pipet with your instructor.

How many significant figures can be reported? Do you need

to repeat the calibration? Should you practice using the pipet

before repeating the calibration? What might cause the

standard deviation to be too large to be acceptable?

http://chem.wisc.edu/deptfiles/genchem/lab/labdocs/modu

es/pipetvol/volpipcal.htm

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