WHEATON 2015 Lab Catalog

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Liquid Handling

> 131

How To Use a Pipette

Abstract

Pipettors are precision tools that, when used properly, will provide years of trouble-free

service. However, many users do not receive training to use this common laboratory

utensil.

Procedure

Socorex

Pipettors are precision tools that will help you efficiently perform your research

and testing tasks. Like any tools, your pipettor should be used and stored with care to

provide the best possible results.

The information below applies to Socorex Pipettors as well as other manufacturers'

pipettors. All air displacement pipettors work on the same principle. A plunger is

used to displace air- when the plunger is returned to its initial position (upper stop),

it creates a vacuum that is used to draw liquid into the disposable tip. The pipette

is calibrated such that the vacuum draws a volume of water as indicated on the

volume setting. Liquids lighter than water are pulled further into the tip, so these liquids

will pipette to a larger volume. Similarly, liquids heavier than water pipette to a lesser

volume. In the case of either lighter or heavier liquids, compared with water, the volume

dispensed will be different than that indicated on the pipettor. Pushing the plunger

pushes air, which then pushes on the liquid in the tip, which is dispensed.

It is important that you use approved tips with your pipettors to assure accuracy. Using

unapproved tips may lead to inconsistent results.

Normal Pipetting

1. Fit the tip, set the volume (variable pipettors only) and press down the plunger to the

first stop (metering stroke) with your thumb. Immerse the tip 2-3mm in the sample

while holding the pipettor vertically.

2. Slowly retract the pipetting plunger while watching the liquid fill the tip. You should

not observe bubbles or turbulence, which indicate gasses being pulled from the

liquid. These gasses affect the vacuum that draws the liquid, reducing the amount of

liquid aspirated into the pipet tip.

3. When the pipetting plunger has been retracted to its upper stop, remove your thumb

as the absence of pressure increases the precision of the pipettor. Slowly withdraw

the pipettor from the liquid. Wipe any drops on the outside of the tip on the wall of the

vessel you are drawing liquid from.

4. To dispense the liquid, hold the tip against the side of the receiving container at a

slight angle. Use your thumb to push down the pipetting plunger to the first stop, and

hold it for one second. After one second, push the button to the second stop. Pushing

to the second stop blows out any liquid left in the tip.

For work with volatile solvents, such as methanol, you may perform this procedure on

a “dummy” sample to saturate the system with vapor. This may improve accuracy, as

pre-saturating the air will reduce the tendency to “blow out” the liquid before you are

ready to dispense the sample. You should saturate the tip in this fashion every time you

change tips.

Reverse Pipetting

Reverse pipetting is used to aspirate an additional volume of liquid. This technique is

useful when working with thick, viscous liquids. This is also useful for volatile solvents.

1. Press the pipetting plunger with your thumb to the second stop. This is different from

the procedure listed for normal pipetting.

2. Holding the pipettor vertically, slowly retract the plunger to its upper stop. Wait for

the liquid to properly fill the tip. With viscous liquids, this will take longer than when

pipetting water. A larger amount of liquid will be aspirated than normal operation

since the plunger was pushed to the second stop.

3. When dispensing, push the plunger only as far as the first stop. Wipe any liquid

hanging on the tip on the side of the receiving container. Any remaining liquid will be

discarded with the tip.

Working Position

When aspirating the sample, the pipettor must be held vertically, or else too much liquid

will be drawn in. Tilting the pipettor by 30 degrees causes nearly 1% more liquid to be

drawn!

When dispensing the sample, the tip should be held at an angle against the container to

draw out the liquid in the tip. Under normal pipetting operations, analytical chemists will

recognize the pipettor as a “to contain” pipettor.

When aspirating the sample, the tip should generally be immersed to 2-3mm. Placing

the tip deeper into the sample allows pressure from the liquid to help push the sample

into the tip, reducing accuracy.

Working Conditions

Under ideal conditions, the sample should have the same temperature used to calibrate

the pipettor. Cold liquids are denser than warm liquids. If the pipettor was calibrated at

room temperature, but used in a cold-room, smaller samples than expected would be

dispensed.

Storage

Pipettors should be stored in an appropriate rack or stand. This reduces the risk of

scratching or damaging the nose cone. Damage to the nose cone could result in a poor

seal to the pipet tip, which will reduce accuracy.

Testing & Calibration

You should periodically check the operation of your pipettor by checking its calibration.

Socorex air-displacement pipettors are warranted for calibration for 2 years. Under

modern quality management such as GLP / GMP, ISO-9000 or regulatory requirements,

you should test your pipettors’ calibration. If a pipettor is dropped, or you suspect any

type of damage, you should check the calibration.

Testing is generally performed gravimetrically, using an analytical balance. Calibration

is usually done at room temperature, away from drafts or direct sunlight. The actual

calibration details vary between pipettors and are listed in the instructions.