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PROFESSOR: Many laboratory procedures,
especially biological and biochemical ones,
require the precise transfer of small quantities of liquids.
Glass pipettes, which are commonly
used in chemistry labs, are simply not
accurate when working with volumes that
are less than 1 milliliter.
The best method of transferring small volumes of liquids
involves the use of an automatic pipette.
An automatic pipette consists of a piston
that displaces air to create a vacuum when
its plunger is pressed.
The tip of the automatic pipette is then
placed into the sample vial and the plunger is slowly released.
This action draws liquid into the tip.
Finally, this liquid is expelled into the desired container
by slowly depressing the plunger a second time.
As shown, a variety of automatic pipettes exist.
This video will demonstrate automatic pipetting technique
using the Pipetman.
Using good technique will ensure accuracy,
which is defined as the closeness
of the dispensed volume to the set volume,
and precision, which is defined as the reproducibility
of individual measurements between your samples.
A Pipetman consists of a plunger button, a tip ejector button,
a volume adjustment knob, a volume indicator,
and a plastic shaft.
Pipetman are available in six different sizes.
Five of them are depicted here.
The number on the plunger button indicates the maximum volume
in microliters that each Pipetman
is capable of transferring.
For example, the P20 has a possible volume range of 0
to 20 microliters.
However, transferring volumes at the low end of the range
is not recommended because the standard deviation is too high
to ensure precision.
Thus the minimum amount of liquid
that can be transferred in microliters is 0.1 for the P2,
0.5 for the P10, two for the P20, 10 for the P100,
50 for the P200, and 100 microliters for the P1000.
Now that we've covered what an automatic pipette is
and how it works, we'll take a detailed look
into how the Pipetman is used in the laboratory.
Let's say, for example, your procedure
requires you to transfer 153.5 microliters of sample.
The first step involves the selection of the Pipetman
with an appropriate volume range.
In this example, a couple of choices exist.
The P200 has a recommended volume range
of 50 to 200 microliters while the P1000
has a recommended range of 100 to 1,000 microliters.
Remember the lower end of a volume range
always has a higher standard deviation.
Therefore the P200 is the better choice.
The second step is the selection of the proper disposable
plastic tip.
Three tip sizes exist--
the large blue tips, which fit the P1000,
the medium yellow tips, which fit the P200, P100,
and the small white tips, which fit the P10 and P2.
Because we're using the P200, we're
going to need the yellow tips.
Using the proper tip is essential for preventing leaks
and promoting accurate delivery of liquids.
After selecting the correct Pipetman end tip,
set the desired transfer volume.
For the smaller Pipetman, P2 to P200,
the volume set on the indicator translates exactly
into the volume that will be delivered.
This P200 is set to deliver a volume of 125.9 microliters.
For the P1000, the volume set on the indicator
must be multiplied by 10 to determine the actual volume
that will be transferred.
This P1000 is set to deliver a volume of 1,259 microliters.
To correctly adjust the volume setting,
turn the volume adjustment knob to one
third of a revolution past the desired volume.
Then slowly move the dial back down to the desired setting.
Always approach the desired volume
by dialing downwards to prevent mechanical backlash
and to ensure precise volume transfer.
When adjusting the volume of a Pipetman,
take care not to rotate the dial past the maximum.
You cannot transfer volumes above the range
of the Pipetman.
Now it's time to transfer your sample.
You will need your appropriately sized Pipetman set
to the desired volume, the correctly
sized tips, a rack, the new container, a chem wipe,
and a waste container.
Attach a disposable tip to the Pipetman
by pressing its plastic shaft firmly
into the tip with a twisting motion.
This action should ensure an airtight seal
and prevent leaks from occurring.
Always use a new sterile tip for each sample
and never let the tip touch anything except sample.
To draw a sample into the plastic tip,
depress the plunger until resistance is felt.
Hold the Pipetman vertically and immerse it into the sample.
After the tip is immersed, slowly release the plunger
to draw liquid into the tip.
Wait a few seconds to ensure that the full volume of sample
is drawn into the tip.
As a guide, wait one second for the P2 to P200 Pipetman
and 2 to 3 seconds for the P1000.
The larger the volume or the more viscous the sample,
the longer you should wait.
Remove the Pipetman from the sample
and carefully wipe excess liquids
from the sides of the tip.
To dispense the liquid in the Pipetman,
touch its tip to the side wall of a new container
and slowly depress the plunger to the first stop.
Wait a few seconds to ensure complete sample flow,
then press the plunger to the second step
to expel any remaining liquid.
With the plunger fully depressed,
carefully remove the tip from the container
by sliding it up the container wall.
The importance of sliding the table along the wall
increases as the volumes become smaller.
Finally, allow the plunger to return to the up position.
If you are done working with a particular sample,
press the tip ejector button to release the tip
into the proper waste receptacle.
Several other guidelines will help
to ensure accuracy and precision when transferring liquids.
First, use a fresh tip for each sample to avoid contamination.
Second, pre-rinse each new tip.
Most liquids leave a film on the inside wall
of a new tip, which affects the actual volume that
can be transferred.
By pre-rinsing a tip, these volume variances
are eliminated.
To pre-rinse a tip, simply draw solution into the tip
and expel it into a waste container.
Third, check the immersion depth.
If the immersion depth is too deep,
liquid may get into the plastic shaft
and damage the mechanics of the Pipetman.
If the immersion depth is too shallow,
air may be drawn into the tip, which greatly affects accuracy.
As a general rule, immerse the weight tips
to a depth of 1 to 2 milliliters,
the yellow tips to a depth of 2 to 3 milliliters,
and the blue tips to a depth of 2 to 4 millimeters.
If you do find an air bubble in the tip,
dispense the sample into a waste container,
check your immersion level, and pipette more slowly.
If you still see an air bubble, discard your current tip
and use a new one.
Fourth, release the plunger button slowly.
Do not let the plunger snap up.
This violent action may force liquid into the plastic shaft.
Fifth, when wiping excess liquid off the sides of the tip,
avoid touching the tip opening because some of the sample
may be lost.
Six.
When liquid is in its tip, never allow the Pipetman
to tilt more than 20 degrees from vertical
and never lay the Pipetman on its side.
For optimum reproducibility, it is
important to be consistent with your samples.
Specifically, use a consistent speed and smoothness
when pressing and releasing the push button.
Use consistent pressure at the first step.
Immerse the tip to a consistent depth.
And hold the petmin at a consistent angle
that is less than 20 degrees from vertical.
If you are confident that you used good technique
and are still seeing errors with accuracy or precision,
the following troubleshooting guidelines
may fix your problem.
To ensure that your Pipetman is actually delivering the desired
volume, you should periodically calibrate it.
To calibrate a Pipetman, determine
how many grams of your liquid equals
1 milliliter from its density.
For example, if water is the solvent,
then 1 milliliter of water equals 1 gram of water.
Tare a small container on an analytical balance
and set the Pipetman at a volume near the top of its range.
Transfer the set volume of liquid
to the tared container on the balance
and note the mass of the liquid.
Repeat this step five more times and determine the mean weight
and standard deviation from your measurements.
Repeat the entire process with the Pipetman
set at a volume near the bottom of its range.
If these values deviate from what you expect,
adjust the petmin accordingly when you transfer liquid.
Occasionally, the Pipetman will begin
to leak due to improperly fitted tips or worn o-rings.
To check for leaks, put a fresh tip on the Pipetman.
Pre-rinse the tip and draw up liquid.
Keep the Pipetman vertical and wait about 20 seconds.
If you see a droplet, then the Pipetman is leaking.
Two final things that decrease the accuracy and precision
of the Pipetman involve pipetting samples
below 4 or above 70 degrees Celsius
and using the Pipetman with acids and corrosives.
If you absolutely must pipette an acid or corrosive,
then you should disassemble the Pipetman
immediately to inspect and clean the piston, shaft,
seal assemblies, and o-rings.
To summarize, using proper technique
with an automatic pipette is essential for ensuring
accuracy and precision.
A few key points to remember include the following.
Be consistent with plunger timing and pressure as well as
immersion time and depth.
Use a fresh, pre-rinsed tip for each new sample.
Check for air bubbles and leaks during each transfer.
And calibrate the automatic pipette periodically.
In addition, take care not to drop the expensive
and easily damaged Pipetman.
Do not allow the tip to touch anything except sample.
Do not introduce liquid into the plastic shaft
by letting the plunger snap up or by laying
the Pipetman on its side.
Finally, avoid using the Pipetman
under extreme temperature conditions
or with acids and corrosives.
When used with consistency and care,
an automatic pipette is an excellent means
of transferring microliter quantities of liquid.
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