Measuring Volume
Biotech I
pg. 301 to 323 in Basic Laboratory Methods
Volume
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Liquid volumes are measured in Liters
(L), milliliters (mL) or microliters (mL)
A liter is slightly more than a quart
A microliter is 1/1000 of a mL or smaller
than a tiny drop
Depending on the volume to be
measured, 3 options: graduated
cylinder, pipet or micropipet
B
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S Rule
Bigger units left
Smaller units right
Move decimal point to the right if
converting big units to small units
Move decimal point to the left is
converting small units to big units
B
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S Rule
1.25 L is equal to how many mLs?

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How many liters is 60 mLs?

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1250 mL
0.06 L
250 uL is how many L?

0.00025 L
Small to Medium Volumes
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Typically 0.1 mL to 25 mL
Plastic disposable or glass pipets
Pipette roller or pipette bulb is needed
Commonly used pipettes are:
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1mL, 5mL, 10mL, and 25 mL
Pipets are named by the
maximum they deliver
Pipetting Terminology
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Graduations: lines marked on volume
measuring devices that indicate
volume
Meniscus: a curve formed by the
surface of liquids confined in a narrow
space, such as in a measuring device
Pipetting Terminology

TC: To Contain; will contain the
specific amount when filled to the
capacity mark. It will not deliver the
amount if the liquid is poured out
because some of the liquid will adhere
to the sides of the container.
Pipetting Terminology

TD: To Deliver; marked so that it will
deliver the specified amount, assuming
the liquid is 20C and is poured out.

Note: plastic containers are considered non-wetting
which means water does not stick to them so there
is no difference between TD and TC
Pipetting Terminology

Tolerance: how much error is allowed
in the calibration or a measuring
device.

Volumetric: the most accurately
calibrated glassware
The “art” of Pipetting
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Learning how to pipette takes practice
and determination
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Patience and careful lab technique will
ensure your results are accurate and
reliable
Steps to Proper Pipetting
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Determine the proper pipette size
Determine the proper pipette roller or bulb
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Blue: 1 mL pipette
Green: 5 or 10 mL pipette
Draw up past your required volume and
dispense to your exact fill volume required
Pay attention to the meniscus
Pipetting Lab: Precautions
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Never mouth pipette
Hold the bottom of the roller and the top
of the pipette when you are assembling
them
Always keep the pipette vertical when
there is fluid in it
Always keep the tip of the pipette sterile
and take care to not touch surfaces
Pipetting Lab: see handout

See your hand out for lab practical
using disposable pipettes
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Pipets with a TD on them indicate they
are designed “To Deliver” and that the
tiny amount left in the bottom of the
pipet after dispensing should NOT be
blown out.
Really Small Volumes
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Most QC and R&D laboratories measure
very small volumes.
Micropipets are used to measure
microliter quantities.
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What is the symbol for microliter?
mL
Micropipets are expensive instruments
which must be handled carefully
Getting to Know your micropipet
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We have 2 types of pipets in our lab
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P20: for 2 to 20 mL
P200: for 20 to 200 mL
Let’s take a look at your micropipet
diagram handout.

Keep this handout out for the micropipet
lab exercise
Micropipetters
Getting to Know your micropipet
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There are 4 parts to a micropipet
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Plunger button
Ejector button
Volume display (setter)
Dispensing Tip
Getting to Know your micropipet
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Plunger button
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Typically there are 2 stops
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The first evacuates the air in the micropipet
The second stop evacuates the volume plus
another 50% or so.
Practice to feel the difference
Picking the proper size micropipet
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Look at your plunger
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It will give you the min. and max. that it
can deliver
3 numbers will be displayed
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The top # is the digit for the maximum
I.e. 2 is the top # on the P200
Interpret the following settings
How many microliters is this
on a m10?
on a m100 on a m1000
3
5
0
3.50 uL
or 0.00350 mL
3
5
35.0 uL
or 0.0350 mL
0
3
5
350.0 uL
or 0.350 mL
0
Setting your required volume
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To set your volume you need to turn
the adjusting knob.
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Rotate slowly to the desired volume
setting
At no time should the knob be turned
past its upper or lower limits.
Do not force the settings, the knob will
turn easily
Sources of Error for micropipet
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Bad pipet tip: leaks, clogged tip
Damaged pipetor
Wrong size tip for pipet size
Bubbles in your tip due to poor
technique
Liquid not at room temperature or it
may be viscous
Multi channel pipets
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In order to increase efficiency, pipets
have been designed to hold 4-16
channels at a single dispense.
Most commonly used when running
ELISA assays

8 channel pipet
Multi Channel pipet
Electronic or Automated pipets
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Refer to your handout for a picture of an
electric pipet.
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Typically they are battery operated
Press a button to pick up, press button to
dispense
Use
to increase or decrease volume
settings
For large volume labs, automated pipetting
instruments are used. Multi channel heads
Burettes
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Long graduated tube with a stop cock
at one end which is used to dispense
known volumes accurately.
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See your handout for a picture. There
was one out during the scavenger hunt
Micropipetter Precautions
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These are expensive and precise
pieces of equipment.
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DO NOT play with them
Handle them with care
Keep them clean
Micropipet Lab Exercise
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Micropipet operating instructions
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Choose the proper pipet for the desired
volume, choose proper pipet tip
Set the volume
Depress the plunger to first stop and draw
up required volume
Wipe tip gently with Kimwipe, do not draw
out liquid
Depress plunger to expel liquid. Go to the
second stop to blow out all of the liquid.
Calibration
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Definition: adjustment of dispensing
devices so they dispense accurate
volumes.
In the case of pipets we are checking
the accuracy and precision of the
pipet.
Calibration
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Accuracy: to deliver the exact quantity
required
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Mean measured volume divided by
Nominal or required volume
Precision: the ability to deliver that
exact quantity time after time

The amount of variation between the
each volume measured

1.01, 1.01, 1.02
Calibration Procedure
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Pre Calibration Activities
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Be sure the Micropipetter is clean
Be sure the balance is calibrated and
working properly (Verification)
Be sure the water you are using is 22 to
25C
Ensure the scale is free of drafts
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Place a wt. on and verify it provides a
consistent reading
Calibration Procedure
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Set the Micropipetter to deliver 2 uL on a 2
to 20 uL pipetor or 25 uL on the 20 to 200 uL
pipetor
Place a weigh boat on the calibrated
balance
Add a few drops of water to the weigh boat
Tare the balance by pressing the zero
button
Calibration Procedure
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Pipet the appropriate volume and
dispense it into the weigh boat.
Record your reading
Zero the balance (good practice to change tips each time)
Pipet the appropriate volume and
dispense it into the weigh boat.
Record your reading
Repeat until you have 5 readings
Calibration Procedure
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Set the Micropipetter to 20 uL for 2-20
uL pipet or 200 for 20 to 200 uL pipet
Zero the balance
Pipet the appropriate volume and
dispense it into the weigh boat.
Record your reading
Repeat until you have 5 readings
Calibration Procedure
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Calculations:

Calculate the mean weight of water
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Density of water at 22C = 1.0031 g/mL
1 uL would weigh how many mgs?
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1.0031 mgs
Convert the mean water weight to the
mean volume weight
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Mean Volume of water = Mean weight /
density
Calibration Procedure
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Mean of 5 weights / 1.0031 mgs
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i.e. if the weights of 2 uL setting were
2.001, 2.001, 2.003, 2.002, 2.002 mg
What is the mean weight?
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What is the mean volume of water?
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2.018
2.018/1.0031 = 2.006
What is the accuracy of the pipetor?
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2.006 / 2.000 x 100 = 100.3% or .3% error
Preventive maintenance of pipets

Wipe the pipet with alcohol if it gets
contaminated with solution

Store properly which is in a stand if
possible as you do not want the shaft
part of the pipet to get bent from laying
on a bench