Measuring Volume

Biotech I
pg. 301 to 323 in Basic Laboratory Methods
 Volume

 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 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 S Rule

 1.25 L is equal to how many mLs?

 1250 mL
 How many liters is 60 mLs?
 0.06 L
 250 uL is how many L?
 0.00025 L
 Small to Medium Volumes

 Typically 0.1 mL to 25 mL
 Plastic disposable or glass pipets
 Pipette roller or pipette bulb is needed
 Commonly used pipettes are:
 1mL, 5mL, 10mL, and 25 mL
 Pipets are named by the
maximum they deliver
Pipetting Terminology

 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

 Learning how to pipette takes practice

and determination

 Patience and careful lab technique will

ensure your results are accurate and
reliable
Steps to Proper Pipetting

 Determine the proper pipette size

 Determine the proper pipette roller or bulb
 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

 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

 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

 Most QC and R&D laboratories measure

very small volumes.
 Micropipets are used to measure
microliter quantities.
 What is the symbol for microliter?
 mL
 Micropipets are expensive instruments
which must be handled carefully
Getting to Know your micropipet

 We have 2 types of pipets in our lab

 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

 There are 4 parts to a micropipet

 Plunger button
 Ejector button
 Volume display (setter)
 Dispensing Tip
Getting to Know your micropipet

 Plunger button
 Typically there are 2 stops
 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

 Look at your plunger

 It will give you the min. and max. that it
can deliver
 3 numbers will be displayed
 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 5 0 3 5 0

3.50 uL 35.0 uL 350.0 uL

or 0.00350 mL or 0.0350 mL or 0.350 mL
Setting your required volume

 To set your volume you need to turn

the adjusting knob.
 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

 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

 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

 Refer to your handout for a picture of an

electric pipet.
 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

 Long graduated tube with a stop cock

at one end which is used to dispense
known volumes accurately.

 See your handout for a picture. There

was one out during the scavenger hunt
Micropipetter Precautions

 These are expensive and precise

pieces of equipment.
 DO NOT play with them
 Handle them with care
 Keep them clean
Micropipet Lab Exercise
 Micropipet operating instructions
 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

 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

 Accuracy: to deliver the exact quantity

required
 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

 Pre Calibration Activities

 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
 Place a wt. on and verify it provides a
consistent reading
Calibration Procedure

 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

 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

 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

 Calculations:
 Calculate the mean weight of water
 Density of water at 22C = 1.0031 g/mL
 1 uL would weigh how many mgs?
 1.0031 mgs
 Convert the mean water weight to the
mean volume weight
 Mean Volume of water = Mean weight /
density
Calibration Procedure

 Mean of 5 weights / 1.0031 mgs

 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?
 2.018
 What is the mean volume of water?
 2.018/1.0031 = 2.006
 What is the accuracy of the pipetor?
 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