Measuring Volume

1
Measuring Volume

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

2
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

3
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

4
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

5
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

6
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

7
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.

8
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

9
Pipetting Terminology

• Tolerance how much error is allowed in the
  calibration or a measuring device.
• Volumetric the most accurately calibrated
  glassware

10
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

11
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

12
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

13
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.

14
Really Small Volumes

• Most QC and RD 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

15
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
• Lets take a look at your micropipet diagram
  handout.
• Keep this handout out for the micropipet lab
  exercise

16
Micropipetters
17
Getting to Know your micropipet

• There are 4 parts to a micropipet
• Plunger button
• Ejector button
• Volume display (setter)
• Dispensing Tip

18
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

19
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

20
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 or 0.00350 mL
35.0 uL or 0.0350 mL
350.0 uL or 0.350 mL
21
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

22
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

23
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

24
Multi Channel pipet
25
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

26
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

27
Micropipetter Precautions

• These are expensive and precise pieces of
  equipment.
• DO NOT play with them
• Handle them with care
• Keep them clean

28
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.

29
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.

30
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

31
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

32
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

33
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

34
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

35
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

36
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

37
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