1. Measuring Volume

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? How many liters is 60 mLs?
250 uL is how many L? 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.
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.
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
We will be using pipette pumps in this shop
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. 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

14. Getting to Know your micropipet
There are 4 parts to a micropipet
Plunger button
Ejector button
Volume display (setter)
Dispensing Tip/ Tip attachment

15. Getting to Know your micropipet

16. Name the parts

17. 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

18. 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

19. Interpret the following settings
How many microliters is this
on a m10? on a m on a m1000 3 5 3 5 3 5
3.50 uL
or mL
35.0 uL
or mL
350.0 uL
or mL

20. 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

21. 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

22. 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

23. Multi Channel pipet

24. Electronic or Automated pipets
Also referred to as a pipette pump
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

25. Burettes
Long graduated tube with a stop cock at one end which is used to dispense known volumes accurately.

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

27. Using a micropipet 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.

28. 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.

29. 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

30. 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

31. 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

32. 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)
Repeat until you have 5 readings

33. 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

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

35. Calibration Procedure
Mean of 5 weights / mgs
i.e. if the weights of 2 uL setting were 2.001, 2.001, 2.003, 2.002, mg
What is the mean weight?
2.018
What is the mean volume of water?
2.018/ = 2.006
What is the accuracy of the pipetor?
2.006 / x 100 = 100.3% or .3% error

36. 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