1. Measurement of Volume
Chapter 20

2. Glassware Calibrations
Capacity marks and graduations are lines marked on volume measuring devices that indicate volume
ASTM methods are used for calibrations
Calibrated to contain (TC) or to deliver (TD)
TC will contain the specified amount at mark
TD will deliver the specified amount at mark

3. Continued
Tolerance is how much error is allowed in the calibration of a volume measuring device
Accurately calibrated glassware is termed volumetric
Tolerance 100ml ml
At mark will measure to ml (A)
ml is to Class B

4. Reading glassware
Meniscus is a curve formed by the surface of liquids confined in narrow spaces such as in measuring devices
Read from bottom of meniscus
Beaker Grad Cylinder Burette

5. Glass and plastic volumetric wares
Graduated Cylinders
Burettes
Pipettes
Including pipette-aids, and micropipettes
Volumetric flasks

6. Proper use of a Volumetric Flask
Choose the proper type of flask for application: TD or TC
Clean flask before use
Read the meniscus with your eyes level with liquid surface
If calibrated to deliver, pour the contents to avoid splashing and then allow to drain for 30 seconds
Never expose to temperatures that may cause expansion or contraction of glass flask

7. Measuring Pipettes
Are calibrated with a series of graduation lines to allow measurement of more than one volume
Sometimes known as serological or Mohr pipettes
Serological pipettes require that the last drop in the tip needs to be blown out
Mohr pipettes are TD based

8. Volumetric Pipettes
Made of borosilicate glass and calibrated to deliver
No afterdrainage period is allowed
The are not blown out
Manual dispensers for reagent bottles deliver set volumes of liquids when the apparatus is used

9. Pasteur pipettes Used to transfer liquids from one place to another
Are not volume measuring devices
Measure less than one ml of volume
The disposable type is so useful it is used when high accuracy is not needed

10. Micropipetting devices
Micropipettor delivers 1 to 1000 ml
Are known by several names
Microliter pipette, piston or plunger operated pipettes,
Two types of micropipettors
Positive displacement – plunger contact
Air displacement – no plunger contact
Digital Microliter pipettors ml pipettes

11. Factors affecting the accuracy of manual micropipettors
Operator’s technique
Physical and chemical properties of the liquid being measured
Measurements are affected by the environment in which they are made
Condition of the micropipettor

12. Contamination and micropipettors
Aerosols are fine liquids that remain suspended in the air
Carryover is contamination from one sample to another
Sooo
Tips have been invented to prevent aerosols
Dedicated micropipettors reduce carryover
Proper operation is critical

13. Procedure for operating a manual micropipettor
Set to the desired volume
Attach the disposable tip
Observe the tip and sample while the plunger to the first stop and place the tip in the liquid
Allow the plunger to slowly return to the undepressed position to draw liquid

14. continued
Place the tip so that it touches the side of the container into which the liquid is to be dispersed
Depress the plunger to the second stop
Eject the tip using the third stop, tip ejector button, or other mechanism

15. Verification of Micropipettors
Are calibrated by the manufacturer
May become uncalibrated and must be verified periodically
Gravimetric method using balances
Liquids are pipetted into a tared container.
The liquid is weighed and the volume dispersed is calculated from density
Nominal volume is the amount dispensed

16. Pipetting methodically
Plan exactly what will go into tubes
Label all tubes before additions
Place tubes in front of rack, move to rear when filled
Check off components as added
Change tips with new material or when contaminated
Mix well after additions are complete

17. Cleaning/maintaining pipettes
Problem conditions
Visual inspection shows that tip does not take up right volume of liquid
Micropipettor leaks or drips
Plunger jams, sticks, or moves erratically
Micropipettor is blocked and will not aspirate liquid
Notes on cleaning