The Measurement of Volume

1. Chapter 20 The Measurement of Volume

2. Principles of Measuring the Volume of Liquids Overview Volume The amount of space a substance occupies Liter Basic unit of volume Measuring devices Depends of amount to measure and accuracy Larger volumes Glass and plastic vessels Graduated cylinders Volumetric flasks Pipettes Preferred for volumes in 1-25ml range Micropipette devices Less than 1ml

3. Basic principles of Glassware Calibration Calibration up to manufacturer Capacity marks/graduation Lines marked on volume measuring devices that indicate volume Lines the way to make the measurements Determined by American Society for Testing and Materials (ATSM) Meniscus Must be considered A curve formed by the surface of liquids confined in narrow spaces (measuring devices) Lowest point of the meniscus used as point of reference for calibration Hold at eye level to be entirely accurate

4. Basic principles of Glassware Calibration Liquid measuring calibrated to either To contain (TC) Will contain the specified amount when filled to the capacity mark Will not deliver that amount Adhesion to sides To deliver (TD) Marked slightly differently so that it does deliver the specified amount, assuming the liquid is water at 20oC and it is poured using specific techniques Volumetric flask Hold specific volume Essential for proper concentrations of buffers, etc Not good for “delivering” proper volume Use of TD device should be used

5. Basic principles of Glassware Calibration Small amount error in calibration inevitable Tolerance How much error is allowed in the calibration of a volume measuring item Most accurately calibrated glassware termed volumetric Has narrowest tolerance allowed Two effects of temperature to consider in high accuracy measurements Expand or contract with changes in temperature Affects accuracy Volume of the solution changes as the temperature changes Devices calibrated at 20oC with water

6. Glass and Plastic Labware used to Measure Volume Beakers and Erlenmeyer flasks Hold liquids Not to measure Calibrated with tolerance of ± 5% 100mL line may be anywhere form 95-105mL Graduated cylinders Cylindrical vessels calibrated with sufficient accuracy for most volume measurements ± 0.6mL tolerance Usually calibrated TD Can be obtained with different set of graduations, depending on need Not designed for mixing or storing Unstable/easily knocked over

7. Glass and Plastic Labware used to Measure Volume Burettes Long graduated tubes with a stopcock at one end that are used to dispense known volumes accurately Volumetric Flasks Vessels used to measure specific volumes where more accuracy is required than is attainable from a graduated cylinder Calibrated either TC OR TD a single volume Several disadvantages Relatively expensive Calibrated for only one volume Used only in situations where high accuracy volume measurements are required

8. Glass and Plastic Labware used to Measure Volume Volumetric Flasks Proper use Choose the proper type of flask for the application TC or TD Class A or Class B Serialized or not Be sure the flask is completely clean before use Read the meniscus with eyes even with liquid surface If calibrated TD, then pour as follows: Incline the flask to pour the liquid; avoid splashing on the walls as much as possible When the main drainage stream has ceased, the flask should be nearly vertical Hold flask in vertical position for 30 sec or so and touch off the drop of water adhering to the top of the flask by touching it to the receiving vessel Never expose volumetric glassware to high temperatures because heat causes expansion and contraction that can alter it calibration

9. Pipettes-Pipettes and Pipette-Aids Pipettes Hollow tubes that allow liquids to be drawn into one end and are generally used to measure volumes in the 01.-25ml range Glass or plastic Disposable or multiple use Pre-sterilize disposable with cotton plugged tops Pipette-aids (fig 29.5, pg 352) Devices used to draw liquid into and expel it from pipettes Mouth pipeting Used to be the norm Now banned by safety regulation

10. Pipettes-Measuring Pipettes Measuring pipette Calibrated with a series of graduation lines to allow the measurement of more than one volume Serological pipettes Usually calibrated so that the last drop in the tip needs to be “blown out” to deliver the full volume to deliver the full volume of the pipette

11. Pipettes-Measuring Pipettes Serological pipettes To use: Check that the pipette is calibrated to be “blown out” by looking for the bands at the top Examine the pipette to be sure the tip is not cracked or chipped Fill the pipette about 10mm above the capacity line desired and remove any water on the outside of the tip by a downward wipe with lint-free tissue Place the tip in contact with a waste beaker and slowly lower the meniscus to the capacity line Deliver the contents into the receiving vessel by placing the tip in contact with the wall of the vessel When the liquid ceases to flow, “blow out” the remaining liquid in the tip with on firm “puff” with the tip in contact with the vessel wall

12. Pipettes-Measuring Pipettes Mohr pipettes Calibrated “to deliver” but, unlike the serological pipettes, the liquid in the tip is mot part of the measurement and the pipette is not blown out

13. Pipettes-Volumetric (Transfer) Pipettes Volumetric (transfer) pipettes Made of borosilicate grass and are calibrated TD a single volume when filled to their capacity line at 20oC Most accurately calibrated pipette Can be serialized Calibrated “Delivery of the contents into the receiving vessel is made with tip in contact with the wall of the vessel and no after-drainage period is allowed Not “blown out”

14. Pipettes-Other Types Pasteur pipettes Used to transfer liquids from one place to another Not volume-measuring devices No calibration lines Good for transferring liquids Manual dispenser for reagent bottles Devices placed in a reagent bottle with a tube that extends to the bottom of the bottle Dispenser has a plunger that is depressed to deliver a set volume of liquid

15. Micropipetting devices-positive displacement and air displacement micropipettors Micropipettors Devices commonly used to measure smaller volumes, in the 1-1000µL range Two types Positive displacement Include syringes and similar devices where the sample comes in contact with the plunger and walls of the pipetting instrument Used for viscous and volatile samples Air displacement micropipettors Designed so that there is an air cushion between the pipette and the sample such that the sample only comes in contact with a disposable tip and does not touch the micropipettor itself Disposable tips-reduce chance of cross contamination Accurately measure volume of aqueous samples Most common instruments used in biotech

16. Micropipetting devices-air displacement micropipettors Obtaining accurate measurements Procedure for operation Plungers Control used by operator for the uptake and expulsion of liquids Different stop levels can be felt (fig 20.13, pg. 357) Factor affecting accuracy Operator’s technique Physical/chemical properties of the liquids Measurements affected by the environment in which they are made Condition of the micropipettor