1. lseidman@matcmadison.edu MEASUREMENT OF VOLUME

2. lseidman@matcmadison.edu OVERVIEW First some general principles Then, more detail about micropipettes

3. lseidman@matcmadison.edu DEFINITION Volume is the amount of space a substance occupies. Liter is the basic unit of volume.

4. lseidman@matcmadison.edu EQUIPMENT FOR VOLUME MEASUREMENT Less than 1mL: micropipette 1 - 25mL: serological pipette More than 25mL: graduated cylinder or volumetric flask

5. lseidman@matcmadison.edu CALIBRATION OF GLASSWARE Glassware is calibrated according to national standards Specifies where calibration lines go  Based on certain temperature  Pure water  Based on methods of pouring

6. lseidman@matcmadison.edu VOLUMETRIC FLASKS Volume markings on ordinary flask or beaker are "sort of" correct – don’t rely on them!

7. lseidman@matcmadison.edu On volumetric flask are much more correct. Why? Standards much more stringent.

8. lseidman@matcmadison.edu MENISCUS Half-moon, curve formed at the surface of liquid. Read from the bottom of meniscus. Why? That is the way they are manufactured according to standard.

9. lseidman@matcmadison.edu CALIBRATION TD vs. TC TD = calibrated to deliver Use to measure out an exact amount of liquid to pour into something else Example: graduated cylinder TC = calibrated to contain Use to see how much liquid is there Example: preparing standards in volumetric flask

10. lseidman@matcmadison.edu PLASTICWARE Glass vs. plastic Glassware will be marked either TD or TC Plasticware is not marked - liquid doesn't stick to plastic like glass  so, plasticware is both TD and TC

11. lseidman@matcmadison.edu USE CORRECT DEVICE Depends on accuracy and volume required.

13. Using a Serological Pipette

14. lseidman@matcmadison.edu MICROPIPETTES Used to dispense microliter volumes. Common in molecular biology. Use “air displacement”  Protects pipette from materials dispensed  Disposable tips

16. lseidman@matcmadison.edu ACCURACY AND PRECISION Depends a lot on operator Depends on instrument’s calibration and maintenance

17. lseidman@matcmadison.edu PROPER USE Lab manual has a lot of information

18. lseidman@matcmadison.edu From Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, Seidman and Moore, 2000

19. lseidman@matcmadison.edu MICROPIPETTE CALIBRATION/ PERFORMANCE VERIFICATION Calibration: performance of micropipette is evaluated and adjusted to bring into conformance with external authority. Performance verification: check only.

20. lseidman@matcmadison.edu BASIS The MASS of 1 mL of water is 1 gram. 1 microliter of water has a mass of 1 mg.

21. lseidman@matcmadison.edu PROCESS Dispense set volumes of water Weigh water dispensed Know how much it should weigh Determine accuracy of volumes dispensed External authority is standards used to calibrate balance  Traceability

22. lseidman@matcmadison.edu FACTORS THAT AFFECT METHOD Operation of micropipette  Smoothness and speed  Depth of immersion  Pre-rinsing or not  New tip or not Evaporation, related to humidity Temperature Balance calibration Thermometer calibration Purity of water Barometric pressure

23. lseidman@matcmadison.edu VALUE OF STANDARD METHOD Correction for mass versus weight ???

24. lseidman@matcmadison.edu ASTM STANDARD GRAVIMETRIC METHOD “The general procedure is based on the determination of the weights of water samples delivered by the instrument. The values are corrected for evaporation, then true mass and volume are calculated simultaneously, based on knowledge of the density of water at specific temperatures and corrections for air buoyancy.”

25. lseidman@matcmadison.edu FORMULA V = (W - E)Z Where: V = mean volume delivered in microliters W = mean weight in mg E = evaporation loss in mg Z = conversion factor in microliters/mg, incorporating density of water when buoyed in air at the test temperature and pressure

26. lseidman@matcmadison.edu PROCESS Dispense water Estimate evaporation Weigh water Average values Plug into formula Get volumes Quantify accuracy and precision Compare to specifications In lab, we simplify considerably

27. lseidman@matcmadison.edu ACCURACY The closeness of agreement between the nominal volume and the mean volume  Nominal volume is what you set on device Can quantify by absolute error or Percent error: % error = nominal volume – measured volume X 100 Nominal volume

28. lseidman@matcmadison.edu PRECISION Consistency Can quantify with standard deviation or Relative standard deviation: RSD = Standard deviation mean