[email protected] measurement of volume. [email protected] overview first some general...
TRANSCRIPT
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DEFINITION
Volume is the amount of space a substance occupies.
Liter is the basic unit of volume.
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EQUIPMENT FOR VOLUME
MEASUREMENT Less than 1mL: micropipette
1 - 25mL:
serological pipette
More than 25mL: graduated cylinder or volumetric flask
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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
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VOLUMETRIC FLASKS
Volume markings on ordinary flask or beaker are "sort of" correct – don’t rely on them!
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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.
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CALIBRATIONTD 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
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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
Using a Serological Pipette
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MICROPIPETTES
Used to dispense microliter volumes.
Common in molecular biology.
Use “air displacement” Protects pipette from materials
dispensed Disposable tips
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ACCURACY AND PRECISION
Depends a lot on operator Depends on instrument’s
calibration and maintenance
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From Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, Seidman and Moore, 2000
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MICROPIPETTECALIBRATION/ PERFORMANCE VERIFICATION
Calibration: performance of micropipette is evaluated and adjusted to bring into conformance with external authority.
Performance verification: check only.
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BASIS
The MASS of 1 mL of water is 1 gram.
1 microliter of water has a mass of 1 mg.
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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
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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
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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.”
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FORMULA
V = (W - E)Z
Where:V = mean volume delivered in microlitersW = mean weight in mgE = evaporation loss in mgZ = conversion factor in microliters/mg,
incorporating density of water when buoyed in air at the test temperature and pressure
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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
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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
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PRECISION
Consistency Can quantify with standard
deviation or Relative standard deviation:
RSD = Standard deviation
mean