Transcript volume - Bio-Link

MEASUREMENT OF
VOLUME
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OVERVIEW

First some general principles

Then, more detail about
micropipettes
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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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

On volumetric flask are much
more correct.
Why? Standards much more
stringent.
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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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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
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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
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USE CORRECT
DEVICE

Depends on accuracy and
volume required.
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Using a Serological Pipette
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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PROPER USE

Lab manual has a lot of
information
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From Basic Laboratory Methods for Biotechnology: Textbook and Laboratory
Reference, Seidman and Moore, 2000
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MICROPIPETTE
CALIBRATION/ 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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VALUE OF STANDARD
METHOD

Correction for mass versus
weight???
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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 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
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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
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