Download Measuring Accurately

Measuring Accurately
Learning to use equipment for liquids
Glassware
• Borosilicate (Pyrex, Kimax)
– High resistance to heat/cold shock and
low metal contaminants.
• Corex
– High resistance to pressure and
scratching- used for centrifuge tubes
• High silica (quartz)
– Excellent optics- cuvettes
Glassware
• Low Actinic
– Red tinted to reduce light exposure.
• Flint
– Soda lime glass containing oxides of sodium,
silicon and calcium. Poor resistance to
temperature changes. Used in disposable
pipettes, test tubes, many containers.
• Some containers/glassware is tinted
brown or amber to light filter.
Plastics
• Polystyrene
• Polypropylene
Low-density polyethylene
High-density polyethylene
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Polycarbonate
Polyethylene
Polyvinyl chloride (PVC)
Teflon
Acrylic
Measuring Review
• Always use either a graduated
cylinder or a volumetric flask to
accurately measure solutions.
• Always use the pipette or cylinder
closest in volume to the volume you
are measuring to ensure greatest
accuracy.
– Use the 10-100 for 100 ul not the 1001000ul
Choice of Materials for
Measuring Liquids
• Glass breaks but is chemically inert
and doesn’t warp.
• Plastic doesn’t break but can be
chemically reactive.
• Always know what plastic you are
using and what it can unsafely react
with.
Specifications
• Specifications are established by the
manufacturer.
• They guarantee, in terms of
accuracy and precision, the
performance of all pipettes of a
given brand and a given model at a
certain volume setting.
Accuracy vs. Precision
• Accuracy is your ability to deliver a
specified amount. Dictionary defines
it as “exactness arising from careful
effort”
• Precision is your ability to
reproducibly deliver the same
amount.
• On the average you can be accurate
and not precise, or precise and not
accurate.
Variation/Error
• Error is emotionally laden, so
statisticians often use the term
variation to express the concept.
• Human variation vs. equipment
variation.
• The most commonly used measures
of data variation are the range, the
variance and the standard deviation.
• Range is the distance between the
biggest and smallest samples.
Variance of a sample:
s2=population variance
Xi= the item or observation
M= sample mean
N = total number of observations in the sample.
Calibration
• An ancient science growing out of
measuring ammunition in time of
war.
• A droplet of reagent or sample so
small it can hardly be seen can have
major implications in clinical,
research and quality control
laboratories.
• Accurate liquid handling and weight
determination are important.
The National Institute of
Standards and
Technology
• NIST is responsible for developing,
maintaining and disseminating
national standards – International
Standards of measurement or SI for the basic measurement
quantities, and for many derived
measurement quantities.
The National Institute of
Standards and
Technology
• NIST is also responsible for assessing the
measurement uncertainties associated
with the values assigned to these
measurement standards.
• Nothing is ever precisely measured
enough, but they get to decide how close
we can come.
• As such, the concept of measurement
traceability is central to NIST’s mission.
Calibration of
Micropipettes
• Weight of 1 ml water= 1 gm.
• (under proper barometric and
humidity conditions of course).
• Must use an accurate scale.
• Check out scale with calibrated
weights first.
The Micropipette:
Friend or Foe?
How to be More
Accurate
• It is critical to get in the habit of looking
at what you are doing.
• The pipette only works as well you do.
Things That Go Wrong
• The tip was not in the liquid deeply
enough to fill it.
• The tip was inserted too deeply and
carried liquid over on the outside of
the tip.
• The liquid was viscous and hard to
pipette accurately (50% glycerol in
enzymes).
Things That Go Wrong
• The pipette was released too quickly
and the tip didn’t fill accurately.
• The tip wasn’t securely fastened and
didn’t measure accurately because
of a leak.
• Using the wrong tip
• Pipette has broken seals or bent
piston.
CAUTION
• Don’t drop the pipettes- the seals
break easily, and micropipettes
become inaccurate.
• If the pipette drops, check out its
accuracy before using it.
Getting a Feeling for
Volumes
• Volumes look different in different
tips.
• Practice using a scale and
measuring water.
• Always look at what you are doing- it
will become second nature
eventually.
Dr. Murray’s Law
• Always use bigger amounts if
possible. They are more accurately
dispensed.
• Make up dilutions and dispense
those OR make a cocktail of all
reagent components and dispense
those.
• Avoid pipetting less than 2 ul.
Dr. Murray’s Laws
• Thaw completely
• Mix Well
Use of SD
• The standard deviation enables us
to determine, with a great deal of
accuracy, where the values of a
frequency distribution are located in
relation to the mean.
• The standard deviation is an
absolute measure of dispersion that
expresses variation in the same
units as the original data.
Limitations of Standard
Deviation
• For example, the unit of standard
deviation of the data set of height of
a group students is centimeter, the
unit of standard deviation of the data
set of their weight is kilogram.
• Can we compare the values of these
standard deviations?
• Unfortunately, no, because they are
in the different units.
Coefficient of Variation
• A relative measure that gives us a
feel for the magnitude of the
deviation relative to the magnitude of
the mean. The coefficient of
variation is a relative measure of
dispersion.
• CV= Standard Deviation/Mean X
100
• The unit of the coefficient of variation
is percent.
Example
• Suppose that each day laboratory
technician A completes 40 analyses
with a standard deviation of 5.
• Technician B completes 160
analyses per day with a standard
deviation of 15. Which employee
shows less absolute variability?
• Who has relatively less variability?
Answer
• For technician A:
cv=5/40 x 100% = 12.5%
• For technician B:
cv=15/60 x 100% = 9.4%.
• So, we find that, technician B who has more
absolute variation in output than technician A,
has less relative variation (cv).
How to Interpret CV
• A coefficient of variation of 1 percent
would indicate that an estimate could vary
slightly due to sampling error, while a
coefficient of variation of 50 percent
means that the estimate is very
imprecise.
• One way to increase CV is to increase
sample size.
• Another is to increase precision of the
measurement.
• Micropipette measuring- CV can be <3%
fairly easily with practice and good habits.
A Word of Warning
• CV measures precision not
accuracy.
• For an objective measure of
accuracy you need calibration.