Measuring Accurately

1
Measuring Accurately

• Learning to use equipment for liquids

2
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

3
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.

4
Plastics

• Polystyrene
• Polypropylene
• Low-density polyethylene
• High-density polyethylene
• Polycarbonate
• Polyethylene
• Polyvinyl chloride (PVC)
• Teflon
• Acrylic

5
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 100-1000ul

6
Choice of Materials for Measuring Liquids

• Glass breaks but is chemically inert and doesnt
  warp.
• Plastic doesnt break but can be chemically
  reactive.
• Always know what plastic you are using and what
  it can unsafely react with.

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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.

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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.

9
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.

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Variance of a sample s2population variance Xi
the item or observation M sample mean N total
number of observations in the sample.
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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.

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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.

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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 NISTs mission.

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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.

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The Micropipette Friend or Foe?
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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.

17
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).

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Things That Go Wrong

• The pipette was released too quickly and the tip
  didnt fill accurately.
• The tip wasnt securely fastened and didnt
  measure accurately because of a leak.
• Using the wrong tip
• Pipette has broken seals or bent piston.

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CAUTION

• Dont drop the pipettes- the seals break easily,
  and micropipettes become inaccurate.
• If the pipette drops, check out its accuracy
  before using it.

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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.

21
Dr. Murrays 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.

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Dr. Murrays Laws

• Thaw completely
• Mix Well

23
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.

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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.

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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.

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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?

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Answer

• For technician A
• cv5/40 x 100 12.5
• For technician B
• cv15/60 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).

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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 lt3 fairly
  easily with practice and good habits.

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A Word of Warning

• CV measures precision not accuracy.
• For an objective measure of accuracy you need
  calibration.