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 Polycarbonate Polyethylene Polyvinyl chloride (PVC) Teflon Acrylic

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 for 100 ul not the ul Measuring Review

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: s 2 =population variance X i = 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.