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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 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.