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Q : WHAT IS A METHOD?A : method is a procedure for the
analysis of a specific analyte (e.g.
determination of Mn in water).
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Q : WHAT IS METHOD VALIDATION AND
HOW DOES IT APPLY TO US (AS ANALYTICAL
CHEMISTS).A :Method Validation is a way of testing a particular
analytical method to see if it is suitable for itsintended purpose. The goal of Method Validation is
to prove that the results obtained are true and toshow that the assay will perform properly underthe conditions in which it is intended to be used.
Method validation is an important part of analyticalchemistry as well as many other fields (softwaredesign, engineering, environmental toxicology, andthe pharmaceutical industry to name a few).
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WHY SHOULD WE VALIDATE METHODS?In order to answer that question, we need to look at how and
why methods are developed in the first place.1.Identify parameter that needs to be known (is it a qualitative
or quantitative application?)
2.Choose type of technology to be used (e.g. HPCL, GC or Massspectrometry) 3.Develop the method
4.Pre-validate the method (how does it perform in its intendedarea of use)
5.Adjust or modify method if necessary (based on results ofpre-validation)
6.Develop a validation plan (determine what criteria will berequired)
7.Validate
8.Report findings (Conclusion: Is this method suitable for itsintended purpose?).
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ACCURACY
Closeness of agreement between the valueobtained by the method and the true value.(this is how teacher calculated your resultsmarks in the lab, if you had 100% Accuracy,you got 10/10 on your Data mark).
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use the procedure on a pure and
characterized reference standard(more later) and calculate therecovery
compare your results using onetechnique to results obtained
with a second, validatedprocedureprepare a placebo matrix withoutany analyte in it and spike itwith varying concentrations of
the standard and analyse
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PRECISION
Expresses the closeness of agreement between aseries of measurements obtained from multiplesampling. Precision is often expressed as thestandard deviation or Relative standarddeviation of replicate measurements (morelater on RSD). Note that a method can beprecise, but not accurate (so all your
measurements may be close together, but theresult is wrong).
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ACCURACY AND PRECISION
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SPECIFICITY
The ability to measure the analyte in thepresence of components which we expect tobe present in the sample matrix. So if youare determining the concentration of Iron and
Chromium in water by UV/Vis spectroscopy, ifthere are small amounts of Fluoride andChloride in the sample, will that affect mymeasurement?
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DETECTION LIMIT
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QUANTITATION LIMIT
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LINEARITY
The range of concentration of analyte for which
the procedure provides test result that are indirect correlation to the amount of analyte in the
sample (remember when you studied Beers law
,that absorbance wasnt linear at higher
concentration of analyte
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RANGE
The same as linearity, except the result must also be
accurate and precise( over the concentration tested)
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LINEARITY/RANGE
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RSD : ( RELATIVE STANDARD DEVIATION),WHAT IS IT
The RSD is just a way of normalizing standard deviation
data so that it is easier to compare the variance in twodifferent sets of numbers that may not be of the samemagnitude. For example, in experiment (Determinationof Nitrite and Nitrate by Anion ExchangeChromatography) you were asked to determine which
measurement was better, peak height or peak area.Data from four injections of the unknown may havelooked like this
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BIAS
difference between the expectation of the
test results and an accepted reference
value
NOTE Bias is the total systematic error as contrasted to random error.
There may be one or more systematic error componentscontributing to the bias. A larger systematic difference from the
accepted reference value is reflected by a larger bias value.
[ISO 3534-1]
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STANDARD UNCERTAINTY
uncertainty of the result of a measurement expressed as a standard deviation
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TRUENESS
trueness closeness of agreement between the average value obtained from a
large set of test results and an accepted reference value
NOTE The measure of trueness is normally expressed in terms of bias. The reference to
trueness as accuracy of the mean is not generally recommended.
[ISO 3534-1]
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ONGOING DEMONSTRATION OF METHOD CONTROL
Date/Time
Measurem
ent
Average
+ 1 std dev
+ 2 std dev
+ 3 std dev
- 1 std dev
- 2 std dev
- 3 std dev
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UNCERTAINTY
measurement parameter, associated with the result of a
measurement, that characterizes the dispersion of the valuesthat could reasonably be attributed to the measurand
NOTE 1 The parameter may be, for example, a standard deviation (or a given multiple
of it), or the half-width of an interval having a stated level of confidence.
NOTE 2 Uncertainty of measurement comprises, in general, many components. Some of
these components may be evaluated from the statistical distribution of the results
of a series of measurements and can be characterized by experimental standard
deviations. Other components, which also can be characterized by standard
deviations, are evaluated from assumed probability distributions based on
experience or other information.
NOTE 3 It is understood that the result of the measurement is the best estimate of thevalue of the measured, and that all components of uncertainty, including those
arising from systematic effects such as components associated with corrections and
reference standards, contribute to the dispersion.
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C.2 DETERMINATION OF MEAT CONTENT
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C.2 DETERMINATION OF MEAT CONTENT
C.2.1 Introduction
Meat products are regulated to ensure that the meat content is accurately declared.Meat content is determined as a combination of nitrogen content (converted to
total protein), and fat content. The present example accordingly shows the
principle of combining different contributions to uncertainty, each of which itself
arises chiefly from reproducibility estimates, as described at Clause 12.
C.2.2 Basic equations
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C.2.3 EXPERIMENTAL STEPS IN MEAT-CONTENT
DETERMINATION
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C.2.4 UNCERTAINTY COMPONENTS
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