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How do you validate HPLC instrumentation?

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Quick Answer

Validating a high-performance liquid chromatography instrument involves determining the instrument's ability to be accurate and precise and to give reproducible results. It also determines the lowest limits of detection. Validation requires the use of samples with known results.

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HPLC is a common laboratory technique for separating the individual parts of a solution. After running a solution through an HPLC machine, researchers obtain a readout showing the components of the mixture and their proportions in the solution. Validation is a means of determining if a method or an instrument meets the requirements of its application. Two of the most important aspects of validation are precision and accuracy. Precision is the closeness of test results to one another, and accuracy is the closeness of results to an accepted true value. Determining precision involves running repeated tests on a substance and determining the standard deviation of those tests; lower standard deviations are best. After testing a mixture containing a known amount of one specific component, the instrument's analysis of that component's presence gives a good measure of its accuracy.

Ruggedness is a measure of the HPLC's ability to produce consistent results. Ruggedness involves testing the same sample under different conditions, such as different labs and researchers. This type of testing requires a collaboration between multiple researchers and facilities.

Researchers also often need to know the lowest amount of analyte that they can detect with a particular instrument. This information comes in the form of a signal-to-noise ratio; the acceptable signal-to-noise ratio for a limit of detection is typically 2:1. This ratio tells researchers the lowest levels of concentration that are detectable despite background noise.

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