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Lecture 7

CHEM 212 Lecture 7: Sept. 21/2016

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University of Victoria
CHEM 212
Hamilton M.Coreen

Chem 212 Lecture 7 Sept. 21/2016 Approaches to Quantification: 1. External Standard 2. Internal Standard 3. Standard Addition External Standard: -Calibration standards -Calibration curves (plot of signal versus concentration) -Use calibration curve to "Read" many unknown samples -avoid single point calibration for deterring Sa =kna or Sa=k[A] -Single point assumes linear relationship over large concentration range => Not good -potential error by assuming calibration curve goes through zero -error in reported concentration is difference in x-axis=>accuracy problem -can not solve this by replicates=>systematic error -Multipoint external standard method: -requires cal curve -hopefully y=mx+b or a non-linear equation -use least squares on spreadsheet -never assume linearity beyond calibration points: -dont assume anything past 1st and last calibration points because you don't know if linearity continues forever -sample signal must be in range if too high then dilute it -Calibration standards must "bracket" unknown concentration -if its not then fix it adding more sample or diluting sample -May need "Matrix Matched" standards or use "method of standard additions" -matrix may have some kind of interference -instead of making standard in reagent water I would also have some other materials to mimic the blood -Internal standard does nothing for matrix interference -if complex matrix you probably won't want to use external standard curve Internal Standard Quantification: -Add a known amount of internal standard to the unknown sample -a related compound that is not the analyte is added to the sample before you do anything else -Signal from analyute is compared to signal from internal standard to determine amount of analyte in sample -Isotope dilution is the ideal way but not only way -Measure Calcium with magnesium as IS -Pick something that has same chemical and physical properties for internal standard I.S. Analyte (75%) 100ng 75ng -measure by ratio compared to standard -if we spill sample the ratio is s
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