Approaches to Quantiﬁcation:
-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!
""""" 100ng" " 75ng!
-measure by ratio compared to standard!
-if we spill sample the ratio is still relevant so we still know how much we started with!
-CON: hard to ﬁnd analyte and internal signal do not always give you the same intensity of signals!