Question

3.  (15 pts) For the following calibration techniques:  (a) Standard addition, (b) External calibration, and (c) Internal calibration:  Draw a picture of what each graph would look like, describe each technique, and give a reason why each might be used for quantification (an advantage of each).

*just wants a general example plot, not data is provided for this problem.*

Answer 1

a) Standard additions' is a generally applicable calibration technique, devised to overcome a particular type of matrix effect that would otherwise give rise to a biased result. This 'rotational effect' is manifested as a change in the slope of the calibration function.

The method is performed by reading the experimental (in this case fluorescent) intensity of the unknown solution and then by measuring the intensity of the unknown with varying amounts of known standard added. The data are plotted as fluorescence intensity vs. the amount of the standard added (the unknown itself, with no standard added, is plotted ON the y-axis). The least squares line intersects the x-axis at the negative of the concentration of the unknown, as shown in Figure 1.

Figure 1. Graphic representation of method of standard addition.

b) With external calibration, a sample containing a synthetic mixture of amino acids at known concentrations and internal standard(s) is run separately from the test samples (or patient samples). The external calibration sample is used to determine the signal-to-concentration relationship for each amino acid.

External calibration is the process of manually calibrating a balance with a predetermined mass. Basically, users set the weighing instrument to calibrate (manually or through menus with digital balances), put the calibration mass on the balance and verify that the weight displayed is correct.

c) Internal calibration is a process that uses the balance's inner mechanism to self-calibrate. ... This option adds cost to the balance price, but users don't need to buy a full calibration set to calibrate the balance. This feature is commonly found on high-grade analytical balances and microbalances.

DInternal standard calibration involves the comparison of the instrument responses from the target compounds in the sample to the responses of reference standards added to the sample or sample extract before injection.