A standard method for the determination of calcium in human serum is available as SP 260-36 (shown at the left) from the National Institute of Standards and Technology. The AA instrument will be set up to determine calcium at 422.7 nm using a stoichiometric air-acetylene flame with the burner head in the conventional position. A list of control materials for calcium in serum can be found on the WWW at http://www.pss.aus.net/products/lgc/section_1/BIOBLOOD.DOC.html. A good control to use is NIST SRM 909b (shown at the right). You prepare standards of 0 µg/mL (reagent blank), 1.000 µg/mL, 2.000 µg/mL, and 3.000 µg/mL and dilute the samples (replicate dilutions of the patient sample and of the SRM) by pipeting 1.000 mL of serum into 50.00-mL volumetric flasks containing water and diluting to the calibration mark. All standards and the reagent blank contain added sodium and potassium to match the serum matrix. A sample blank is prepared in the same manner as the diluted serum. A preliminary analysis using a calibration line is done to establish the approximate concentration of the diluted serum sample. The following signals (absorbance x 1000) are obtained.

Reagent blank: 5
1.000 µg/mL Ca standard: 241
2.000 µg/mL Ca standard: 441
3.000 µg/mL Ca standard: 620
Patient samples: 530
SRM 909b: 439
Sample blank: 0

From these values, determine the approximate concentration of the diluted serum sample. Then determine how to apply the method of bracketing to perform the analysis in the most accurate manner possible.

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