Quantitative Analysis Laboratory: A New Approach Funded by the National Science Foundation

Determination of Calcium by Spectrophotometry

Background: Calcium is one the most important cations in any natural sample matrix. Calcium is the principle cation in limestone and is essential to many body functions including myocardial contraction.

Biological/Clinical Significance Calcium is essential to many functions in the human body including myocardial contraction. Over 99% of the calcium present in the human body is found in the bones while the majority of the remaining 1% is found in the blood. Blood calcium is distributed among several forms including free calcium ions, calcium ions bound to proteins like albumin, and calcium ions bound to anions like bicarbonate, lactate, phosphate, etc. Ionized serum calcium levels are regulated by three hormones whose secretion rate changes in response to the level of calcium. Ionized calcium, which may be the most important parameter for assessment of renal diseases and the monitoring of neonates, is difficult to measure owing to the difficulty in maintaining pH levels if carbon dioxide escapes from the sample. In addition, levels of many of the anions mentioned above can vary during illness making the assessment a normal calcium range difficult. A typical range for serum calcium levels is 2.18 -2.55 mmol/L in adults and 2.15 - 2.65 mmol/L in children.

Environmental Significance Calcium is the cation usually found in highest concentrations in fresh water systems. The primary source of calcium in fresh water are minerals (primarily gypsum CaSO4*2H2O, dolomite CaMg(CO3)2, and calcite and aragonite which are forms of CaCO3. Calcium plays a key role in many geochemical processes and is one of the principle cations involved in water hardness.

Emphasis and Technique: This experiment emphasizes the use of spectrophotometric methods methods and the practical application of Beers Law. The analysis involves the use complexation agents and illustrates the relative strengths of these agents. In addition, the determination is actually an indirect method as the amount of one of the complexation agents is measured..
1 Dorey and Draves University of Central Arkansas Department of Chemistry Conway, AR 72035 Update:5/98

Quantitative Analysis Laboratory: A New Approach Funded by the National Science Foundation

Chemistry of the Analysis Spectrophotometric analysis relies on the interaction of electromagnetic radiation (light) with the matter of interest. Strictly speaking, every compound has a distinct absorption spectrum which allows its identification, in many cases, in the presence of other compounds. In addition to the identification of a compound, it is also possible to determine quantitatively the concentration of that compound. The relationship between absorbance and concentration is given by the Lambert-Beer Law and is written mathematically as: A = c where A is the absorbance, a unit-less quantity; is the molar absorptivity constant ( a constant of the compound having units of L*mol-1*cm-1); and is the path length over which the light interacts with the sample in cm. In a more practical sense, the absorbance is defined as the negative logarithm of the transmittance This is given mathematically as: A = log T = log I I0

where I is the intensity of the light beam when the sample is present; and I0 is the intensity of the light beam when everything but the sample is present. ( in some cases this is called a background or a blank) In analytical spectrophotometry, the molar absorptivity of the unknown compound may not be known. It is therefore a common practice to generate a calibration or standard curve. A standard curve is generated by measuring the absorbance of a series of samples for which the concentration is known. Then, since the Lambert -Beer Law shows a linear relationship between absorbance and concentration, a linear least squares fit of the standard curve will yield a mathematical relationship between the absorbance and concentration. This relationship in turn can them be used to calculate the concentration of the unknown sample.

2 Dorey and Draves University of Central Arkansas Department of Chemistry Conway, AR 72035 Update:5/98

Quantitative Analysis Laboratory: A New Approach Funded by the National Science Foundation

In this analysis, the calcium is initially bound by o-creosolphthalein complexone (CPC). CPC has a max at 575 nm, which shifts upon complexation to a metal ion. The reaction proceeds as follows: CPC Ca-CPC (575 nm) Measurement of the absorbance at 575 nm of a solution containing calcium and CPC will thus yield the absorbance of only the free CPC. The structure of CPC is: Ca2+ +
-

CH2CO2 HO2CH2C N+ H

OH CH3 CH2CO2 CH3 H2C N+ H

-

H2 C

CH2CO2H

H3C OH O CH3 O

o-Cresolphthalein Complexone

Upon the addition of the EGTA, the calcium will preferentially bind the EGTA releasing free CPC. This reaction, whose equilibrium lies far to the right, proceeds as follows: Ca-CPC + EGTA Ca-EGTA + CPC

Measurement of the absorbance after the addition of the EGTA is then a measurement of the total free CPC. The difference between the two absorbance measurements is related to the amount of CPC which was bond to the calcium and thus, to the concentration of the calcium ions in the sample. Since we are measuring the absorbance of the complexation agent CPC, we are indirectly determine the amount of calcium present in the sample. Indirect determinations of analytes are useful when there are no reliable direct methods. The structure of EGTA is:
HOOCH2C N HOOCH2C CH2CH2OCH2CH2OCH2CH2 N CH2COOH CH2COOH

EGTA [ethyleneglycol bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid

3 Dorey and Draves University of Central Arkansas Department of Chemistry Conway, AR 72035 Update:5/98

Quantitative Analysis Laboratory: A New Approach Funded by the National Science Foundation

Reagents: 1. Hydrochloric acid, concentrated. 2. Stock calcium standard, 100 mg/dL. Dissolve 0.625 g of calcium carbonate in 25 mL of distilled water and add 2 mL of concentrated HCl. When all calcium carbonate is dissolved, dilute to 100 mL in a volumetric flask with distilled water. 3. Dimethyl sulfoxide (DMSO), reagent grade. 4. 8-hydroxyquinoline 5. Cresolphthalein complexone (CPC). Dissolve 0.625 g of 8-hydroyquinoline in 25 mL of DMSO. Add 10.0 mg of CPC and shake until dissolved. Add 75 mL of water and mix. Add 250 L of concentrated HCl, mix, and dilute to 250 mL in a volumetric flask with distilled water. 6. Potassium cyanide, reagent grade. 7. Diethylamine buffer. Dissolve 0.025 g of KCN in 40 mL of distilled water. Add 2.0 mL of diethylamine and dilute to 50 mL in a volumetric with distilled water. Store in a polyethylene bottle. 8. Ethylene glycol, bis-(2-aminoethyl ether) tetraacetic acid (EGTA). Prepare by dissolving 0.5 g of EGTA in 90 mL of water. Dilute to 100 mL in a volumetric flask with distilled water.

Procedure: 1) Generate a series of working standards by diluting the stock calcium standard. Youll need the following concentrations: 2 mg/dL, 4 mg/dL, 6 mg/dL, 8 mg/dL and 10 mg/dL. 2) Prepare 7 cuvettes, the first five containing 10 L of a working standard, one containing 10L of water (reagent blank), and one containing 10 L of serum unknown. 3) Add 1.0 mL of CPC reagent to each of the cuvettes. 4) Add 1.0 mL of diethylamine buffer to each cuvette and 1.0 mL of water, mix by inversion let stand five minutes. 5) Zero the Spec 20 at 575 nm using the reagent blank. Measure the absorbance of each of the remaining cuvettes. 6) Add 50 L of EGTA solution to each cuvette, mix by inversion. 7) Again, zero the Spec 20 at 575 nm using the reagent blank. Measure the absorbance of each of the remaining cuvettes. 8) Take the difference between the absorbance before the addition of the EGTA and the absorbance after the addition of EGTA. Perform a linear least squares fit to the plot of the difference in absorbance vs concentration. Use this line to determine the concentration of the unknown.

4 Dorey and Draves University of Central Arkansas Department of Chemistry Conway, AR 72035 Update:5/98