Abstract
Phosphorus is a vital nutrient involved in microbial growth. Its dissolved forms in water are generally classified in three groups: orthophosphates, condensed phosphates (polyphosphates), and organic phosphates. Analysis of dissolved phosphate concentrations can be performed using spectrophotometry by measuring the absorbance or transmittance of the sample solution. In this experiment, a standard curve for phosphate was generated through spectrophotometry to determine the phosphate concentrations in two water samples. These concentrations were found to be 0.8 mg/L and 1.5 mg/L.
Introduction
Phosphorus (P) is an essential nutrient in the growth of microorganisms. Typically, domestic wastewater contains between 4-16 mg/L of P (Metcalf, 2003). Excessive amounts in water may lead to eutrophication, giving rise to algal blooms.
Dissolved P is mostly in the form of phosphates (PO4-3). The principal forms of PO4-3 in aqueous solutions are categorized in three groups: orthophosphates, condensed phosphates or polyphosphates, and organic phosphate. Orthophosphate is readily available for microbial uptake. Polyphosphate contains at least two atoms of P attached to atoms of oxygen (and sometimes also with hydrogen) forming a complex molecule. It hydrolyzes back to the orthophosphate from which it is derived; however, the hydrolysis occurs at very slow rates (Metcalf 2003, Sawyer 1994). Organic phosphate is more important in sludges from industrial and wastewater treatment rather than in domestic wastewater.
Phosphates may come from anthropogenic and natural sources. Toilet flushings and laundry soaps account for majority of the PO4-3in domestic wastewater. Fertilizers and some pesticides contain polyphosphates and organic PO4-3, respectively, that eventually end up in agricultural run-offs. A number of inorganic phosphate compounds are also used to control scaling in boilers and pipes. Natural sources include decayed organic matter and mineral deposits. In sediments, adsorbed PO4-3may be released with changes in temperature and pH.
The concentration of phosphates in aqueous solutions is typically expressed in milligrams per liter of total P (or mg/L total P). Analytical techniques for determining PO4-3 concentrations include volumetric, gravimetric, and colorimetric methods. Spectrophotometry is a colorimetric method that is premised on the conformity of the analyte solution to Lambert’s and Beer’s laws. These laws, when combined, relate the concentration of a chemical specie in solution to the absorbance and transmittance of light energy that is passed through the solution. A standard curve (also known as reference or calibration curve) is first plotted using known concentrations of phosphates. The absorbance and transmittance of samples containing unknown concentrations of phosphates may then be determined using the standard curve.
This experiment makes use of spectrophotometry in determining the orthophosphate concentration in artificially prepared water samples. By the end of the experiment, the student is expected to gain familiarity with the functions of the spectrophotometer and with the spectrophotometric method of phosphate determination. Further, the student should be able to identify the limitations on the use of the standard curves generated in this experiment for future phosphate analyses.
Materials and Methods
The following materials were used: Hach® spectrophotometer, cuvettes or 10 mL small test tubes with caps, autopipettes, disposable pipette tips, small beaker or flash, organic-free deionized (DI) water, 10 mg/L standard phosphate solution, and Phosver® ascorbic acid pillows.
Preparation of Phosphate Dilutions and Analytes
Dilutions of 10-mL standard phosphate solutions were prepared in increasing concentrations of 0.0, 0.5, 1.5, and 2.5 mg/L PO4-3. Using autopipettes, varying proportions (refer to Table 1) of DI
water and standard PO4-3 solution were prepared in 10-ml cuvettes / tubes. 10 mL of the unknown samples was also placed in separate cuvettes / tubes.
Table 1: Standard Dilutions’ Compositions
| Sample Name | Volume water, mL | Volume standard PO4-3 |
| (Concentration, mg/L) | solution, mL | |
| Reagent Blank (0.0) | 10 | 0.0 |
| Dilution 1 (0.5) | 9.5 | 0.5 |
| Dilution 2 (1.5) | 8.5 | 1.5 |
| Dilution 3 (2.5) | 7.5 | 2.5 |
One Phosver® ascorbic acid pillow was dissolved into each cuvette / tube to develop the blue color of the solution. The samples was allowed to stand at least two minutes before transmittance measurements were taken to allow excess ascorbic acid to settle to the bottom of the cuvette / tube. The solution containing 0.0 mg/L phosphate was designated as the reagent blank…..CONTINUED
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