Introduction

Orthophosphates, pyro-, meta-, and other polyphosphates and organophosphates are phosphorus compounds found in natural waters and wastewaters as phosphates. They are found in solution, in particulate or powder form, or in the form of aquatic organisms.

Different forms of phosphate arise from different sources. Small amounts of orthophosphates or some concentrated phosphates are added to some water supplies during treatment. Large amounts of similar compounds may be added during washing or cleaning, as they are major ingredients in many commercial detergents. Phosphates are widely used in boiler water treatment. Orthophosphates used as fertilizers on agricultural or residential land enter surface waters with stormwater runoff or snowmelt. Organic phosphates are formed by biological processes. They help to remove waste water and food residues and may also be produced from orthophosphates during biological processes or by water source biota.

Phosphorus is essential for the growth of organisms and can act as a nutrient that limits the initial fertility of a body of water. Where phosphate is a growth-limiting nutrient, discharges of raw or treated sewage, agricultural runoff, or some industrial wastes into the water can promote the growth of photosynthetic aquatic micro- and macro-organisms to levels that are disruptive.

Phosphates are also present in bottom sediments and in environmental sludges, both as precipitated mineral acids and as organic compounds.

Phosphorus analysis involves two general steps:

• Conversion of the phosphorus form to soluble orthophosphate
• Colorimetric determination of soluble orthophosphate.

Separation of phosphorus into its various forms is defined by analytical methods, but analytical separations are used for interpretative purposes.

Filtration through a 0.45 μm-pore diam membrane filter separates soluble forms of phosphorus from suspended forms. There is no claim that filtration through 0.45 µm filters is a correct separation method for separating the soluble and suspended forms of phosphorus. It is simply a convenient and reproducible analytical method designed for general separation. Pre-filtration through a glass fiber filter can be used to increase the filtration rate.

Phosphates that respond to colorimetric tests without prior hydrolysis and oxidative digestion are referred to as “reactive phosphorus.” While reactive phosphorus is primarily a measure of orthophosphate, a small fraction of dense phosphate is usually hydrolyzed in this method. Reactive phosphorus exists in both dissolved and suspended forms.

Acid hydrolysis at boiling water temperatures converts dissolved phosphates and phosphate particles to orthophosphate. Hydrolysis inevitably releases some phosphate from organic compounds, which can be minimized by proper selection of acid strength and hydrolysis time and temperature. The term “acid-hydrolyzable phosphorus” is more commonly used for this phosphorus than “condensed phosphate.”

The breakdown of phosphate to orthophosphate, which occurs by the breakdown of organic matter, is known as “organic” phosphorus. The intensity of the oxidation required for this conversion depends on the form and, to some extent, the amount of organic phosphorus present. Like reactive phosphorus and acid-hydrolyzable phosphorus, organic phosphorus exists in both dissolved and suspended forms.

Total phosphorus, as well as soluble phosphorus and suspended phosphorus, may each be broken down into three chemical classes: reactive, acid-hydrolyzable, and organic phosphorus. As noted, measurements are usually made only on unfiltered and filtered samples. Suspended phosphorus is generally determined by its differences. However, it can be determined directly by digestion of material retained on a glass fiber filter.

Method Selection

a) Digestion Methods: Since phosphorus may be present in combination with organic matter, the digestion method for the determination of total phosphorus must be capable of effectively oxidizing the organic matter to release phosphorus as orthophosphate. The perchloric acid method is the most hazardous and time-consuming method, which is recommended only for very hard samples such as sediment. The nitric acid-sulfuric acid method is recommended for most samples. The simplest method is the persulfate oxidation method. Persulfate oxidation is used in conjunction with ultraviolet light for a more efficient digestion in an automated in-line digestion/determination, with flow injection analysis.

The persulfate oxidation method is a digestion method that can be used for both total nitrogen and total phosphorus analysis. This method can be used for both parameters, as it occurs over a wide pH range. In the initial stage of digestion, the pH of the sample is alkaline (pH > 12); in the final stage, the pH of the sample becomes acidic. As a result, nitrogenous compounds are oxidized to nitrate and phosphate to orthophosphate.

It is recommended that persulfate oxidation methods be compared against one or more more robust digestion methods and used if similar results are obtained.

b) Colorimetric method: Three methods for the determination of orthophosphate are described. The choice of method depends mainly on the concentration range of orthophosphate. The vanadomolybdophosphoric acid method (4500-PC) is useful for routine analysis in the range of 1 to 20 mg P/L. The zinc chloride method (4500-P.D) or the ascorbic acid method (4500-P.E) are suitable for the range of 0.01 to 6 mg P/L. An extraction step is recommended for levels below this range to eliminate the effect of interfering substances. Automated versions of the ascorbic acid method (4500-P.F, G, and H) are also provided. Careful attention to the method may make it possible to use these methods for very low levels of phosphorus, such as those found in freshwater systems.

Ion chromatography (Section 4110) and capillary ion electrophoresis (Section 4140) are used to determine orthophosphate in immobilized samples.

Sampling and Storage

If soluble phosphorus forms need to be distinguished, filter the sample immediately after collection. Store frozen and below 10°C. In some cases, 40 mg HgCl2/L may be added to the sample, especially when stored for long periods before analysis. WARNING: HgCl2 is a hazardous substance; take appropriate precautions. The use of HgCl2 is not recommended. When determining the phosphorus form, do not add acid or CHCl3 as a preservative. If total phosphorus only needs to be determined, add H2SO4 or HCl to pH <2 and cool to 4°C or freeze without any additions.

Do not store samples containing low concentrations of phosphorus in plastic bottles unless stored frozen because phosphates may adsorb to the walls of plastic bottles.

Wash all glassware with strong dilute HCl, then rinse several times in water. Never use commercial detergents containing phosphates to clean containers used in phosphate analysis. More specific washing methods may be used.

Required equipment:

Spectrophotometer, sensitive electrical balance, required glassware

Reagents:

1. Aqueous solution of phenolphthalein indicator
2. Concentrated sulfuric acid
3. Ammonium molybdate reagent: Dissolve 25 grams of ammonium molybdate tetrahydrate in 175 ml of distilled water and carefully add 280 ml of concentrated sulfuric acid to 400 ml of distilled water, cool, then add it to the molybdate solution and dilute to one liter.
4. Stannous chloride reagent: Dissolve 2.5 grams of stannous chloride tetrahydrate SnCl2 2H2O in 100 ml of glycerol and then place it in a hot water bath and stir to dissolve more quickly. This reagent is stable and does not require special storage or preservatives.
5. Phosphate Standard Stock Solution: Dissolve 219 mg of anhydrous KH2Po4 in distilled water and make up to 1 liter. 1mil = 50 μgP-PO
6. Prepare 2-tenths mg/liter phosphate standards.

If high sensitivity is desired or if interferences are high, the sample should be extracted according to the Standard Method (2005) instructions.

Phosphorus test method in water:

Add one drop of phenolphthalein to 100 ml of sample or a diluted volume of sample to 100 ml. If the solution turns pink, add sulfuric acid drop by drop until it becomes colorless. If more than 5 drops are used, select a smaller volume of sample. In the next step, add 0 ml of stannous chloride to each of the standards and samples, and after each 4 ml of molybdate reagent and mix 5 increments. The intensity of the color depends on the temperature of the solution (each degree of increase is one percent increase in color). Therefore, samples, standards and reagents should be stored at 2 degrees apart from each other in the range between 20 and 30 degrees Celsius.

After ten minutes and less than 12 minutes, read the light absorption at a wavelength of 690 nm against the blank. A distilled water blank should be used. The light path length is proportional to the phosphorus concentration range according to the table below.

Calculation from the standard curve, based on the phosphorus absorption rate, we obtain the phosphorus concentration and determine the phosphorus concentration in the sample using the following formula.