Phosphate molybdate blue method

Fig. 4.17 is a plot of phosphate concentration given by the enzymatic method vs that found by the molybdate blue method. The line fit to these points had a slope of 0.999 0.046 and an intercept of 0.421 1.222 with a correlation coefficient of 0.996. High performance liquid chromatography has also been linked to an inductively coupled plasma atomic spectrometric detector [322,323] to determine orthophosphate, pyrophosphate and tripolyphosphate. 

Colorimetric Techniques The most familiar colorimetric technique is the molybdate blue method (Koch and Koch-Dedic 1964). Phosphate ion (P04 ), when in solution, reacts with a molybdate ion to form a heteropoly acid complex, H7[P(Mo207)5], that after use of suitable reduction agent is reduced to a phosphoro-molybdate blue complex, with an absorption maximum at 820-830 nm. The most popular reduction agents are chlorostannous (tin II dichloride) acid reductant, sodium bisulfite (monosodium, monohydrogen sulfate (IV)) and ascorbic acid. 

Molybdenum blue method. When arsenic, as arsenate, is treated with ammonium molybdate solution and the resulting heteropolymolybdoarsenate (arseno-molybdate) is reduced with hydrazinium sulphate or with tin(II) chloride, a blue soluble complex molybdenum blue is formed. The constitution is uncertain, but it is evident that the molybdenum is present in a lower oxidation state. The stable blue colour has a maximum absorption at about 840 nm and shows no appreciable change in 24 hours. Various techniques for carrying out the determination are available, but only one can be given here. Phosphate reacts in the same manner as arsenate (and with about the same sensitivity) and must be absent. 

A. Molybdenum blue method Discussion. Orthophosphate and molybdate ions condense in acidic solution to give molybdophosphoric acid (phosphomolybdic acid), which upon selective reduction (say, with hydrazinium sulphate) produces a blue colour, due to molybdenum blue of uncertain composition. The intensity of the blue colour is proportional to the amount of phosphate initially incorporated in the heteropoly acid. If the acidity at the time of reduction is 0.5M in sulphuric acid and hydrazinium sulphate is the reductant, the resulting blue complex exhibits maximum absorption at 820-830 nm. 

B. Phosphovanadomolybdate method Discussion. This second method is considered to be slightly less sensitive than the previous molybdenum blue method, but it has been particularly useful for phosphorus determinations carried out by means of the Schoniger oxygen flask method (Section 3.31). The phosphovanadomolybdate complex formed between the phosphate, ammonium vanadate, and ammonium molybdate is bright yellow in colour and its absorbance can be measured between 460 and 480 nm. 

The accelerating effect of US on the determination of phosphate by the Molybdenum Blue method was ascribed to depolymerization of molybdate, which was thought to speed up its reaction with phosphate and inorease the sensitivity [32]. 

Early colorimetric methods for arsenic analysis used the reaction of arsine gas with either mercuric bromide captured on filter paper to produce a yellow-brown stain (Gutzeit method) or with silver diethyl dithiocarbamate (SDDC) to produce a red dye. The SDDC method is still widely used in developing countries. The molybdate blue spectrophotometric method that is widely used for phosphate determination can be used for As(V), but the correction for P interference is difficult. Methods based on atomic absorption spectrometry (AAS) linked to hydride generation (HG) or a graphite furnace (GF) have become widely used. Other sensitive and specihc arsenic detectors (e.g., AFS, ICP-MS, and ICP-AES) are becoming increasingly available. HG-AES, in particular, is now widely used for routine arsenic determinations because of its sensitivity, reliability, and relatively low capital cost. 

Standard methods for phosphates, polyphosphates, and organic phosphates in environmental samples are predominantly nonchromatographic methods, which are based upon the molybdenum blue method. Within this colorimetric method ammonium molybdate and antimony potassium tartrate react under acidic conditions with dilute solution of phosphorous to form an antimony-phospho-molybdate complex which is then reduced to an intensely blue-colored complex by ascorbic acid. U.S. EPA Methods 365.1 to 365.4 are based upon this chemistry. 

A prerequisite for the molybdenum blue method is that all the arsenic has to be present as arsenate. After digestion with oxidizing acids, such as nitric acid, all the arsenic is converted into arsenate when appropriate heating time and temperatures are applied. The principle of this determination is the reaction of arsenate with ammonium molybdate in acidic medium to form an arsenate containing molybdenum heteropolyacid that can be reduced to molybdenum blue with stannous chloride, hydrazine, or ascorbic acid. Best results are obtained with hydrazine sulfate. The absorption maximum of the blue solution is between 840-860 nm (15). The most severe interferences for this method derive from phosphates and silicates. To remove interfering ions, distillation of arsenic as AsCb or AsBrs is often recommended (12,15). 

Total phospholipids are quantitated by determination of the phosphorus content of lipid extracts from which non-lipid phosphorus has been removed by purification procedures. For this purpose chloroform-methanol extracts subjected to diffusion purification (Folch et al. 1951, Sperry 1955) are suitable. Lipid phosphorus is determined in aliquots of these extracts after digestion. Most procedures for determination of phosphorus are based on the method of Fiske and Subbarow (1925) which utilizes conversion of phosphate to phosphomolybdate and its subsequent reduction to molybdic blue. Modifications of this method were reviewed by Lindberg andERNSTER (1956). Avery convenient phosphorus assay was described by Bartlett (1959). Total phospholipids are calculated by multiplication of the lipid phosphorus values with 25. These values are only approximations since phosphorus does not represent exactly 4% of each phospholipid molecule. 

Phosphate in water may be determined according to a procedure outlined in [9], known as the "molybdenum blue method" It involves the complexation of phosphate with molybdate, with subsequent reduction of the complex with ascorbic acid The result is a complex having an intense blue color The overall reaction rate is limited by the complexation step, with maximum conversion of phosphate to the reduced complex requiring about 10 minutes This analytical procedure has been adapted by many groups for phosphate analysis in flow systems (see, for instance, [2, 10]) In one instance, a system was developed to monitor phosphate concentrations in fermentation broths [11] The flow manifold employed in that application is the model for the phosphate analysis using a stacked system described m this paper... 

Yoshimura et al. [193] carried out microdeterminations of phosphate by gel-phase colorimetry with molybdenum blue. In this method phosphate reacted with molybdate in acidic conditions to produce 12-phosphomolybdate. The blue species of phosphomolybdate were reduced by ascorbic acid in the presence of antimonyl ions and adsorbed on to Sephadex G-25 gel beads. Attenuation at 836 and 416 nm (adsorption maximum and minimum wavelengths) was measured, and the difference was used to determine trace levels of phosphate. The effect of nitrate, sulfate, silicic acid, arsenate, aluminium, titanium, iron, manganese, copper, and humic acid on the determination were examined. 

Olsen et al. [62] have described a method for the determination of pH8.5 sodium bicarbonate extractable phosphorus in soils. The concentration of the blue complex produced by the reduction, with ascorbic acid, of the phosphomolybdate formed when acid ammonium molybdate reacts with phosphate is measured spectrophotometrically at 880 nm [63]. 

A similar method is used for the determination of inorganic phosphates in urine. 1 to 5 c.e. of the urine, containing about 0-5 milligram of phosphorus, are diluted and treated with a solution of ammonium molybdate in 15 per cent, sulphuric acid (5 c.c.), 1 c.c. of 1 per cent, hydroquinone solution and 1 c.c. of 20 per cent, sodium sulphite solution. The blue colour is compared in Nessler glasses with that developed by the same solutions when mixed with a standard phosphate solution of which 5 c.c. contain 0-5 milligram of phosphorus. 

Riley, 1962 Oscarson et al 1980 Strickland and Parson, 1968). This method is based on the spectrophotometric measurement of the blue As(V) and P(V) molybdate complexes. Phosphate is measured in one sample after reducing As(V) to As(III), which does not form the color complex. In a second sample, both P(V) and As(V) are measured and the As(V) concentration determined by subtracting the P(V) concentration. A third sample is oxidized to convert As(III) to As(V), and the As(lll) concentration obtained by subtracting As(V) and P(V). 

In this method for the determination of phosphorus in solution, phosphorus (in the form of phosphate) combines with acid ammonium molybdate, forming phosphomolybdic acid. Ascorbic acid reduces the phosphomolybdic acid to give a blue-coloured complex. The antimony salt acts as a catalyst, speeding up the formation of the coloured complex. 

A more complex but sensitive IC method was developed by Antony et al. based upon separation using a Dionex AS4A-SC column and postcolumn derivitization of phosphate using a solution containing 0.5% w/v ammonium molybdate and 0.5% w/v bismuth nitrate, in 1.75 M H2SO4 and 0.75% ascorbic acid. The resultant reduced ion association complex absorbed strongly at 700 nm and a detection limit for phosphate (P) of an impressive 0.8 /rg/1. The above chemistry has also been exploited in a recent publication by Haberer and Brandes " who carried out precolumn derivitization of phosphate within freshwater and saltwater samples and then solvent extracted the resultant molybdenum blue complex prior to separation and detection (at 700 nm) using reversed-phase HPLC. 

A method of phosphate determination described in the German Standard Methods and in DIN 38405 similarly uses molybdate ions, in the presence of antimony ions, to form a complex which is reduced to phosphorus molybdenum blue by ascorbic acid and can be measured photometrically. Experience with this method has so far been good. 

Phosphate and other salts may be measured by various titrimetric and colorimetric methods. Phosphate is measured titrimetrically by precipitation of quinoline phosphomolybdate that is collected and dissolved in a small known excess of alkali and then back-titrated with standard acid. A colorimetric method based on the reaction of the phosphate with ammonium molybdate followed by partial reduction to give molybdenum blue has been developed for use in auto-analyzers. 

Generally silicate is a interference in the methodologies described previously. Methods have been developed for simultaneous determination of phosphate and silicate, based on kinetic information or using multivariate calibration. A kinetic separation method is based on the reaction of molybdenum blue and involves monitoring at different times. A multivariate calibration method proposed is also based on the reaction of molybdenum blue. Ascorbic acid and oxalic acid are added to the sample (molybdate/ antimony) for the formation of two molybdate... 

The benzidine reaction forms the basis of a very sensitive spot test. A spot of acidified phosphate solution is placed on a filter paper followed by a drop of ammonium molybdate solution and then the benzidine solution. The filter paper is held over ammonia to neutralise the free acid and a blue stain appears. Under these conditions there is no interference from silicate or arsenate if they happen to be present. The method is sensitive to about 1 part in 50,000. 

Another test for phosphorus (as phosphate) is based on the fact that the phosphomolybdates (and phosphotungstates) form deeply coloured insoluble lakes with triphenylmethane dyes such as methyl violet or malachite green (Chapter 12.8). The procedure is as follows a drop of test solution is placed on a filter paper which is then sprayed with a 1% solution of methyl violet. After about half a minute the paper is then sprayed with a solution of acidified ammonium molybdate and a blue spot develops. This method is sensitive to 1 part in 500,000. 

All methods for the determination of inorganic phosphate in seawater are based on the reaction of the ions with an acidified molybdate reagent to yield a phosphomolybdate heteropoly acid, which is then reduced to a highly coloured blue compound. In early work, tin(//) chloride was used as the reductant in flow-analysis (Hager et al, 1968). However, this reductant has several disadvantages, including the appreciable temperature dependence of the reduction rate and the pronounced salt error. 

Silicate, arsenate, and germanate also form heteropoly acids, which on reduction yield molybdenum blue species with similar absorption maxima [97]. This positive interference in the determination of phosphate is particularly pronounced for silicate because of its relatively high concentration in many waters. However, the formation of silicomolyb-date may be suppressed by the addition of tartaric or oxalic acid to the molybdate reagent [98]. If, however, the organic acid is added after the formation of the heteropoly acid, the phosphomolybdate is destroyed, and this is used as the basis for determination of silicate in the presence of phosphate. Kinetic discrimination between phosphate and silicate, arsenate and germanate is also possible because of the faster rate of formation of phosphomolybdate. Thus, the widely adopted Murphy and Riley method employs a reagent mixture of acidic molybdate and antimonyl tartrate [83] at concentrations which are known to enhance the kinetics of phosphomolybdate and suppress the formation of silicomolybdate. 

All methods for phosphate in sea water rely on the formation of a phospho-molybdate complex and its subsequent reduction to highly coloured blue compounds. Methods using stannous chloride as a reductant at room temperature have been fevoured as they arc most sensitive and give less interference from easily hydrolysable organic compounds than do other techniques. There are complexities in these methods due to interference from arsenic and to concealed blanks arising from the reduction of molybdate in sea water in the absence of phosphate. An excellent program of comparative tests has been described by Jones and Spencer (7. Marine Bid. Assoc. U.K., 43 251, 1963). 

Specific chemical methods can be used to reveal phospholipids, glycolipids, sterols or their esters as well as compounds with quaternary nitrogens or vicinal diols. Particularly useful for many membrane extracts is the reaction of ammonium molybdate with inorganic phosphate released from phospholipids. The phosphomolybdic acid so produced is then reduced to give an intense blue colour. The method can be adapted to a spray reagent or, more often, used to detect as little as 1 jug of phosphorus in scraped samples. 

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