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A sensitive, rapid determination of inorganic phosphate in presence of labile phosphate esters
SHORTCOMMUNICATIONS
295
A few typical analyses obtained by the above method are given in Table I, from which it will be seen that the accuracy attainable is quite satisfactory. The coefficient of variation when analysing a solution of 0.543 j&ml was 1.7 which compares very favourably with the figures for the other methods*. ACKNOWLEDGEMENT We are grateful to the Tata Iron & Steel Company for permission to publish this paper. G. V. L. N. MURTY T. S. VISWANATHAN
Chemical Laboratory, Tata Iron G Steel Comfiany, Jamshed$ur(India)
W. N. ALDRIDGE, Analyst, 70 (1945) 474. J. EPSTEIN, Anal. Chem., 19 (1947) 273. 3 E. ASMUS AND H.GARscHAGEN,Z.~~~C.C~~~~., 1 2
4 F.J.LuDzAcK,W.
A.MOOREANDC.C.RUCKHOFP,
138 (1953)414. Anal. Chem..26(1954)
1784.
Received February zoth, 1961 Anal. Chim. Acta, 25 (1961) 293-295
A sensitive, rapid determination of inorganic phosphate in presence of labile phosphate esters In a previous paper1 a determination of phosphate by a modified method based on that of BERENBLUM ANDCHAINwas described. The method is very sensitive and a large excess of sulphate does not interfere, while labile phosphate esters are not hydrolysed because of the short duration of the action of the acid ammonium molybdate solution (less than 30 set). A disadvantage of the method is the elaborate procedure, as the phosphate has to be extracted first with butanol-benzene, after which the mixture is washed with water and finally reduced by shaking with stannous chloride. The separatory funnel must therefore be opened, and the stopper rinsed, three times. The present paper describes a more convenient procedure. In order to avoid rinsing, the stopper of the separatory funnel is altered to a small funnel. The reduction by shaking with a stannous chloride solution is replaced by reduction with this reagent in a homogeneous alcoholic phase. If a large number (e.g. IO) of separatory funnels are attached to a revolving bar, IO samples can be shaken simultaneously, which renders the method suitable for routine determinations. The objection of MORI AND NAKAMURA~ to the method of BERENBLUM AND CHAIN, i.e. its being complicated and time-consuming, is met by these three improvements. Therefore, the method of BERENBLUM AND CHAIN remains preferable to that involving a single phase, because of the hydrolysis which occurs in the single phase, and because of the great susceptibility to interference by sulphate ions. Anal. Chim. Acta. 25 (1961) 295-297
296
SHORTCOMMUNICATIONS
Apparatus In the neck of the separatory funnel (Fig. I) a hole (I) is made; opposite this hole lies a small groove(IV), similar to those in dropping bottles. The small funnel has a similar groove (II) in its solid lower half, and a hole (III) leading to the interior of the separatory funnel. When the two holes are placed opposite the grooves, liquid can be poured into the separatory funnel (via arrow B), while the air can escape (via arrow A). When the small funnel is turned through 90’ the separatory funnel is closed and can be shaken. Degassing by turning the lower cock is no longer necessary. The whole apparatus is made water-repellent by treatment with Desicote.
IV
II
-isib OpS”
closed
Fig. I.
Reugertts (1) Dissolve 5 g of (NH&Mor0~-4HsO’p. a. in IOO ml of 2.5 N sulphuric acid. 5 g of (NH&Mo,OLU-~HZO p. a. in IOO ml of water. (3) Dissolve z g of hydrazine sulphate in I 1 of 0.6 N sulphuric acid, cool to about IO’, and add I g of stannous chloride. This solution becomes clear after about 12 h in a refrigerator and can be kept for about 4 weeks, (4) Isobutanol p.a. or distilled (fraction 107-108.5”). (2) Dissolve
Procedwe TO 25 or 35 ml of the sample add 5 ml of reagent (I), when the sample contains less than 12.5 mequiv. of sulphuric acid; add 5 ml of reagent (2) when it contains 12.5-24 mequiv. of sulphuric acid. Then introduce 15 ml of isobutanol into the separaAnal.
Chim.
Acta,
25 (1961)
295-297
297
SHORTCOMMUNICATIONS
tory funnel. Close the funnel by turning the stopper through go’, and shake. Then turn the stopper back and remove the lower layer. Shake the upper layer with 15 ml of 0.6 N sulphuric acid and subsequently rinse the upper layer with ethanol (96%) into a volumetric flask of a ml. While shaking the flask add OC/IOOml of the stannous solution (3) and dilute the contents to volume. Measure the extinction e.g. with a Zeiss Colorimeter ELK0 III, filter 72. Depending on the amount of phosphate, 0~r= 25, 50 or 100 ml. When the concentration of phosphorus as phosphate is less than about 2 pug/q ml, a larger sample must be used along with a proportionally larger amount of reagent (I) or (2).In this case 15 ml more of isobutanol than is required for saturation of the sample must be added. The other solutions are used in normal quantities. When, for example in limnology, determinations are carried out on samples of 500 ml, alcohols such as +z-hexanol (or mixtures) are preferable, because they are less soluble in water. However, the time needed for the separation is then much longer. When more than 24 mequiv. of sulphuric acid is present, the surplus must be neutralised with sodium hydroxide. If more than I g of sodium sulphate is formed per 25 ml of sample, the sample must be extracted twice. The method has been applied succesfully to sea-water. Even 5% of sodium chloride added to a phosphate stock solution in water does not interfere with the determination. The amount of phosphate that can be determined in this way is equivalent to 1-200 ,ugof phosphorus. The calibration graph is straight in this range. The experimental error is about 1.5%.
The method is biochemically attractive because no interference arises from the hydrolysis of labile phosphate esters, nor from a large excess of sulphate or chloride. The latter quality and the great sensitivity (2pugof phosphate-phosphorus in 500 ml) render the method suitable for limnology and oceanography. Hydrobiological Institzcte, Nieuwersluis
(The Netherlands)
1 H.L. GOLTERMAN,A&Z Bolan.,~ (1960) I. s K. MORI AND M. NAKAMURA. Bull. Agric. Chem. Soc.Japan,
H.L. GOLTERMAN I. M. WURTZ
23 (1959) 272.
Received June 3rd, 1961 Anal.Chim. Acta. 25 (1~61)295-2~7
295
A few typical analyses obtained by the above method are given in Table I, from which it will be seen that the accuracy attainable is quite satisfactory. The coefficient of variation when analysing a solution of 0.543 j&ml was 1.7 which compares very favourably with the figures for the other methods*. ACKNOWLEDGEMENT We are grateful to the Tata Iron & Steel Company for permission to publish this paper. G. V. L. N. MURTY T. S. VISWANATHAN
Chemical Laboratory, Tata Iron G Steel Comfiany, Jamshed$ur(India)
W. N. ALDRIDGE, Analyst, 70 (1945) 474. J. EPSTEIN, Anal. Chem., 19 (1947) 273. 3 E. ASMUS AND H.GARscHAGEN,Z.~~~C.C~~~~., 1 2
4 F.J.LuDzAcK,W.
A.MOOREANDC.C.RUCKHOFP,
138 (1953)414. Anal. Chem..26(1954)
1784.
Received February zoth, 1961 Anal. Chim. Acta, 25 (1961) 293-295
A sensitive, rapid determination of inorganic phosphate in presence of labile phosphate esters In a previous paper1 a determination of phosphate by a modified method based on that of BERENBLUM ANDCHAINwas described. The method is very sensitive and a large excess of sulphate does not interfere, while labile phosphate esters are not hydrolysed because of the short duration of the action of the acid ammonium molybdate solution (less than 30 set). A disadvantage of the method is the elaborate procedure, as the phosphate has to be extracted first with butanol-benzene, after which the mixture is washed with water and finally reduced by shaking with stannous chloride. The separatory funnel must therefore be opened, and the stopper rinsed, three times. The present paper describes a more convenient procedure. In order to avoid rinsing, the stopper of the separatory funnel is altered to a small funnel. The reduction by shaking with a stannous chloride solution is replaced by reduction with this reagent in a homogeneous alcoholic phase. If a large number (e.g. IO) of separatory funnels are attached to a revolving bar, IO samples can be shaken simultaneously, which renders the method suitable for routine determinations. The objection of MORI AND NAKAMURA~ to the method of BERENBLUM AND CHAIN, i.e. its being complicated and time-consuming, is met by these three improvements. Therefore, the method of BERENBLUM AND CHAIN remains preferable to that involving a single phase, because of the hydrolysis which occurs in the single phase, and because of the great susceptibility to interference by sulphate ions. Anal. Chim. Acta. 25 (1961) 295-297
296
SHORTCOMMUNICATIONS
Apparatus In the neck of the separatory funnel (Fig. I) a hole (I) is made; opposite this hole lies a small groove(IV), similar to those in dropping bottles. The small funnel has a similar groove (II) in its solid lower half, and a hole (III) leading to the interior of the separatory funnel. When the two holes are placed opposite the grooves, liquid can be poured into the separatory funnel (via arrow B), while the air can escape (via arrow A). When the small funnel is turned through 90’ the separatory funnel is closed and can be shaken. Degassing by turning the lower cock is no longer necessary. The whole apparatus is made water-repellent by treatment with Desicote.
IV
II
-isib OpS”
closed
Fig. I.
Reugertts (1) Dissolve 5 g of (NH&Mor0~-4HsO’p. a. in IOO ml of 2.5 N sulphuric acid. 5 g of (NH&Mo,OLU-~HZO p. a. in IOO ml of water. (3) Dissolve z g of hydrazine sulphate in I 1 of 0.6 N sulphuric acid, cool to about IO’, and add I g of stannous chloride. This solution becomes clear after about 12 h in a refrigerator and can be kept for about 4 weeks, (4) Isobutanol p.a. or distilled (fraction 107-108.5”). (2) Dissolve
Procedwe TO 25 or 35 ml of the sample add 5 ml of reagent (I), when the sample contains less than 12.5 mequiv. of sulphuric acid; add 5 ml of reagent (2) when it contains 12.5-24 mequiv. of sulphuric acid. Then introduce 15 ml of isobutanol into the separaAnal.
Chim.
Acta,
25 (1961)
295-297
297
SHORTCOMMUNICATIONS
tory funnel. Close the funnel by turning the stopper through go’, and shake. Then turn the stopper back and remove the lower layer. Shake the upper layer with 15 ml of 0.6 N sulphuric acid and subsequently rinse the upper layer with ethanol (96%) into a volumetric flask of a ml. While shaking the flask add OC/IOOml of the stannous solution (3) and dilute the contents to volume. Measure the extinction e.g. with a Zeiss Colorimeter ELK0 III, filter 72. Depending on the amount of phosphate, 0~r= 25, 50 or 100 ml. When the concentration of phosphorus as phosphate is less than about 2 pug/q ml, a larger sample must be used along with a proportionally larger amount of reagent (I) or (2).In this case 15 ml more of isobutanol than is required for saturation of the sample must be added. The other solutions are used in normal quantities. When, for example in limnology, determinations are carried out on samples of 500 ml, alcohols such as +z-hexanol (or mixtures) are preferable, because they are less soluble in water. However, the time needed for the separation is then much longer. When more than 24 mequiv. of sulphuric acid is present, the surplus must be neutralised with sodium hydroxide. If more than I g of sodium sulphate is formed per 25 ml of sample, the sample must be extracted twice. The method has been applied succesfully to sea-water. Even 5% of sodium chloride added to a phosphate stock solution in water does not interfere with the determination. The amount of phosphate that can be determined in this way is equivalent to 1-200 ,ugof phosphorus. The calibration graph is straight in this range. The experimental error is about 1.5%.
The method is biochemically attractive because no interference arises from the hydrolysis of labile phosphate esters, nor from a large excess of sulphate or chloride. The latter quality and the great sensitivity (2pugof phosphate-phosphorus in 500 ml) render the method suitable for limnology and oceanography. Hydrobiological Institzcte, Nieuwersluis
(The Netherlands)
1 H.L. GOLTERMAN,A&Z Bolan.,~ (1960) I. s K. MORI AND M. NAKAMURA. Bull. Agric. Chem. Soc.Japan,
H.L. GOLTERMAN I. M. WURTZ
23 (1959) 272.
Received June 3rd, 1961 Anal.Chim. Acta. 25 (1~61)295-2~7