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The analytical separation of phosphate from natural water by ion exchange
D2ater Research Vo|. 8. pp. 467 to -1.70. Pergamon Press 1974. Printed in Great Britain.
THE ANALYTICAL SEPARATION OF PHOSPHATE FROM N A T U R A L WATER BY ION E X C H A N G E ALAN I). WESTLANDand ISAnELLEBOISCLAIR Department of Chemistry, University of Ottawa, Ottawa, Canada KIN 6N5
(Received 3 December 1973) Abstract--An ion exchange method has been developed for separating inorganic orthophosphate from natural water samples. The technique makes it possible to determine orthophosphate by the molybdophosphate blue procedure without interference from phosphate esters or certain other forms of bound phosphorus. Experiments to examine the conditions under which phosphate is found in natural ~aters suggest that adsorption by high molecular weight organic substances such as lignins may account for a large fraction of the soluble phosphorus.
INTRODUCTION The various forms in which phosphate can occur in natural water has been the subject of considerable speculation. Strickland and Parsons (1960) have classified them with some arbitrariness on the basis ofwhether they pass a Millipore HA filter and whether they react with acid molybdate within a 3 rain period. The molybdophosphate blue reaction is the basis of practically all methods of trace phosphorus determination and when applied directly to a filtered water sample it has usually been assumed to provide a measure of dissolved inorganic ~rthophosphate (DIP). However, Harvey (1948) has pointed out that under the acid conditions of the reaction, organic phosphate may be hydrolyzed. Chamberlain and Shapiro (1969) examined several methods of determination in order to ascertain whether one of the chemical methods would give results similar to a bioassay using algal cells. With most of the watc'rs tested, good agreement was found between the bioassay and a method in which the sample was treated with acid molybdate and immediately extracted into isobutanol. Since arsenic is not extracted under these conditions, the results led these workers to conclude that arsenic rather than hydrolysis of organic phosphate was responsible for poor agreement between most chemical analyses and the bioassay. However, it had earlier been shown (Martin and Doty, 1949) that the removal of protein prior to analysis by the molybdophosphate blue method resulted in a reduction of the indicated phosphorus levels. This suggests that the hydrolysis of organic phosphorus is a matter of importance. Westland and Langi'ord (1956) have shown that treatment of the water sample with an anion exchange resin leads to a discrimination between anionic nitrate and phosphate and other bound forms of these ions.
Other workers (Jones and Spencer, 1963: Rigler, 1968) had similar experience with Amberlite IRA-400. Rigler showed in addition that it is probable that the DIP constitutes only a fraction of the soluble reactive phosphorus (SRP) in certain lakewaters. We felt that a definitive answer to the question concerning which forms of phosphorus are bio-available to phytoplankton must be preceded by establishing a method for determining DIP specifically. The shortcoming of the standard methods is the uncertainty about whether hydrolysis of phosphate-containing compounds takes place. As the adsorption of anionic constituents on an ion exchange resin is not accompanied by drastic changes in solution conditions, particularly pH, we felt that this separation technique offered considerable promise as the basis for a more absolute method of determining DIP. METHODS
Water preparation Reagent solutions were prepared from twice distilled water. The second distillation was carried out in an all glass still. Synthetic water samples were prepared either from twice distilled water or distilled water which had been passed through Dowex l-X8 resin in the chloride form. This latter is referred to hereafter as "anion-frce" water, although it, of course, contained chloride ion. Natural water samples were filtered by suction through MiUipore HA (0A5 /am) membrane filters within a few minutes of collection in the case of river samples and within a few hours in the case of lake samples.
Colorimetric determination of phosphate. The procedure for molybdophosphate blue forma467
.*68
AL,~'qD. WF.STLANDand LSABELLEBOISCLAIR
tion was similar to that described by Harvey (1948). One ml of a 2.5 per cent solution of ammonium molybdate in 13.5 N H,SO,L was added to 50 ml of sample. This was followed by 0.15 ml of a 10 per cent solution of SnCI_, . 2 H , O in concentrated HC1 (s.g. 1.18). The final acid concentration was 03 N. The absorbance was determined after 5 rain in ceils having a 10 cm. light path. The color of natural samples was compensated by using such water as the reference solution. The reagent blanks were compared against twice distilled or anion-free water.
Ion exchange separations Col umns of Dowex I-X8 having a 20--50 mesh particle size were prepared with a depth of 8 cm in Pyrex tubes having a 11 nun i.d. Capillary tubing joined to the bottom of the columns carried the effluent back up to the level of the top of the resin bed before it was delivered into a receiver. In this way, the columns were prevented from running dry. The beds were initially washed with 200 mi of 19/o NaCI solution and 100 ml of anion-free water. Samples were passed at a rate of 1-2 rnl min -x and eluted at the same rate with 45 ml of 1% NaCI solution. The eluate was made up to 50 ml volume and analyzed by the colorimetric procedure. A band of dark material formed at the top of the resin bed when natural water was passed through. This coloured material was not removed during the elution step and when, after several runs, the band had extended midway down the bed, the resin was discarded. RESULTS AND DISCUSSION
In a procedure to concentrate phosphate samples (Pavlik, 1965), microgram quantities were recovered from an ion exchange resin. In order to establish that adsorption and elution of microgram quantities of phosphate would be quantitative in our hands, we added aliquots of standard phosphate solution to 500 ml portions of double distilled or anion-free water and passed these solutions through columns of Dowex 1-X8 anion exchange resin. The columns were eluted and the phosphate in the eluate determined. The procedures were as described in the section on methods. The recoveries were essentially quantitative as shown in Table 1. Exploratory analyses of natural water taken from the Rideau River at Ottawa showed that less phosphate was separated and recovered from the samples by the ion exchange procedure, than was indicated by a direct analysis done in the conventional manner. Table 2 shows that the discrepancy may be as high as .500 per cent. Thus, the particular water used in this study shows
Table 1. Recovery of orthophosphate adsorbed onto
Dowex l-X8 Type of water 500 ml double distilled 500 ml double distilled 500 ml double distilled S00 ml double distilled 250 ml anion-free
PO,-P added #g
PO,-P recovered ,ag
2.18 2.18 2.18 2.18 2.18
2.21 2.46 2.19 2.10 2.19
Table 2. Comparison of the determination of phosphate by direct analysis and after separation on Dowex I-X8 Date of sample
SRP in influent /ag PO,rP 1- t
Analysis by ion exchange /ag PO,t-P 1- l
SRP in effluent /ag PO,-P 1- t
11.8 11.3 8.2 7.4
2.9 4.2 2.9 2.8
6.5 2.0 2.2 2.2
24.07.72 26.07.72 27.07.72 28.07.72
A sample volume of 250 ml was used for each run. higher ratios of unrecoverable to recoverable phosphate than the water examined by Rigler. It is interesting to note that the total oftha phosphate recovered from the resin and that found in the column effluent is less than the SRP in the influent. We believe that much of the bound phosphate is absorbed by the anion exchange resin but is not in turn eluted by 1% NaCI solution. High molecular weight material bearing polar groups would behave in this manner. The fact that material from filtered water was so retained was evident from the appearance of a dark band at the top of the resin bed which remained throughout the elution step. In an attempt to determine whether the ion exchange technique separated DIP from natural water, a quantity of filtered fiver water was passed through a bed of Dowex 1-X8. 250 ml of the effluent were salted with 12.5 /zg of PO4-P and this solution passed through a fresh column of resirL This was eluted with IVo NaCI and the eluate was found by analysis to contain 12.0/ag of PO4-P. In another experiment, 3.0 #g PO,,-P were added to 200 rnl of column effluent and 3.0/ag were recovered.
Samples of river and lake water were salted with additional orthophosphate without prior treatment other than filtration through Millipore filters. Assuming ideal behaviour, the analysis of the salted samples would indicate the recoverable natural phosphate content plus the amount added. Results of these experiments are given in Table 3. The entries in columns (4) and (5) of the table are the analytical results by the ion exchange method for PO4-P
Analytical separation of phosphate
469
Table 3. Recovery of added phosphate from river and lake water
Date of sample*
(t)
(2)
(3)
(4)
(5)
(6)
Initial SRP in sample /~g PO,-P 1- t
PO,-P added /~g 1- I
SRP efltuent ~g PO,-P 1- t
Analysis of salted sample by ion exchange
Analysis of
Difference
#g PO,-P l- t
26.07.72 09.08.72 02.08.72' 27.07.72 01.08.72 * 28.07.72 I 1.08.72
28.08.72 02.08.72
I 1-! 12.7 10.5 8.3 n.d. 7.2 7- I 5.6
10-5
5.2 0 5.2 0 6.5 0 8.7 0 8.7 0 17.5
3.0 2.0 1.7 1.3 2.2 1.3 - 3.1 • 2-8 1-7 1.1 1.7
0 17.5 0 ! 7-5
, 2.2 3.7 n.d. 0
0 26.2 0
0 2-6 3.1
unsalted s~ple
(4)-(5)
by ion exchange /~g PO,-P 1=t
/~g PO,-P 1- t
5.3
9.5 4.2
5-4
10.8 5.4
3-8
9.9 6-1
8.4
11.3 2-9
7.6
16.8 9.2
12.7
! 5.5 2-8
13-2
20- I
5.9 10.1
11.4
i.3 23.2
28.8 5.6
* Samples were Rideau River water unless indicated by tt Wolfe Lake water. n.d. Not determined. in the salted and unsalted samples respectively. Column (6) contains values of recovered PO4-P which ideally should equal the amounts added and shown in colum (2). With one exception, the recovery of added phosphate up to 8.7 /~g PO,-PI - t was nearly quantitative. The recovery was far from complete for larger quantities, however. It is evident that DIP was lost by conversion to another form in the time between salting and completion of the ion exchange process. The phosphate so lost did n o t appear in the effluent as SRP, as direct analysis of effluents from salted samples gave essentially the same result as analysis of effluents from unsalted samples. As before, the sum..of SRP in column eluate and effluent was less than the sum of colurans (1) and (2), i.e, the SRP in the influent. Because the salted phosphate which was lost did not appear in the effluent, we conclude that it was retained by the resin. The brown adsorbed material referred to earlier would appear to he a likely cause of the losses. However, one must coil.sider the possibility that some of the added phosphate was converted to a soluble, unreactive form (SUP). It has been shown that 32p added to lake water did not remain totally in the form of orthophosphate (Rifler, 1966). This observation alone does not indicate that the concentration of orthophosphate was affected because it is normal in chemical processes for radioactive tracers to be distributed
amongst the various chemical species in equilibrium, often in exceedingly short times, Our results suggest that the lower concentrations of orthophosphate are fairly constant during the time required for analysis but higher concentrations cause a transfer of phosphate from the recoverable form into a high molecular weight formwhichis irreversibly bound to the resin. Substances such as lignins have many polar sites capable of binding to the resin. A set of experiments was performed using water which had been boiled for 5-8 rain. The heating was an attempt to denature any constituent of the water such as Jn vitro e n z y m e which may have been liberated by rupture of cells during the suction filtration. Samples of 250 ml volume were filtered, boiled and re-filtered. Aliquots containing 4.4/~g of PO4-P were used for the salting. The results are presented in Table 4. The results reveal no essential difference in behaviour as a consequence of boiling. There was an apparent trend toward increased loss of phosphate extending over the 11 day period. It is not certain, however, whether this trend is an indication of a fundamental alteration in the constitution of the water over this period of time such as an increase in dissolved organic matter. A further possible cause of phosphate loss would he a slow reaction such as esterification. As a slow reaction
470
ALAN D. WESTLANDand ISABELLEBOISCLAIR
Table 4. Recovery of phosphate from boiled water samples Date of sample
17.08.72 18,08,72 21.08.72 28.08.72
(t)
(2)
(3)
(4)
(5)
SRP by direct analysis #g of PO,-P 1- t
PO,-P added #g 1-1
Analysis of salted sample by ion exchange
Analysis of unsalted sample by ion exchange
Difference (3)-(4)
/~g PO,-P 1- l
#g P O , - P I- l
17-5 17.5 17-5
17-4 17.1 14.0
17-5
11,0
2-1 2.2 1.9 2-2
7.0 10.6 5.9
should be slowed down further by decreasing the temperature, s.alting followed by ion exchange separation was carried out at c a 3°C. River water samples were taken within 5 min of sampling into a refrigerated room. The water was filtered through a membrane filter and a salted sample as well as an unsalted sample was treated by ion exchange in the usual way. The eluates were warmed to room temperature and the colorimetric analysis carried out. A parallel and concurrent pair of analyses were performed at room temperature. The analysis carried out in the cold room indicated a recovery of 2.9/~g P O , - P I - t out of 4.4 #g P O , - P I - t added. The recovery at room temperature was very nearly the same, viz, 2.8 #g P O , - P I - t. The results of these experiments lead us to believe that the mechanism which is chiefly responsible for the loss of DIP is a process which is not normally considered. Adsorption of PO 3- ions onto colloidal matter seems to be the most likely possibility. It has been shown (Drabent, 1971) that adsorption of PO 3- onto soils, AlzO 3, activated charcoal, etc., occurs very readily, and it seems almost certain that colloidal organic matter would serve as an adsorbent. Drabent has made the interesting observation that orthophosphate may condense to pyrophosphate when it undergoes adsorption at low pH. Whether this can occur at the higher pH values of natural water is open to question. In order to examine the behaviour of condensed phosphate, a standard solution of sodium metaphosphate was prepared. This was added to anion-free water to give a solution containing 17.3 gg of PO3-PI-~, When 250 ml. samples of this were taken through the ion exchange procedure, recoveries of 2.1 and 2.8 #g of PO,-PI-1 were obtained. A direct analysis indicated only 0.1 #g of PO,t-PI - t . The adsorption on the resin from neutral solution seems to catalyse slightly the hydrolysis of metaphosphate. In order to determine whether the metaphosphate was in the column efltuent, this was boiled for 10 min. and analyzed for P O ,3-. None was found so it was concluded that metaphosphate was strongly absorbed by the resin and it was not eluted by the NaCI solution.
15-3 14-9 12.1 8"8
The near-quantitative recoveries of small quantities of phosphate from natural water and ion exchange effluents suggests that release of bound (absorbed?) phosphate does not occur rapidly with respect to the time required for the ion exchange process to be carried out on 250 ml samples. We therefore believe that the phosphate determination carried out with ion exchange separation provides a fairly good estimate of DIP. The ion exchange resin should reject simple phosphate esters and irreversibly adsorb the high molecular weight material referred to earlier. Apart from orthophosphate one further type of species may be separated. This is partially esterified phosphate, i.e. RPO 2-, R P O , H - or R2PO2 where R is a fairly small organic group. Such species would presumably react with hydrolysis to give molybdenum blue.
R~E~NCES
Chamberlain W. and Shapiro J. (1969) On the biological significance of phosphate analysis; comparison of standard and new methods with a bioassay. Limnol. Oceanoor. 14, 921-927. Drabent Z. (1971) Mechanism of phosphate ion sorption in model systems. Zesz. Nctak. Wyzsz. Szk. Roln. Olsztynie. Ser. A, (Suppl. 8) 3--63. Harvey H. W. 0948) The estimation of phosphate and of total phosphorus in sea waters. J. Mar. Biol. Assoc. U.K. 27, 337-359. Martin J. B. and Doty D. M. (1949) Determination of inorganic phosphate. Modification of the isobutyl alcohol procedure. Anal. Chem. 21, 965-967. Pavlik M. (1965) Determination of phosphates in underground, surface and waste waters. Vodni Hospodarstvi 15, 403-404. Rifler F. H. (1966) Radiobioloflcal analysis of inorganic phosphorus in lake water. Intern. Vet. Theoret. Angew. Linmol. Yerhandl. 16, 465--470. Rifler F. H. (1968) Further observations inconsistent with the hypothesis that the molybdenum blue method measures orthophosphate in lake water. Linmol. Oceanogr. 13, 7-13. Westland A. D. and Langford R. R. (1956) Determination of nitrate in fresh water. Concentration of samples by an ion exchange procedure. Anal. Chem. 28, 1996-1998.
THE ANALYTICAL SEPARATION OF PHOSPHATE FROM N A T U R A L WATER BY ION E X C H A N G E ALAN I). WESTLANDand ISAnELLEBOISCLAIR Department of Chemistry, University of Ottawa, Ottawa, Canada KIN 6N5
(Received 3 December 1973) Abstract--An ion exchange method has been developed for separating inorganic orthophosphate from natural water samples. The technique makes it possible to determine orthophosphate by the molybdophosphate blue procedure without interference from phosphate esters or certain other forms of bound phosphorus. Experiments to examine the conditions under which phosphate is found in natural ~aters suggest that adsorption by high molecular weight organic substances such as lignins may account for a large fraction of the soluble phosphorus.
INTRODUCTION The various forms in which phosphate can occur in natural water has been the subject of considerable speculation. Strickland and Parsons (1960) have classified them with some arbitrariness on the basis ofwhether they pass a Millipore HA filter and whether they react with acid molybdate within a 3 rain period. The molybdophosphate blue reaction is the basis of practically all methods of trace phosphorus determination and when applied directly to a filtered water sample it has usually been assumed to provide a measure of dissolved inorganic ~rthophosphate (DIP). However, Harvey (1948) has pointed out that under the acid conditions of the reaction, organic phosphate may be hydrolyzed. Chamberlain and Shapiro (1969) examined several methods of determination in order to ascertain whether one of the chemical methods would give results similar to a bioassay using algal cells. With most of the watc'rs tested, good agreement was found between the bioassay and a method in which the sample was treated with acid molybdate and immediately extracted into isobutanol. Since arsenic is not extracted under these conditions, the results led these workers to conclude that arsenic rather than hydrolysis of organic phosphate was responsible for poor agreement between most chemical analyses and the bioassay. However, it had earlier been shown (Martin and Doty, 1949) that the removal of protein prior to analysis by the molybdophosphate blue method resulted in a reduction of the indicated phosphorus levels. This suggests that the hydrolysis of organic phosphorus is a matter of importance. Westland and Langi'ord (1956) have shown that treatment of the water sample with an anion exchange resin leads to a discrimination between anionic nitrate and phosphate and other bound forms of these ions.
Other workers (Jones and Spencer, 1963: Rigler, 1968) had similar experience with Amberlite IRA-400. Rigler showed in addition that it is probable that the DIP constitutes only a fraction of the soluble reactive phosphorus (SRP) in certain lakewaters. We felt that a definitive answer to the question concerning which forms of phosphorus are bio-available to phytoplankton must be preceded by establishing a method for determining DIP specifically. The shortcoming of the standard methods is the uncertainty about whether hydrolysis of phosphate-containing compounds takes place. As the adsorption of anionic constituents on an ion exchange resin is not accompanied by drastic changes in solution conditions, particularly pH, we felt that this separation technique offered considerable promise as the basis for a more absolute method of determining DIP. METHODS
Water preparation Reagent solutions were prepared from twice distilled water. The second distillation was carried out in an all glass still. Synthetic water samples were prepared either from twice distilled water or distilled water which had been passed through Dowex l-X8 resin in the chloride form. This latter is referred to hereafter as "anion-frce" water, although it, of course, contained chloride ion. Natural water samples were filtered by suction through MiUipore HA (0A5 /am) membrane filters within a few minutes of collection in the case of river samples and within a few hours in the case of lake samples.
Colorimetric determination of phosphate. The procedure for molybdophosphate blue forma467
.*68
AL,~'qD. WF.STLANDand LSABELLEBOISCLAIR
tion was similar to that described by Harvey (1948). One ml of a 2.5 per cent solution of ammonium molybdate in 13.5 N H,SO,L was added to 50 ml of sample. This was followed by 0.15 ml of a 10 per cent solution of SnCI_, . 2 H , O in concentrated HC1 (s.g. 1.18). The final acid concentration was 03 N. The absorbance was determined after 5 rain in ceils having a 10 cm. light path. The color of natural samples was compensated by using such water as the reference solution. The reagent blanks were compared against twice distilled or anion-free water.
Ion exchange separations Col umns of Dowex I-X8 having a 20--50 mesh particle size were prepared with a depth of 8 cm in Pyrex tubes having a 11 nun i.d. Capillary tubing joined to the bottom of the columns carried the effluent back up to the level of the top of the resin bed before it was delivered into a receiver. In this way, the columns were prevented from running dry. The beds were initially washed with 200 mi of 19/o NaCI solution and 100 ml of anion-free water. Samples were passed at a rate of 1-2 rnl min -x and eluted at the same rate with 45 ml of 1% NaCI solution. The eluate was made up to 50 ml volume and analyzed by the colorimetric procedure. A band of dark material formed at the top of the resin bed when natural water was passed through. This coloured material was not removed during the elution step and when, after several runs, the band had extended midway down the bed, the resin was discarded. RESULTS AND DISCUSSION
In a procedure to concentrate phosphate samples (Pavlik, 1965), microgram quantities were recovered from an ion exchange resin. In order to establish that adsorption and elution of microgram quantities of phosphate would be quantitative in our hands, we added aliquots of standard phosphate solution to 500 ml portions of double distilled or anion-free water and passed these solutions through columns of Dowex 1-X8 anion exchange resin. The columns were eluted and the phosphate in the eluate determined. The procedures were as described in the section on methods. The recoveries were essentially quantitative as shown in Table 1. Exploratory analyses of natural water taken from the Rideau River at Ottawa showed that less phosphate was separated and recovered from the samples by the ion exchange procedure, than was indicated by a direct analysis done in the conventional manner. Table 2 shows that the discrepancy may be as high as .500 per cent. Thus, the particular water used in this study shows
Table 1. Recovery of orthophosphate adsorbed onto
Dowex l-X8 Type of water 500 ml double distilled 500 ml double distilled 500 ml double distilled S00 ml double distilled 250 ml anion-free
PO,-P added #g
PO,-P recovered ,ag
2.18 2.18 2.18 2.18 2.18
2.21 2.46 2.19 2.10 2.19
Table 2. Comparison of the determination of phosphate by direct analysis and after separation on Dowex I-X8 Date of sample
SRP in influent /ag PO,rP 1- t
Analysis by ion exchange /ag PO,t-P 1- l
SRP in effluent /ag PO,-P 1- t
11.8 11.3 8.2 7.4
2.9 4.2 2.9 2.8
6.5 2.0 2.2 2.2
24.07.72 26.07.72 27.07.72 28.07.72
A sample volume of 250 ml was used for each run. higher ratios of unrecoverable to recoverable phosphate than the water examined by Rigler. It is interesting to note that the total oftha phosphate recovered from the resin and that found in the column effluent is less than the SRP in the influent. We believe that much of the bound phosphate is absorbed by the anion exchange resin but is not in turn eluted by 1% NaCI solution. High molecular weight material bearing polar groups would behave in this manner. The fact that material from filtered water was so retained was evident from the appearance of a dark band at the top of the resin bed which remained throughout the elution step. In an attempt to determine whether the ion exchange technique separated DIP from natural water, a quantity of filtered fiver water was passed through a bed of Dowex 1-X8. 250 ml of the effluent were salted with 12.5 /zg of PO4-P and this solution passed through a fresh column of resirL This was eluted with IVo NaCI and the eluate was found by analysis to contain 12.0/ag of PO4-P. In another experiment, 3.0 #g PO,,-P were added to 200 rnl of column effluent and 3.0/ag were recovered.
Samples of river and lake water were salted with additional orthophosphate without prior treatment other than filtration through Millipore filters. Assuming ideal behaviour, the analysis of the salted samples would indicate the recoverable natural phosphate content plus the amount added. Results of these experiments are given in Table 3. The entries in columns (4) and (5) of the table are the analytical results by the ion exchange method for PO4-P
Analytical separation of phosphate
469
Table 3. Recovery of added phosphate from river and lake water
Date of sample*
(t)
(2)
(3)
(4)
(5)
(6)
Initial SRP in sample /~g PO,-P 1- t
PO,-P added /~g 1- I
SRP efltuent ~g PO,-P 1- t
Analysis of salted sample by ion exchange
Analysis of
Difference
#g PO,-P l- t
26.07.72 09.08.72 02.08.72' 27.07.72 01.08.72 * 28.07.72 I 1.08.72
28.08.72 02.08.72
I 1-! 12.7 10.5 8.3 n.d. 7.2 7- I 5.6
10-5
5.2 0 5.2 0 6.5 0 8.7 0 8.7 0 17.5
3.0 2.0 1.7 1.3 2.2 1.3 - 3.1 • 2-8 1-7 1.1 1.7
0 17.5 0 ! 7-5
, 2.2 3.7 n.d. 0
0 26.2 0
0 2-6 3.1
unsalted s~ple
(4)-(5)
by ion exchange /~g PO,-P 1=t
/~g PO,-P 1- t
5.3
9.5 4.2
5-4
10.8 5.4
3-8
9.9 6-1
8.4
11.3 2-9
7.6
16.8 9.2
12.7
! 5.5 2-8
13-2
20- I
5.9 10.1
11.4
i.3 23.2
28.8 5.6
* Samples were Rideau River water unless indicated by tt Wolfe Lake water. n.d. Not determined. in the salted and unsalted samples respectively. Column (6) contains values of recovered PO4-P which ideally should equal the amounts added and shown in colum (2). With one exception, the recovery of added phosphate up to 8.7 /~g PO,-PI - t was nearly quantitative. The recovery was far from complete for larger quantities, however. It is evident that DIP was lost by conversion to another form in the time between salting and completion of the ion exchange process. The phosphate so lost did n o t appear in the effluent as SRP, as direct analysis of effluents from salted samples gave essentially the same result as analysis of effluents from unsalted samples. As before, the sum..of SRP in column eluate and effluent was less than the sum of colurans (1) and (2), i.e, the SRP in the influent. Because the salted phosphate which was lost did not appear in the effluent, we conclude that it was retained by the resin. The brown adsorbed material referred to earlier would appear to he a likely cause of the losses. However, one must coil.sider the possibility that some of the added phosphate was converted to a soluble, unreactive form (SUP). It has been shown that 32p added to lake water did not remain totally in the form of orthophosphate (Rifler, 1966). This observation alone does not indicate that the concentration of orthophosphate was affected because it is normal in chemical processes for radioactive tracers to be distributed
amongst the various chemical species in equilibrium, often in exceedingly short times, Our results suggest that the lower concentrations of orthophosphate are fairly constant during the time required for analysis but higher concentrations cause a transfer of phosphate from the recoverable form into a high molecular weight formwhichis irreversibly bound to the resin. Substances such as lignins have many polar sites capable of binding to the resin. A set of experiments was performed using water which had been boiled for 5-8 rain. The heating was an attempt to denature any constituent of the water such as Jn vitro e n z y m e which may have been liberated by rupture of cells during the suction filtration. Samples of 250 ml volume were filtered, boiled and re-filtered. Aliquots containing 4.4/~g of PO4-P were used for the salting. The results are presented in Table 4. The results reveal no essential difference in behaviour as a consequence of boiling. There was an apparent trend toward increased loss of phosphate extending over the 11 day period. It is not certain, however, whether this trend is an indication of a fundamental alteration in the constitution of the water over this period of time such as an increase in dissolved organic matter. A further possible cause of phosphate loss would he a slow reaction such as esterification. As a slow reaction
470
ALAN D. WESTLANDand ISABELLEBOISCLAIR
Table 4. Recovery of phosphate from boiled water samples Date of sample
17.08.72 18,08,72 21.08.72 28.08.72
(t)
(2)
(3)
(4)
(5)
SRP by direct analysis #g of PO,-P 1- t
PO,-P added #g 1-1
Analysis of salted sample by ion exchange
Analysis of unsalted sample by ion exchange
Difference (3)-(4)
/~g PO,-P 1- l
#g P O , - P I- l
17-5 17.5 17-5
17-4 17.1 14.0
17-5
11,0
2-1 2.2 1.9 2-2
7.0 10.6 5.9
should be slowed down further by decreasing the temperature, s.alting followed by ion exchange separation was carried out at c a 3°C. River water samples were taken within 5 min of sampling into a refrigerated room. The water was filtered through a membrane filter and a salted sample as well as an unsalted sample was treated by ion exchange in the usual way. The eluates were warmed to room temperature and the colorimetric analysis carried out. A parallel and concurrent pair of analyses were performed at room temperature. The analysis carried out in the cold room indicated a recovery of 2.9/~g P O , - P I - t out of 4.4 #g P O , - P I - t added. The recovery at room temperature was very nearly the same, viz, 2.8 #g P O , - P I - t. The results of these experiments lead us to believe that the mechanism which is chiefly responsible for the loss of DIP is a process which is not normally considered. Adsorption of PO 3- ions onto colloidal matter seems to be the most likely possibility. It has been shown (Drabent, 1971) that adsorption of PO 3- onto soils, AlzO 3, activated charcoal, etc., occurs very readily, and it seems almost certain that colloidal organic matter would serve as an adsorbent. Drabent has made the interesting observation that orthophosphate may condense to pyrophosphate when it undergoes adsorption at low pH. Whether this can occur at the higher pH values of natural water is open to question. In order to examine the behaviour of condensed phosphate, a standard solution of sodium metaphosphate was prepared. This was added to anion-free water to give a solution containing 17.3 gg of PO3-PI-~, When 250 ml. samples of this were taken through the ion exchange procedure, recoveries of 2.1 and 2.8 #g of PO,-PI-1 were obtained. A direct analysis indicated only 0.1 #g of PO,t-PI - t . The adsorption on the resin from neutral solution seems to catalyse slightly the hydrolysis of metaphosphate. In order to determine whether the metaphosphate was in the column efltuent, this was boiled for 10 min. and analyzed for P O ,3-. None was found so it was concluded that metaphosphate was strongly absorbed by the resin and it was not eluted by the NaCI solution.
15-3 14-9 12.1 8"8
The near-quantitative recoveries of small quantities of phosphate from natural water and ion exchange effluents suggests that release of bound (absorbed?) phosphate does not occur rapidly with respect to the time required for the ion exchange process to be carried out on 250 ml samples. We therefore believe that the phosphate determination carried out with ion exchange separation provides a fairly good estimate of DIP. The ion exchange resin should reject simple phosphate esters and irreversibly adsorb the high molecular weight material referred to earlier. Apart from orthophosphate one further type of species may be separated. This is partially esterified phosphate, i.e. RPO 2-, R P O , H - or R2PO2 where R is a fairly small organic group. Such species would presumably react with hydrolysis to give molybdenum blue.
R~E~NCES
Chamberlain W. and Shapiro J. (1969) On the biological significance of phosphate analysis; comparison of standard and new methods with a bioassay. Limnol. Oceanoor. 14, 921-927. Drabent Z. (1971) Mechanism of phosphate ion sorption in model systems. Zesz. Nctak. Wyzsz. Szk. Roln. Olsztynie. Ser. A, (Suppl. 8) 3--63. Harvey H. W. 0948) The estimation of phosphate and of total phosphorus in sea waters. J. Mar. Biol. Assoc. U.K. 27, 337-359. Martin J. B. and Doty D. M. (1949) Determination of inorganic phosphate. Modification of the isobutyl alcohol procedure. Anal. Chem. 21, 965-967. Pavlik M. (1965) Determination of phosphates in underground, surface and waste waters. Vodni Hospodarstvi 15, 403-404. Rifler F. H. (1966) Radiobioloflcal analysis of inorganic phosphorus in lake water. Intern. Vet. Theoret. Angew. Linmol. Yerhandl. 16, 465--470. Rifler F. H. (1968) Further observations inconsistent with the hypothesis that the molybdenum blue method measures orthophosphate in lake water. Linmol. Oceanogr. 13, 7-13. Westland A. D. and Langford R. R. (1956) Determination of nitrate in fresh water. Concentration of samples by an ion exchange procedure. Anal. Chem. 28, 1996-1998.