Analytica Chimica Acta, 260 (1992) 113-121 Elsevier Science Publishers B.V., Amsterdam

113

Techniques for extraction of dissolved inorganic and organic phosphorus from large volumes of sea water

T. Lee, E. Barg and D. La1 *

Scripps In&&m of Oceanography, GRD-0220, La Jolla, CA 92093 (USA)

(Received 22nd August 1991)

Abstract

Studies of marine phosphorus biodynamics using cosmogenic 32P as a radio-tracer necessitated the determination of its specific activities C3*P/P ratios) in the dissolved inorganic (DIP) and organic phosphorus (DOP) pools. Owing to the low atmospheric injection rate of 32P atoms (ca. 6 X lo-’ atoms cm-’ min -l), its analysis involves extraction of DIP and DOP from large volumes of sea water (> 1 m3). When this work was undertaken, no suitable methods were available for their extraction, especially from large volumes of sea water. Two techniques are presented, one for the exclusive extraction of DIP and the other for both DIP and DOP from up to several m3 of sea water. The extraction of DIP is based on the selective adsorption of DIP-molybdenum blue complex using a cation-form acrylic fiber. The total dissolved phosphorus is extracted efficiently by adsorbing it on to iron(II1) hydroxide surfaces [iron(III) hydroxide-coated acrylic fiber], following a technique used earlier for the extraction of dissolved silicon and other trace elements from sea water.

Keywords: Extraction; Phosphorus; Sea water; Waters

Two naturally occurring cosmogenic radionu- elides, 32P and 33P, half-lives 14.3 and 25.3 days, respectively, have recently been used as tracers for studying the biogeochemistry of phosphorus in the upper ocean [l]. Their measurement neces- sitates the extraction of dissolved phosphorus from 2 1 m3 of sea water because of the low atmospheric injection rate of 32P atoms (about 6 x 10m3 atoms cm-* min-’ [2]). The concentra- tions of dissolved inorganic (DIP) and organic phosphorus (DOP) in the mixed layer are usually 0.2-0.3 PM, whereby the specific activities of 32P (32P/P) can be measured in DIP and DOP.

Dissolved phosphorus in aquatic environments falls into two broad categories, DIP and DOP. Each group is composed of diverse chemical species which remain to be properly character- ized, especially for DOP in natural waters 131. DOP consists of a multitude of chemical com- pounds, complicating any single method of ex-

traction. Several methods reported earlier for the extraction of DOP were tried. Adsorbents with a hydrophobic surface such as activated charcoal [4], textured carbon fiber and Amberlite XAD resins [5] were tested. They do not adsorb DIP and they have low adsorption efficiencies of less than 25%, even at a space velocity of 50 (space velocity is defined as the number of column vol- umes of liquid passed through column per hour). Although activated charcoal has a relatively high extraction efficiency, it has appreciable intrinsic phosphorus which cannot be removed completely even with repeated cleansing with hydrochloric acid and ammonia solution.

Metal ions known to complex easily with dis- solved organic matter, such as Cu*+ and Fe3+, were tested for DOP extraction [6]. These ions were loaded on to chelating resin, Chelex 100, to make iminodiacetate-metal pairs and then tested for DOP-metal complex formation. Laboratory

0003-2670/92/$05.00 0 1992 - Elsevier Science Publishers B.V. All rights reserved

114 i? Lee et al. /Ad. Chim. Acta 260 (1992) 113-121

experiments showed the DOP-metal complexa- tion to be a slow process; further, it displays poor discrimination between DIP and DOP. The iron-iminodiacetate complex was found to be unstable at the pH of sea water; it changes gradu- ally to Fe(OH), after several column volumes of sea water have been passed through the resin column. Activated alumina and silica were also tested for exclusive adsorption of DIP or DOP. Both show a limited adsorption capacity and a lack of ability to discriminate between DIP and DOP.

Laboratory experiments confirmed that iron (III) hydroxide-coated fibers [7] have the ability to adsorb ATP, DNA, glucose-l-phosphate and glucosed-phosphate quantitatively. In view of the diversity of DOP compounds in sea water, it was decided not to attempt to develop a technique exclusively for the extraction of organic phospho- rus compounds and be content with the extrac- tion of both DIP and DOP in one step using iron(II1) hydroxide [7] and the extraction of DIP alone by another technique. Both are described in this paper. Determination of the relative con- centrations of DIP and DOP in sea water, cou- pled with 32P specific activity measurements sep- arately in the total dissolved phosphorus (TDP) and DIP, then allows calculation of the 32P spe- cific activity of DOP.

EXPERIMENTAL

First the experimental methods developed for the measurement of DIP and DOP concentra- tions in sea water. are discussed, followed by techniques for their extraction from large vol- umes of sea water.

DIP-molybdenum blue complex formation and pseudo-detection of DOP

Soluble reactive phosphorus (SRP) refers to the portion of redissolved phosphorus that forms a molybdenum blue (MB) complex in the pres- ence of ascorbic acid [8]. The term SRP is often used interchangeably with DIP, but in a strict sense it is different. Because the MB method uses acid and molybdate, which have catalytic ability,

it can overestimate the DIP concentration by including hydrolyzed labile DOP and metalphos- phorus complex in natural waters [9J. Despite the problem of hydrolyzing DOP, the MB complex method offers a unique advantage as the MB can be easily extracted by liquid-liquid extraction [8], adsorption [lO,ll] and ion-exchange methods. Di- rect application of these methods for the extrac- tion of DIP from large volumes of sea water, however, is not practical. The extraction is slow and the capacity of the adsorbents is also limited.

Laboratory experiments with ion-exchange resins in the cation form (5OW X8) and anion form (1 X8) and and adsorbent polymer (XAD-7) showed that they can extract MB quantitatively with decreasing adsorption capacity in the order 1 X8, 5OW X8 and XAD-7. The principal disad- vantage of the resin method, however, is that the amount of resin needed is large, ca. 10 kg for 1 m3 of surface sea water. As the cation-exchange resin removes MB efficiently, extraction with syn- thetic acrylic fiber (Acrilan) converted to the cation form was tried 1121. It was found that the Na-form Acrilan surpasses all resins tested both in capacity and extraction efficiency, with high speeds of extraction. The saturation capacity of Na-form Acrilan is about 10 mg P 1-l fiber vol- ume. The extraction efficiency measured by com- paring the absorbance at 880 nm of inflow and outflow from the column exceeded 95% even at space velocities greater than 300. This allows the convenient extraction of MB from large volumes of sea water; only 2 1 of Na-form Acrilan fiber suffice to extract MB from 1 m3 of most sea waters.

The reagents used to make the DIP-MB com- plex are essentially the same as used in the Ko- roleff method [13]. As the amount of reagent required for the present scale of experiments is large, attempts were made to reduce the concen- tration of reagents on the following basis: the methods of Koroleff [13] and Strickland and Par- sons [8] are capable of measuring 30 PM DIP whereas the average concentration of DIP in the oceans is ca. 2 mM; the acidity (0.1% in reacted solution) is kept high primarily to avoid silicate interference, which is not a serious problem for extraction purposes; and, as mentioned earlier,

T. Lee et al. /Anal. Chim. Acta 260 (1992) 113-121 115

molybdate and acid can cause instantaneous hy- drolysis of labile DOP.

The measured absorbance of sea-water sam- ples spiked with up to 10 PM of orthophosphate with 60% of the sulfuric acid in the Koroleff method [13] showed good linearity, although there was about 10% increase in the absorbance com- pared with the case when the normal concentra- tion of sulfuric acid was used (Fig. 1). The ab- sorbance increased linearly with phosphate con- centration. Also, the linearity over a wide range of DIP concentration suggests no appreciable in- terference by other compounds.

To check on the effect of hydrolysis of organic phosphorus compounds, and their consequent de- tection as SRP, the absorbance at 880 mu of sea-water samples spiked with up to 10 PM of glucose-l-phosphate, glucosed-phosphate and ATP was studied. Absorbances measured at 880 mn over the entire range of concentrations showed that 3%, 1% and 2% glucose-l-phos- phate, glucosed-phosphate and ATP, respec- tively, are detected as SRP using either the nor- mal or a reduced concentration of sulfuric acid (60%). The color was stable for up to 2 h, the period of time over which measurements were made. No evidence was found for reduced hydro- lysis of organic phosphorus compounds with a lower concentration of sulfuric acid as expected. This could be because the concentration of sulfu- ric acid used in the reduced reagents is still high enough to hydrolyze the acid-labile DOP com-

0 * 4 6 8 10 v.

Connn~rtioaoforthophorphrtcUIM)

Fig. 1. Comparison of measured absorbances of MB complex at 880 nm for sea water spiked with up to 10 PM of or- thophosphate. Data for (0) nomal reagent and (W) diluted reagent containing 60% of sulfuric acid.

0 0.5 1 15 2

concentrrtion ofg-1-p@M)

Fig. 2. Measured absorbances of MB complex at 880 nm for distilled water spiked with glucose-l-phosphate and aged in the laboratory for 1 day. The measured absorbances are due to the hydrolysis of glucose-l-phosphate by the reagents used as no DIP was present. Data for (0) normal reagent and (m) diluted reagent containing 60% of sulfuric acid.

pounds, or because freshly reconstituted organic compounds from frozen stock solution were not as labile as natural DOP. The latter seems to be case because in experiments with solutions of organic phosphorus compounds aged in the labo- ratory for 1 day at room temperature, DOP de- tection as DIP was appreciably lower with the reduced acid concentration (Fig. 2).

Measurements of DIP and DOP concentrations in sutface ocean waters

The experiments carried out to date on the measurement of concentrations of DIP and DOP and 32P in DIP and DOP pools in surface waters from the Southern California Bight are described. DIP concentrations were measured at two sta- tions. The concentrations of DOP were inferred from the difference between total phosphorus measured by the acid peroxodisulfate oxidation technique [13] and of DIP measured using both normal and reduced (60%) concentrations of sul- furic acid in the reagents (Fig. 3). Overestimation (up to 50%) of DIP was clearly observed when the normal concentration of sulfuric acid was used.

The concentrations of acidified ascorbic acid and mixed reagents were reduced further until DIP measurement of natural sea water (contain- ing 1 PM of DIP) could be made reproducibly. It was found that with 50% and 25% concentrations

116 T. Lee et aL /Anal Chim. Acta 240 (1592) 113-121

of the mixed and acidified ascorbic acid solutions, order of m3). With these reduced concentrations, the extraction of DIP could be carried out suc- the molybdenum blue complexation reached a cessfully from’large volumes of sea water (of the plateau after 5 min at room temperature and the

DIP concentration (phi) DIP concentration (PM)

0.0 0.4 0.8 1.2 1.6 2.0 0.0 0.4 0.8 1.2 1.6 2.0

20

30

h 40

.c

2 50

e

n 60

70

80

90

DOP concentration (PM) DOP concentration ($4)

0.0 0.2 0.4 0.6 0.8 1.0 0

10

20

30

5 50

fl?

CJ 60

0

10

20

30

40

3 50

f 2 60 n

70

80

90

100

110

0.0 0.2 0.4 0.6 0.8 1.0 -I

30

40

f ,a 60 !a

70

70 80

80 90

90 100

100 110

Fig. 3. Measured depth profiles of (a and b) dissohred inorganic phosphorus and (c and d) dissohred organic phosphorus at two stations in the Southern California Bight. Data based on (0) normal reagent and (*) diluted reagent containing 60% of suffiric acid.

T. Lee et al. /Anal. Chim. Acta 260 (1992) 113-121

absorbances after 10 min were comparable with those measured using normal reagents. The ab- sorbance, however, increased with time at the rate of 20-30% during first 3 h, possibly owing to interference from other molybdate-reactive ele- ments from sea water such as silicon. Therefore, the reduced concentrations of reagents adopted in this study are not recommended either in an analytical method to measure the DIP or for the extraction of DOP from solutions containing more than 1 PM of DIP.

Extraction of total dissolved phosphorus and radionuclides from large volumes of sea water

Dissolved phosphorus can be conveniently ex- tracted by iron(II1) hydroxide coprecipitation or by adsorption on to iron(II1) hydroxide surfaces converted from cation-exchange resin [14] or from Acrilan [12]. For a large-volume extraction on- board ship, the adsorption technique is more convenient than the coprecipitation technique. The ion-exchange resin and the Acrilan fiber have comparable capacities for loading of iron( about 10% on a dry weight basis, but the fiber matrix presents a much larger active surface area. Also, the fibers, because of their elasticity, are expected to adjust to a change in pressure during flow of sea water, so the chances

117

of channeling are non-existent for the fiber col- umn. Because of these characteristics, of the iron(II1) hydroxide-coated Acrilan allows high-ef- ficiency extraction at high speed.

Initial tests with Fe(OH),-Acrilan using a few column volumes of sea water spiked with or- thophosphate and three organic phosphorus com- pounds (DNA, ATP and glucose-l-phosphate) showed that both inorganic and organic phospho- rus were adsorbed with close to 100% efficiency. The adsorption efficiency and capacity of Fe (OH),-Acrilan were measured up to 450 column volumes of flow. The concentration of DIP was high enough (0.9 PM) to measure the adsorption efficiency; however, that of DOP was too low (0.15 PM) to measure. For DOP adsorption ex- periments, natural sea water spiked with glucose- l-phosphate was used instead. The adsorption efficiency for DIP was measured during the first 200 column volumes with natural sea water and then that of DOP was measured with 25 column volumes of glucose-l-phosphate-spiked sea water (20 PM). Subsequently, 175 and 25 column vol- umes of natural sea water and glucose-l-phos- phate-spiked sea water (5 ,uM), respectively, were passed through the same column. The results, plotted in Fig. 4, show that the adsorption effi- ciencies for DIP and DOP of Fe(OH),-Acrilan

100

90 4 0 100 200 300 400

Column volumes

Fig. 4. Measured adsorption efficiencies of the Fe(OHl,-Acrilan column for DIP (0, natural sea water) and DOP (+, glucose-l-phosphate-spiked sea water) plotted as a function of the cumulative column volumes of flow. The concentration of DIP in natural sea water was 0.9 PM. The concentration of DOP between 200 and 225 column volumes (+I and that between 400 and 425 column volumes (+) were 25 and 5 PM, respectively.

118 T. Lee et aL /Anal. Chim. Acta 240 (1992) 1X3-121

are comparable. The decrease in adsorption effi- ciency after 225 column volume indicates satura- tion of the column; the saturation capacity is estimated to be 15 pg P ml-’ Fe(OH),-Acrilan.

In field trials, the total dissolved phosphorus (DIP + DOP) was extracted from 0.1 pm filtered sea water by passing it through a column of iron(II1) hydroxide-coated Acrilan [2]. About 2 m3 of sea water were passed through each col- umn at a space velocity about 300. As iron0111 hydroxide has a high scavenging ability for other dissolved metal ions [12], the phosphorus adsorp- tion efficiency decreases slowly with the volume of sea water processed. After about 2000 column volumes of sea water had passed through the column, the adsorption efficiency for DIP had decreased to about 60% (cf. Fig. lb in [2]). Of particular interest among others extracted are the naturally occurring radionuclides, ‘Be (half-life 53.3 days) and 2?I’h (half-life 24.1 days), which are relevant to the study of phosphorus biogeo- chemistry [151. ‘Be is produced continuously in the atmosphere by the nuclear spallation of nitro- gen and oxygen by cosmic rays. 234Th is produced continuously in situ from the disintegration of =‘U in sea water. The former nuclide shares the same pathway from the atmosphere to surface ocean with 32P but is not involved in biological processes as is 32P* it is therefore a good tracer for the mixing process. The latter is particulate- reactive which can constrain the ‘Be flux associ- ated with sinking particles [15].

The extraction efficiency of various adsorbed molecules can be easily determined by using two identical columns in series if they are not driven to saturation, as was the case in the present experiments. Assuming the same extraction effi- ciency for the two columns, the extraction effi- ciency for an adsorbed species, E, is obtained from the amounts adsorbed in the first (A,) and second (A,) columns:

E = (4 -A,)/4 (1)

Extraction of dissolved inorganic phosphorus from large volumes of sea water

The developed MB complex technique was tested in the field for the extraction of DIP from

large volumes of sea water during two cruises in the Southern California Bight. About 1 m3 of sea water was filtered through a O.l-pm filter car- tridge of 25 cm length into a polyethylene tank. The acidified ascorbic acid solution and the mixed reagent solution, with 25% and 50% concentra- tions of the conventional MB reagents [13], re- spectively, were added to the sea water. After allowing 30 min for the development of the DIP- MB complex it was adsorbed on an Na-form Acrilan column of 1 1 volume at a space velocity of about 300. The extraction efficiency of the MB complex was > 95% up to 400 column volumes. The saturation capacity of the column seemed to be about 1000 column volumes in this field mea- surement, which is equivalent to 10 mg P 1-l of Na-form Acrilan.

Radiochemical and counting procedures Phosphorus was extracted from the ash of Na-

form Acrilan and Fe(OH),-Acrilan fibers follow- ing the procedures outlined in Fig. 5. The analyti- cal procedure for phosphorus is essentially the same used previously [16], utilizing ammonium molybdophosphate and magnesium ammonium orthophosphate precipitations. Two reagent solu- tions used in the procedure were prepared as follows. Ammonium molybdate solution was pre- pared by dissolving 100 g of ammonium molyb- date [(NH,),Mo,O, - 4H,O] in distilled water and adding 80 ml of concentrated ammonia solu- tion. This solution was added to 1 1 of 6 M HNO, with rapid mixing. The magnesia solution was prepared by dissolving 50 g of MgCl, * 6H2O and 100 g of NH,Cl in 500 ml of distilled water, adding 10 ml of concentrated ammonia solution and diluting to 1 1. Phosphorus extracts prepared as magnesium pyrophosphate were counted for their 32P activities using low-level gas-propor- tional p-counters [2].

The activities of ‘Be and u4Th were measured by y-counting 477.6 and 63.3 keV, respectively [171. The samples were prepared by concentrating ‘Be and 234Th from the sea water-treated Fe(OH),-Acrilan fibers. The fibers were ashed and excess of iron was removed from the ash by repeated extractions with diethyl ether. Aqueous extracts were reprecipitated with ammonia solu-

i? Lee et al./Anal. Chim. Acta 260 (1992) 113-121 119

Ndmsn Acrib I

Fe(OH),-Acrh I

cornbust I

elutewitbNH,OH

I evrponte

I cornbust

I elute with HNO,

I

.ddHNO,j , i’“” 1 y;iard

add ammonium molybdaie solution

centrage

discard incinerate at 1050’ c

I MQ’P,

Fig. 5. Schematic diagram of the analytical procedure used for the purification of phosphorus adsorbed on Na-form Acrilan and Fe(OH),-Acrilan.

tion at pH 9. The precipitates were dried and counted for 234Th and ‘Be activities directly using a well-type high-purity germanium photon detec- tor with a lOtI-ml active volume in J-configuration (Ortec) [153.

RESULTS AND DISCUSSION

Laboratory studies were made of the extent of the hydrolysis of organic phosphorus compounds (ATP, DNA, glucose-l-phosphate) and glucose- 6-phosphate using the MB method [13] used for the determination of DIP in natural waters. The results with glucose l-phosphate showed that when organic phosphorus stock solutions were aged for about 1 day, an appreciable amount of DOP was hydrolyzed by the reagent and conse- quently detected as DIP (Fig. 2). Several experi- ments were made with different concentrations of

the reagents and it was found that the extent of hydrolysis can be minimized by using a smaller amount of sulfuric acid in the reagents without losing the analytical sensitivity of the method (Figs. 1 and 2). The differences between the measured DIP concentrations with the original concentration and with 40% less sulfuric acid in reagents at two stations in the Southern Califor- nia Bight were measured. These measurements clearly show that the normal reagent consistently overestimated the DIP concentrations, especially at depths (Fig. 3). This and the result for the aged glucose-l-phosphate solution implies that the bulk of DOP at depths in the ocean is labile to acid owing to its degradation in the water column with age.

The distinctive feature of the DIP-MB com- plex that it can be extracted quantitatively by several adsorbents was exploited. The reagent concentrations were adjusted to correspond to DIP concentrations of about 1 I.LM in the sea water. From laboratory tests, it was found that 25% and 50% acidified ascorbic acid solution and the mixed reagent solution in current method [13], respectively, yielded reproducible absorb- antes. Several adsorbents were compared for their capacity and efficiency for the DIP-MB complex, and it was found that the acrylic fiber converted to the cation form (Na-form Acrilan) was the most effective. Na-form Acrilan is not expected to adsorb DOP from sea water because the sur- face charge of DOP will be neutralized at pH l-2 as encountered in MB formulations, unless DOP is hydrolyzed and makes the DIP-MB complex.

DIP and DOP were successfully extracted from two sea-water samples and their 32P activities were measured. The rationale for the measure- ments is discussed elsewhere [15]. The measured 32P concentrations of DIP and DOP, and the dissolved concentrations of cosmogenic ‘Be and 234Th , are given in Table 1. In the surface waters (O-55 m), most of the 32P activity measured is present in the DIP phase. Reduction of the 32P/P activities in the DOP, 32P concentrations in DIP and TDP and the DIP and DOP concentrations (Fig. 3) yielded essentially zero 32P concentra- tions in the DOP (Table 1). This result implies that the mean time period of DOP utilization in

120 T. Lee ef al. /AnaL Chim Acra 260 (1992) 113-121

TABLE 1

Measured activities (mean f 1 S.D.) of radionuclides 32P, ‘Be and u4Th in surface sea water in the Southern California Bight

Date Station No. Depth Measured 32P , ‘Be and 234Th activities (dpm mm31 (m) 32P (TDP) 32P (DIP) 32P (DOP) a ‘Be 234Th

26 July 1990 CALCOFI 93.60 10 0.33 f 0.09 0.31 f 0.11 0.02 f 0.15 22.6 f 2 788 f 36 35 0.17 f 0.09 0.15 f 0.09 0.02 f 0.12 21.9 f 2 683 f 27

1 December 1990 CALCOFI 93.45 10 0.68 f 0.10 0.67 rt 0.08 0.02 f 0.13 37.3 f 3 1693 f 83 35 0.46 f 0.12 0.37 f 0.12 0.09 f 0.17 30.7 f 3 1784 f 89 55 0.08 f 0.05 0.10 f 0.06 0 f 0.08 7.0 f 3 1567 f 84

a The activities of 32P in DOP are inferred from the measured activities of 32P in DIP and total dissolved phosphorus (see text).

the food chain is much longer than 6 weeks. This result can be understood because in the marine ecosystem nitrogen not phosphorus, is the limit- ing nutrient.

It should be noted that that the iron(II1) hy- droxide-loaded Acrilan fiber acts as very efficient adsorbent not only for the total dissolved phos- phorus but also for other surface-reactive ions in sea water. The scavenging ability of iron(II1) hy- droxide is well known [14], but the exact mecha- nism is not yet understood. Also based on our observations, the adsorption of dissolved phos- phorus compounds and other metals such as tho- rium and beryllium is instantaneous, on contact. The observed adsorption efficiencies for the three elements studied were Th > Be > P0,3-. It is speculated that iron(II1) hydroxide is in the form of goethite (a-FeOOH), whose hydroxyl ligand facilitates instantaneous complexation [181.

In conclusion, two techniques have been suc- cessfully developed for the extraction of dissolved inorganic phosphorus and total dissolved phos- phorus from large volumes of sea water, which can be conveniently executed on-board ship. The latter utilizes the scavenging ability of Fe(OH), and the former the surface characteristics of the DIP-MB complex. With the developments of these techniques, and using the cosmogenic ra- dionuclide 32P as a tracer for phosphorus, it should be possible to quantify the processes and time scales involved in the recycling of phospho- rus between the different phosphorus pools in surface waters.

The authors are grateful to Professor G. Ar- rhenius, Mr. P. Arrhenius and Professor F. Azam

for invaluable discussions and advice throughout this work. They also thank to Drs. S. Krish- naswami and M.P. Bacon for suggestions on the quantitative extraction of 7Be from sea water using the Fe(OH),-loaded Acrilan fibers. Mr. S.P. Wallace of Monsanto Chemical is thanked for providing the Acrilan fibers for this research and A. Spivack for helpful criticisms. This work was supported in part by NSF OCE-8812985 and in part by a donation received from Mr. Myron Eichen.

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