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Speciation of dissolved phosphorus in environmental samples by gel filtration and flow-injection analysis
Tuhta, Vol. 40, No. 12, pp. 1981-1993, 1993 Printed in Great Britain.All rightsreserved
llo39-914Oj93 s6.00 + 0.00
CopyrightQ I993Fcrgamon Press Ltd
SPECIATION OF DISSOLVED PHOSPHORUS IN ENVIRONMENTAL SAMPLES BY GEL FILTRATION AND FLOW-INJECTION ANALYSIS I. D. MCKELVIE* and B. T.
HART
Water Studies Centre and Chemistry Department, Monash University, Caulfield East 3145, Victoria, Australia T. J.
CARDWELL and R. W. CA~TRALL
Centre for Scientific Instrumentation, La Trobc University, Bundoora 3083, Victoria, Australia (Received 16 February 1993. Revired 29
March
1993. Accepted 30
March
1993)
Summary-A study of the factors afkting separation and detection of dissolved organic and inorganic phosphorus species found in waters and sediients is reported. The system involved the use of gel titration and flow injection analysis (FIA). Orthophosphate and myo-inositol hexakisphosphate. as model solutes representative of low molecular weight P (LMWP) and high molecular weight P (HMWP). were separated on a Sephadex G25 cclumn incorporated into a flow-injection manifold which utilixed photo-oxidation and spectrophotometry for detection of dissolved reactive phosphorus (DRP) and dissolved organic phosphorus (DOP). The influence of eluent pH and ionic strength on adsorption and anionic exclusion of the model solutes is described, and the optimum eluent composition and sample size ate described. The method was used to determine LMWP and HMWP in natural and waste waters, and in sediment extracts. Potential limitations of this approach are discussed.
Phosphorus in natural waters and sediment is known to occur as orthophosphate and a variety of organic and condensed phosphorus species with a wide range of molecular weights.’ Some components of the dissolved organic phosphorus (DOP) fraction are thought to hydrolyse rapidly to orthophosphate when acidic molybate is used in the spectrophotometric analysis of orthophosphate as phosphomolybdenum blue, and for this reason the term “dissolved reactive phosphorus” (DRP) is used in reference to “orthophosphate”. Consequently, DRP concentrations may overestimate orthophosphateZ which is undesirable because orthophosphate is currently the best chemical measure of the dissolved phosphorus concentration biologically available for photosynthesis. In soils and sediments, phosphorus may occur as organic compounds such as sugar phosphates, phosphatidyl phosphates, inositol phosphates, phosphoamides, phosphoprotein, aminophosphonic acids, phosphorus-containing pesticides and organic condensed phosphates.3 Of this group, myo-inositol hexakisphosphate *Author for correspondence.
(also called phytic acid), is among the most stable;’ this compound occurs in free and ironcomplexed forms in soils and marine and lake sediments.5 It has been conjectured that this species and its hydrolysis products will be present in natural waters through groundwater input,’ and may comprise a significant proportion of dissolved organic phosphorus (DOP).47 However, only a few studies have actually demonstrated the existence of inositol phosphates in natural waters,8*9and to date little is known of the transport of these species from sediments and soils into stream and lake waters. Analysis of the DRP concentration in natural and wastewaters is commonly performed in an attempt to estimate the concentration of bioavailable phosphorus in the dissolved fraction. While orthophosphate has been shown in a number of studies to be the most bioavailable phosphorus species, lo*higher molecular weight phosphorus (HMWP) species display varying degrees of bioavailability.‘**‘* In order to determine the concentration of both “true” orthophosphate and HMWP in natural water samples, we attempted to separate these species according to differences in molecular weight using gel filtration followed by photo-oxidation
1981
I. D.
1982
MCKELVIE et al.
and subsequent phosphomolybdenum blue detection of the liberated orthophosphate using FIA. Separation of orthophosphate and HMWP in natural waters by gel filtration chromatography has been described by a number of authors”*‘1*‘~‘5using a variety of eluent conditions, large gel columns, slow flow rates and correspondingly long (30 mm-2 hr) elution times. The aim of the work described here was to develop a rapid technique allowing discrimination between these two molecular weight fractions. Small Sephadex G25 columns were investigated for the separation of model solutes representative of LMWP and HMWP, viz; orthophosphate and myo-inositol hexakisphosphate. Significant irreversible, or very slowly reversible, adsorption of phytic acid was found to occur when previously documented eluent conditions were used. In an attempt to overcome these problems, the influence of both ionic strength and pH on elution of orthophosphate and phytic acid was investigated.
EXPERIMENTAL
photolysed carrier/reagent solutions prior to the addition of chromogenic reagents. Reagents Alkaline persulphate reagent. A 0.14&U potassium persulphate (BDH, AnalaR) solution in 0.025M sodium tetraborate (May and Baker, Pronalys) was used. Acid molybdate reagent. Ammonium molybdate (BDH, AnalaR) (8.1 x 10w3it4) in 0.63M sulphuric acid (Ajax Chemicals). Tin(II) chloride reagent. A 8.9 x 10e4M solution of tin(I1) chloride (May and Baker, Pronalys) and 0.015M hydrazine sulphate (Ajax Chemicals, LR) in 0.5OM sulphuric acid were prepared. Model phosphorus compounds
Potassium dihydrogen orthophosphate (BDH, Pronalys) (mol. wt. of anion ca. 97.0); myo-inositolhexakis (dihydrogenphosphate), disodium salt dihydrate (i.e., phytic acid), 78% pure (Aldrich) [mol. wt. of phytate anion (- 8) ca. 837.81; both prepared as 100mg P/l. stock solutions.
Flow injection manifold
Gel jiltration conditions
LMWP and HMWP were determined postseparation using a flow injection manifold for determination of DRP + DOP similar to that described by McKelvie et a1.16 and shown in Fig. 1. An Applied Biosystems ABI 757 W-Vis chromatographic detector was used. For the high range standards, a detector sensitivity of 0.1 AUFS (absorbance units full scale) was selected, whereas 0.02 AUFS was used for the lower range. Accurel S6/2 (ENKA AG) microporous tubing was used for debubbling
Two Sephadex G25 columns were used: Column A. A ClO/lO column equipped with an AC10 adaptor was packed with pre-swollen Sephadex G25 Superfine (all from Pharmacia Fine Chemicals AB) to a bed height of 42 mm (bed volume = 3.3 ml), and used for eluent optimization and sediment extract separations. Column B. A Fast Desalting HRl O/IO high resolution column of bed height 100 mm (bed volume ca. 7.8 ml) (Pharmacia Fine Chemicals AB) was used for analysis of water samples.
Sephadex G25 column
uv
300x x 0.5 mm 600
_
Eluent AlkdinB persulphate Acid moiybdate Tin(M)
Peristaltic pump
Fig. 1. Flow injection manifold for gel filtration separation with post-column DRF + DOP detection. Pump flow rates shown as those used for the Sephadex HRlO/lO column. Flow rates were halved when column A was used.
Speciation of dissolved phosphorus
Columns were stored refrigerated in 0.02% (w/v) sodium azide when not in use. Injection volumes of either 100,350 or 600 ,ul were used depending on the size of column and concentration range of the samples. For the separation optimization studies using column A, 350-~1 injections were made throughout. Gel filtration separations of phytic acid and orthophosphate were investigated using eluents of either 0.034-0.137M NaCl (pH 5-9),‘1*14 0.025M sodium tetraborate (pH 9.1),15 or Tris-HCl buffers adjusted with O.lM NaCl to give total ionic strength (I) values of between 0.001 and 0.007M as required. All eluents were flltered through 0.45~pm Sartorius Type SM membrane fllters and allowed to degas for 15 min prior to measurement of pH and use. To investigate the feasibility of separating orthophosphate and HMWP by gel filtration, columns were calibrated using a range of solutes of varying molecular weights. Elution of these solutes was detected by measurement of W absorbance, or in the case of phytic acid and phosphate, by detection as DOP and DRP, respectively. Blue Dextran (Pharmacia Fine Chemicals AB), with a nominal molecular weight of 2 x lo6 Dalton was used to determine the void volume, V,, and nitrate ion (FW = 62.0) was used to estimate the total pore volume, Vi, of the gel co1umn.‘7 In NaCl and borate eluent solutions, direct W detection at 220 nm was used to determine nitrate. However, the Tris buffer used in later experiments had a large background absorbance, and a nitrate ion selective electrode (Activon) was used for detection of nitrate. Sample preparation Water samples were collected from several lotic and lentic environments in Victoria, a sewage treatment works and a leachate pond at a rural landfill tip. Samples were filtered on-site using a 0.45~pm Lida disposable syringe filter and stored at ~4” prior to analysis, which usually occurred within 48 hr. Filtered water samples were injected in triplicate (600 ~1) onto a Sephadex G25 HRlO/lO column (B) and eluted with a Tris/HCl eluent of I = 0.005M at pH 4.6-4.7 at a flow rate of 1.2 ml/mm. Lacustrine sediments (l-2 g) were extracted in 0.025M disodium tetraborate (ca. pH 9.2) and mixed end-over-end for 1 hr at room temperature. Diluted (1: 10) sediments extracts were analysed using the same Sephadex G25 Superfine column (A) used for the elution
0.8
I: : ;....... ‘hale.....+..............~..............~ ...... ........+a, e ........ 1............. .......
I)$_
.....
l.o;.
y”
1983
..........
\
.....
. ............
i 0.4_.
..............
0.*_
...........
i.4
..>..............i.
............
P’-F+-“w~;‘e ...
;
....aJTA ..I..............;...............>
..i’.........
..PhyUCeCki
. 1.0
; 2.0
.
......
.j.. ............ ;
0.0.
. .......
j.. .....
y-i”
;
;
3.0
4.0
-?-+
\ 5.0
log(MW)
Fig. 2. Molecular weight calibration of different Sephadex G25 columns under differing elution conditions: (0) Column A, eluent: 0.2% NaCl @H 8.5) at 0.62 ml/min; injection volume 100 pl, (m) G25 bed height 103 mm, 10 mm id. eluent: 0.5% NaCl (pH 6.2) at 1.24 ml/min. Injection volume 350 pl, (A) Column B, eluent: 5 mM Tris (PH 4.5) at 1.24 ml/min. Injection volume 350 ~1.
optimization. Standard solutions of phytic acid and orthophosphate were prepared in the same matrix as the extracts. A Tris/HCl eluent of I = 0.005M and pH 4.65 at a flow rate of 0.65 ml/min was used, and triplicate 350~~1 injections were made. DRP and DOP+ DRP analyses were also performed on all water and sediment samples for comparative purposes. RESULT!3 AND DISCUSSION
ikiolecular weight calibration From molecular weight calibration data (Fig. 2), it is evident that an essentially linear relationship between the distribution coefficient, &, and log(molecular weight, MW) applies over the MW range which includes phytic acid and orthophosphate. Those which are significantly larger (aprotinin, myoglobin) fall outside this linear portion. Extrapolation of this linear portion of the calibration graph indicates that the operational exclusion limit is approximately lo3 Dalton, rather than the value of 5 x lo3 Dalton for globular proteins normally quoted.17 The observed k;l values of ca. 0.2 and 0.8 for phytic acid and orthophosphate respectively, are close to those previously reported for separation of these solutes on Sephadex G25 under similar elution conditions,‘8 and it is evident that phytic acid is eluting with a yd consistent with that of a non-aggregated, monomeric form. The calibration data, while indicating molecular weight-dependent elution behaviour, do not show resolution of the solutes. For the fast gel filtration separations on column (A), phytic
I. D. MCKELWE et al.
1984
acid and orthophosphate were incompletely resolved, but nevertheless quantifiable. However, separation of solutes with intermediate molecular weights was not feasible, and consequently analytical results are reported non-specifically as high molecular weight phosphorus (HMWP) and low molecular weight phosphorus (LMWP), rather than as DGP of a particular MW and orthophosphate. Eluen t selection Initial attempts to separate phytic acid and orthophosphate on even quite large Sephadex G25 columns (200 mm x 10 mm i.d.) using 0.2% (w/v) NaCl resulted in poor recoveries of phytic acid, and it was evident that some specific interaction, or slow reversible adsorption was occurring which was only slightly affected by pH. Despite poor recovery, phytic acid eluted with a I’,/ V, value which was consistent with its molecular weight, as shown in Fig. 2. It was observed that when either standard or sample was prepared in deionized water and injected into an eluent of higher ionic strength, notably better recovery of phytic acid was achieved compared with the situation where sample or standard was prepared in a matrix of the same ionic strength as the eluent. This is clearly seen in Fig. 3(a) and (b) where injections of mixtures of phytic acid and orthophosphate have been made in triplicate. Figure (3a) shows that when sample
04 Fig. 3. Triplicate injections of 300 pg P/I each of phytic acid and orthophosphate, showing the influence of sample zone ionic strength on elution of both species (a) eluent and sample zone: 0.2% NaCl (w/v) pH 9.7 with 5 min between injections, and (b) eluent: 0.2% NaCl (w/v) pH 9.7 with 4 min between injections, sample zone: deionized water. Elution conditions: column A, flow rate 0.62 ml/min, injection volume 350 pl. The numerals shown on the chromatograms are retention times in minutes. In both cases, the phytic acid peak elutes first.
zone and carrier had the same ionic strength, significant adsorption of phytic acid occurred (peaks at 3.44, 8.49 and 1344min). However, when the sample zone matrix was of much lower ionic strength, markedly improved recovery of phytic acid was noted [Fig. 3(b)], as indicated by the peaks at 3.19, 7.24 and 11.29 min. This effect is similar in some respects to the salt boundary elution effect previously described by Posner. ‘OPosner suggested that preparing a sample with a higher ionic strength would cause solute adsorption when the sample was added to a gel column. Elution with distilled water would then cause the progressive desorption of the adsorbed solute and this would move down the column as a concentrated zone at the distilled water-saline eluent interface. In the case illustrated by Fig. 3(b), the sample was dissolved in deionized water, and injected into a carrier/ eluent of 0.2% (w/v) NaCl. However, despite these conditions being the opposite of those described by Posner, the same type of elution persisted; i.e., phytic acid in a deionized water matrix showed much less adsorption than the same sample prepared in eluent. It is possible that the adsorptive process occurs at the head of the sample zone where there is considerable dispersion and hence mixing, with the saline eluent. Given that a large injection volume of 350 ~1 (10.6% of total column volume, V,) was used, it could be expected that the relatively undispersed mid-region of the sample zone would have a lower ionic strength than that of the eluent, and it is conceivable that it is this low ionic strength zone which causes the partial desorption of phytic acid which is observed. Although the mechanism of this process is unclear, it is quite evident from the relative sizes of the peaks, that better, albeit not complete recovery of phytic acid occurs when an ionic strength gradient exists across the sample zone, and for this reason the influence or ionic strength and pH on separation of orthophosphate and phytic acid in a deionized water matrix were studied systematically to obtain the optimum condition for separation and recovery of phytic acid. Mixed standards of phytic acid and orthophosphate in deionized water were injected onto the same column and eluted with carrier having a variety of pH and ionic strength conditions using Tris-HCl-NaCl solutions ranging from I = 0.001 to 0.071U. The results of these experiments are shown in Fig. 4 from
1985
Speciation of dissolved phosphorus
J”c4~z5.~6.~z7.~z9.4
I = 0.001
L\Vflo= 0.00 AVe/Vo = 0.00
AVcjVo = 0.00 AVe/Vo = 0.00
AVelvo = 0.00
~H~4x~4.7~~6.x~7~*.6
I =
0.003
AVe/‘.‘o = 0.38 I=o.oo5
AVe/Vo = 0.29 AVeJVo = 0.23 AVe/Vo = 0.00
AVe/Vo = 0.00
~=3~=4.5~6.5~=7.~=9.7
AVelVo = 0.46
AVeJVo = 0.40
AVejVo = 0.33 AVeJVo = 0.22 AVJvo
pH = 6.0
pH = 7.2
pH = 7.5
= 0.17 pH = 9.9
I = 0.01 _.JAAA AVePfo = 0.50 AVeJVo = 0.35 AVeJVo = 0.34 pH = 5.8
I =
pH = 7.2
AVe/Vo = 0.33
pH = 7.5
pH = 10.0
0.03 _ALfLJA AVe/Vo = 0.58
AVe/Vo = 0.55 AVeJVo = 0.52
pH =5.9
pH = 7.2
AVe/;go = 0.47
pH = 7.5
pH = 10.0
I = 0.05 _-JLJLJJ AVe/Vo = 0.59 0.05
AVeJVo = 0.58
pH = 6.2
AVe/Vo = 0.56
pH = 7.2
AVe/Vo = 0.46
pH = 7.5
pH = 9.7
I = 0.07 % <
0L
-JJJi\ 5 minutes
AVelvo = 0.57 AVJvo
= 0.61 AVe/Vo = 0.59
AVe/Vo = 0.50
Fig. 4. Variation in the elution of orthophosphate and phytic acid species as a function of total ionic strength and pH. Peak separation is expressed as AVJV,, where V, is the solute elution volume and V, is the void volume of the gel bed.
which a number of features of the chromatography are evident: (i) At low ionic strength (I = O.OOlM), phytic acid and orthophosphate co-elute under all pH conditions tested.
(ii) At slightly greater total ionic strength (I = O.O03M), orthophosphate shows partial resolution from phytic acid (AVJV,, = 0.23-0.38). However, at pH > 6.4, loss of separation is observed.
I. D. MCKELVIE et al.
1986
that of Blue Dextran, and even at high ionic strength (e.g., Z = O.O7M), with a large amount of adsorption occurring, there is only a small shift in the value of VJV,,. Despite the high charge on the phytic acid species, it appears that its elution is dictated by steric factors rather than charge considerations. This is consistent with the findings of Neddermeyer and Rogers” who suggested that larger charged ions would not be influenced by the Donnan exclusion effect to the same extent as smaller ions because they would be sterically excluded from the gel interior containing most of the charge. This may provide an explanation for the apparent absence of anion exclusion behaviour of phytic acid, despite its high charge. Neddermeyer and Rogers have also noted that solutes subject to the influence of anion exclusion display broadened front and sharp back edges, behaviour which is the converse of that normally observed in chromatography.*’ Examination of orthophosphate and phytic acid peaks reveals that while the orthophosphate peaks show broadened leading edges, phytic acid peaks do not, providing further evidence in support of the hypothesis that steric effects control the behaviour of phytic acid. Maximum recovery of phytic acid with respect to orthophosphate, based on peak height, was achieved for the specified column and flow conditions using eluents with Z = 0.005M and pH 4.5-6.5. Under optimal elution conditions (I = O.O05M, pH 6.5, cf, Fig. 4) the % recovery of phytic acid with respect to orthophosphate calculated using peak height was 97.4% (0, _ , = 0.2%, n = 3), but when calculated on the basis of peak area, was only 78.2% (c,_ , = 3.2%, n = 3). Given that a common phosphomolybdenum blue detection chemistry 2.0 was used, it is evident that significant adsorption pH range: 1.3 _7.5 of phytic acid had occurred even at this sol called optimum condition. Furthermore, when columns were cleaned with dilute sodium hydroxide solution (pH > 10) after the injection of several samples of phytic acid, a large desorp1 T I tion peak was observed; however this was not 1.2 d Q P O the case for orthophosphate. It was also appar0 ent from the uniformity of peak area data, 1.0 0 obtained over a wide range of ionic strength t and pH conditions, that little orthophosphate I 0.8 ! 0.08 0.06 0.04 0.00 0.02 adsorption occurred. It is unfortunate that even under salt-zone Total Ionic Strength elution conditions adsorption of phytic acid perFig. 5. Variation of VJV, for orthophosphate (0) and sisted. It would have been preferable to use only phytic acid (0) species as a function of total ionic strength one calibration substance, i.e., orthophosphate within the pH range 7.3-7.5.
(iii) At total ionic strength in excess of O.OOSM, significant adsorption of phytic acid was found to occur. Very slow desorption of some species (presumably phytic acid), indicated by a gradual return to baseline, was observed after elution of the orthophosphate peak (not shown by the data in Fig. 4). The co-elution of orthophosphate and phytic acid found to occur at the lowest ionic strength conditions tested (I = O.OOlM) was probably due to anionic exclusion of the orthophosphate. At Z = 0.003M some separation of the two species occurred at lower pH, but at pH > 6, the double- and triple-charged phosphate species are more prevalent, and the anion exclusion effect overcame the influence of increased ionic strength; a similar trend to this was observed for an eluent with Z = 0.005M. This anion exclusion effect appears to have been completely suppressed when the ionic strength was increased to O.OlM, and the phytic acid and orthophosphate peaks are as well separated as might be expected given the short column used. Predictably, however, there is significant adsorption of the phytic acid under these higher ionic strength conditions.20 Figure 5 shows the variation of VJV,, of both orthophosphate and phytic acid species with increasing ionic strength within the pH range 7.3-7.5. The orthophosphate shows a pronounced increase in VJV,, but a much smaller effect of ionic strength on phytic acid. This is perhaps surprising, given the highly charged nature of the phytic acid species (within the pH range used, the average charge on the phytic acid would be cu. - 8). The value of VJV,, for phytic acid is, however, always larger than
?
4
I
I
I
Speck&ion of dissolved phosphorus
1987
Table 1. Calculated limits of detection (lad) and sensitivity data for phytic acid and orthophosphate for the 42-mm bed height column (A)
Analyte Phytic acid Phosohate
High range (~OO-~~ 1(8 P/l.) Sensitivity Limit of detection (AU/M P/l.) w PP.) 44 12
1.338 x IO-’ 1.318 x IO-’
and a common peak-height : concentration calibration equation for all eluting species. This would only be possible in the absence of solute-gel interactions, given that all eluting species are converted from DOP to DRP before detection. There was, however, only a narrow range of pH and ionic strength conditions for which the separation and O/Orecovery of phytic acid (by peak height) was maximal. Even slight departure from the optimum ionic strength and pH conditions (I = O.OOSiV,pH 4.5-6.5) would cause the % recovery of phytic acid with respect to orthophosphate to change markedly. Such variations in the ionic strength could occur in the preparation of reagents, or may arise from some hydrodynamic artefact of the FIA manifold causing a variation in the degree of dispersion of the sample zone, and hence altering the ionic strength regime of the sample zone. Such artefacts may involve changes as simple as a fluctuation in carrier flow rate, or a variation in the sample volume. For these reasons, mixed standards of phytic acid and orthophosphate were used for quantitation of HMWP and LMWP, in preference to quantitation using orthophosphate alone and applying a response factor. It is recognized that this calibration approach may be deficient if an HMWP species elutes from the gel in a manner different to that of phytic acid. Linearity, sensitivity and limit of detection of mixed standard calibrations Mixed standards of phytic acid and orthophosphate were prepared over the ranges 50-1000 c(g P/l. and 10-100 pg P/l. Triplicate injections of 350 ~1 of these standards were made onto the same Sephadex G25 Superfine column that was used for all previous experiments under optimal separation conditions. At concentrations greater than approx 200 pg P/l., phytic acid and orthophosphate graphs were essentially co-linear, giving regression equations of A = (-2.63 x lo-‘) + (1.34 x lo-4)pg P/l, r* = 0.999,
Low range (10-100 l(g P/l.) Limit of detection Sensitivity ocg P/l.) (Auk? P/l.) 27 8.88 x 10-s 1.1 1.323 x lo-’
and A = (3.00 x 10-4) + (1.32 x 10-4)c(g P/l., r2 = 1.000, respectively. However, below ca. 100 pg P/l., the orthophosphate calibration is linear, but the phytic acid exhibits non-linear behaviour. At 50 pg P/l., there is a loss of approximately 50% of the phytic acid, while at < 10 p g P/l., phytic acid peaks were scarcely detectable, even though orthophosphate signals were still clearly discernible. Clearly, at concentrations of less than 100 pg P/l., the specific adsorption of phytic acid onto the gel poses a difficulty. Limits of detection (lod) have been calculated for these data using the linear regression method described by Miller and Milled and are listed with calibration sensitivity (slope) values in Table 1. Eflect of sample volume on peak response, resolution and O/O recovery The influence of sample volumes ranging from 100 to 600 ,ul on peak response, resolution, and % recovery (based on comparison with orthophosphate peak height) was investigated. A linear relationship between injection volume (S,) and peak height (Absorbance, A) was observed over the concentration range tested. For the dilute solutions analysed here, it can be shown that the relationship between the absorbances of undispersed (A”) and dispersed (A) sample, -$=
1 -exp(--kS,)
quoted by Ruzicka and Hansenz3 approximates to: A z k S, for a range of A values from 0.001 to 0.1. S1,2,the injection volume required to give a dispersion coefficient of 2, can be determined from 0.693 s l/2=k Using this relationship, and the k (slope) value from an A vs. S, plot, very high SI,2 values of
I. D. MCKELVIE
1988
ef cd.
0.6
0.6 0.4
o.o0
200
400
600
0
200
400
600
Injection Volume (pL)
Injection Volume (pL)
Fig. 6. Influence of sample volume on (a) resolution (for 500 pg P/l. each of phytic acid and orthophosphate), and (b) % recovery of 50 fig P/l. (-A-) and 500 pg P/l. (-A-) phytic acid with respect to orthophosphate. Column A, eluent flow rate 0.62 ml/min.
>20,000 ~1 at 50 pg P/l. and >4,000 ~1 at 500 pg P/l. were obtained. The very large values of & at 50 pg P/l. reflect the consideration dispersion that occurs in the gel column. Clearly, use of a smaller gel-filtration column would be preferable and more in agreement with conventional FIAchromatographic practice. Smaller columns would necessitate the use of small injection volumes if column overloading and attendent loss of resolution are to be avoided. However, the combination of small injection volumes and the high dispersion of the DOP + DRP detection manifold would result in poor sensitivity, and consequently the use of small columns and small injection volumes is precluded in this context. Figures 6(a) and (b) show the improvement in peak resolution and the corresponding decrease in % recovery of phytic acid (calculated from peak height data) as the injection volume is decreased to 100 ~1. The lower recovery of the 50 pg P/l. standard with respect to the 500 pg P/l. standard is probably due to a combination of preferential adsorption of phytic acid to the gel and dispersion. These results highlight the interrelationship between phytic acid adsorption and factors such as eluent-sample zone ionic strength factors like difference, and hydrodynamic sample injection volume, column size, and flow rate which influence dispersion.
trations of both HMWP and LMWP calculated from peak height may be mutually influenced by peak overlap. To determine the magnitude of this effect, several mixed standards all containing 300 pg PO,-P/l. and phytic acid concentrations varying between 50 and 400 pg phytic-P/l. were analysed. Figure 7 shows that the absorbance of the 300 pg PO,-P/l. standard increased linearly, by as much as 10.3%, as the concentration of phytic acid was increased from 50 to 400 c(g phytic-P/l. Similarly, the absorbance as. concentration plot for phytic acid displayed increasing deviation from linearity at higher concentrations because of overlap from the leading edge of the orthophosphate peak. Because this effect proved problematic in the analysis of some sediment extracts, a larger, high resolution column was employed for the analysis of water samples, as described in the following section. 0.05
g
0.03
1 a
0.02
0
100
200
300
IO
pg Phytic acid-P/L
Influence of phytic acid concentration on apparent concentration of orthophosphate for short columns
Phytic acid and orthophosphate peaks were only partly resolved on the short G25 Superfine column (A). Under these circumstances, the measured peak heights, and hence concen-
Fig. 7. Effect of differing phytic acid concentrations (0) on peak height of 300 cg P/l. orthophosphate (0) in short column gel filtration chromatography. Tris/HCl eluent at pH 4.75 and I = 0.005. Column A, injection volume 350 ~1. Eluent flow rate 0.62 ml/min. Lines of best fit for phytic acid and orthophosphate given by: y = (7.49 x IO-‘) + (7.77 x 1O-5)x + (1.04 x IO-‘)x2 r2 = 0.998 and y = (3.20 x 10-2) + (9.47 x 10e6)x r2 = 0.923, respectively.
Speciation of dissolved phosphorus
Analytical results-concentrations of HMWP and LMWP found in sediment extracts and waters Preliminary extractions of sediments with deionized water gave low yields of HMWP. Minea?” has noted that only about 40% of DOP was extracted from particulate material and Chiamydomonas cultures by distilled water, but that 85-100% was extracted when alkaline conditions (ca. pH 13) were used. Extraction of phosphorus from sediments was tested using Olsen’s reagent (OSM NaHCO, at pH 8.5);*’ however, the high bicarbonate concentration was found to interfere with the photo-oxidation of DOP, presumably through hydroxyl radical scavenging. Aluminium may also interfere in the photo-oxidation of DOP at concentrations > 100 pg A1/l.26 and for this reason acidic sediment extractions were avoided. HMWP and LMWP concentrations of the sediment extracts obtained by separation on the short (42 mm x 10 mm id) Sephadex G25 column with subsequent DOP analysis are shown with corresponding DOP and DRP data, expressed on a dry weight basis, in Fig. 8(a)-(c). The concentration of LMWP was significantly greater than the corresponding DRP data (P = 0.0058, paired t-value 3.91, d.f. = 7). Two possible explanations can be tendered, oiz; (i) Desorption of phytic acid, or other late eluting species, may enhance the LMWP peak. While the separation has been optimized to give maximum recovery of phytic acid, some phytic acid appears to undergo very slow desorption. Other phosphate-species, such as Vitamin B12 and 4nitrophenylphosphate, exhibited reversible adsorption and late elution when injected under these elution conditions. (ii) Organic phosphorus species of similar molecular weight range to orthophosphate (e.g., lower phosphate esters of myo-inositol) may be present and elute with the orthophosphate. Given the fairly poor resolution of the small column used, discrimination between orthophosphate and other low molecular weight phosphorus species would not be possible. From Fig. 8(a), it is evident that DOP values are significantly greater than those for HMWP (P = 7.0 x 10m4,paired t-value = 5.76, d.f. = 7). Between 65 and 97% of the DOP is represented by the HMWP of similar MW to myo-inositolhexakisphosphate, and this is consistent with suggestions regarding the stability of this phosphate ester and its probable role in the storage
1989
of phosphorus in sediments.’ Other researchers have reported similar recoveries of inositol phosphate esters from soils*’ and lake sediments.** The fact that the DOP values are all greater than the HMWP values [Fig. 8(b)] also lends some credence to any one of the explanations provided for the apparent discrepancy between LMWP and DRP. Moreover, it can be seen that when (DRP+DOP) and (HMWP+LMWP) data are compared [Fig. 8(c)], there is excellent agreement, as shown by the regression equation: (HMWP + LMWP) = 1.05 (DRP + DOP) - 1.34,
r = 0.987
(r significantly different from zero at P > 99%). The agreement between (HMWP + LMWP) and (DRP + DOP) suggests that there are no major recovery losses associated with the gel filtration separation that cannot be accounted for by calibration. It also supports the hypothesis that differences between DRP and LMWP can be explained by the co-elution of lower MW organic phosphorus species with orthophosphate. For water samples, LMWP and HMWP were separated on the larger, high resolution HRlO/lO column (B). Sample injections of 600 ~1 were necessary to compensate for the added dispersion associated with use of a large column, but this was compensated for by the complete resolution of phytic acid and orthophosphate, and the ability to use high eluent flow rates with this column. Flow rates of up to 6 ml/min are permissible, but in experiments reported here, flow rates in excess of 1.2 ml/min were avoided because the debubbler in the DOP photo-oxidation manifold did not function efficiently at these higher flow rates. Results of the analyses of the water samples are shown in Fig. 9(a)-(c). The importance of HMWP as a constituent of some waters is noted, as it comprised from 0 to 97% of the total HMWP + LMWP detected. There was no significant difference between the (HMWP + LMWP) gel filtration data and the (DRP + DOP) data for the same samples (P = 0.79, paired t = 0.27, d.f. = 9). However, HMWP was significantly greater than the DOP fraction (P = 0.03, paired t-value = 2.67, d.f. = 9) and LMWP is significantly less than the DRP fraction (P = 0.02, paired t-value = 2.85, d.f. = 9). These trends are opposite to those found for the extractable phosphorus in the
I. D. MCKELVIEet al.
1990
2.1176, R-8quw.d:
.9SOlX -
y I
20
3606
16 16 14
12 10
6 6 4 2 o+
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_:
:-.
.
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,
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:
.
s
:
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.
:
.
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:
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.
:
.
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:
.
1s
:
te
.
)
20
DOP significantly > HMWP (P = 7.0 x 1O4, paired t-value 5: 5.76, d.f, = 7) y r
.9364x + 1.6634. R-squared:
,681
6
04 d
W
.
I
I 4
i
I lo
I
li
LMWP significantly > DRP (P = 0.0058, paired t -value = 3.91, d.f. = 7) y
=
1.0446x -
lb
Cc)
RI(IDR%-PIkg
I
e
1.3407, R-squared:
3738
2-5
2’0 lb mg(DRP+DOP).P/kg
No significant difference between HMWP+LMWP
and DRP+DOP
30
(P = 0.14,
paired
t-value = 1.68, d.f. = 7)
Fig. 8. Comparison of (a) HMWP and DOP, (b) LMWP and DRP and (c) HMWP + LMWP and DRP+ DOP for sediments, expressed on a dry weight basis. The broken tines indicate the 95% confidence
intervals for the true mean of the y values.
sediment samples. One possible explanation for the higher DRP than LMWP data is that some of the DRP measured includes the products of rapidly hydrolysed dissolved organic phosphorus, a problem which has been reported
extensively.2*2g*M As DOP is determined by subtracting DRP from the total DRP + DOP, the effect of an inflated DRP value will result in significantly reduced DOP values. Chamberlain and Shapiro, for example, in a batch method,
Speciation of dissolved phosphorus
limited mixing and reaction between sample, molybdate and reductant to 6 set, but noted that some hydrolysis of DOP occurred even under these rapid reaction conditions.3’ For the flow injection DRP method used here, the time Y
(a)
I
+
between injection and emergence of the peak maximum was only 20-25 set, but it is feasible that some hydrolysis of DOP could occur even in this short period. Six of the samples analysed had LMWP 2.2045,
R-squwrd:
.SSBO
HMWP signif’i&nUy > DOP (P = 0.03. paired t-value = 2.67, d.f. = 9) Y
0
lb)
1.4033~
1991
,-20
DRP significantly
.936X
=
40
8.9436.
-
R-wurrod:
AI006
100
60
140
120
160
> LMWP (P = 0.02, paired l-value = 2.85, d.f. = 9) y
o
.7322x
+
17.0794,
R-squared:
.a092
160 160 140 120 100 60 60 40
0 0
w
20
40
No significant difference t-value = 0.27. d.f. = 9)
60
100 60 w(DRP+DOP)-P/L
between
120
140
160
160
HMWP+LMWP and DRP+DOP (P = 0.79. paired
Fig. 9. Comparison of (a) HMWP and DOP, (b) LMWP and DRP and (c) HMWP + LMWP and DRP + DOP for water samples. The broken lines indicate the 95% confidence intervals for the true mean of the y values.
1992
I. D. MCKELVIEer al.
values which were significantly lower than the DRP concentrations. However, there is no significant difference between (LMWP + HMWP) and (DRP + DOP) for most samples, and it is arguable that measurement of LMWP and HMWP provide better measures of nonhydrolysed organic phosphorus and orthophosphate than do DOP and DRP. In two diluted effluent samples the HMWP was undetectable; however, these samples were high in DRP and required dilution of 1: 50 prior to analysis in order to get them into the same concentration range as other samples. It is probable that this was sutIlcient to dilute the low concentration of HMWP to below the detection limit, or that the low concentrations of DOP (14 and 5 pg Pi/l. respectively) were comprised of late-eluting species. This study has highlighted some of the problems associated with the use of Sephadex G25 for the fractionation of dissolved phosphorus species in natural waters and sediment extracts, viz. partial adsorption of phytic acid species and late elution of other higher molecular weight species. This gel filtration approach to the separation of HMWP and LMWP is apparently the most rapid yet reported with an analysis rate of 12-15/hr, but it is not sufficiently sensitive for the analysis of dilute or pristine waters. It is envisaged that further research in this area could involve the use of complexing eluents to eliminate adsorption of HMWP species through complexation by metals adsorbed to the gel, along similar lines to that described by Town and Powell.32 In this study, only Sephadex G25 has been used, and it would be useful to repeat the ionic strength-pH studies on a wide range of gels, including the macro/microreticular Ultrogels, to determine optimum separation conditions. This would be labour-intensive by conventional gel filtration, but could be performed within a reasonable time using the gel filtration-FIA approach described. Separation of HMWP and LMWP in waters with a range of gels of different MW cut-off would provide further information on the concentration and speciation of dissolved organic phosphorus in natural waters. Detection of low concentrations of DOP compounds using the gel filtration-FIA system is clearly a problem. However, it was shown that phytic acid and phosphate could be concentrated onto an ion exchange precolumn prior to injection” and DOP analysis. This approach could be applied to the preconcentration of at
least the anionic HMWP species prior to gel filtration. While this study has identified that waters and sediment extracts contain HMWP in the same molecular weight range as phytic acid, and it is probably reasonable to assume that phytic acid is a major constituent of the HMWP, no information about the presence of other phosphate esters of myo-inositol can be obtained because of the inability of the gel to resolve the small differences in molecular weight involved. Low pressure anion exchange chromatography has been used extensively to separate myo-inositol phosphate esters,6J8*” and from the experience of this study, it should not be difficult to couple an anion exchange column with an FIA-DOP manifold to achieve rapid separations. Finally, the relevance of the HMWP and LMWP data in waters needs to be evaluated in terms of their bioavailability by comparison with appropriate algal bioassay techniques. Acknowledgenrenr-Support for this research by the Australian Research Council is gratefully acknowledged.
REFERENCES
1. E. M. Thurman, Organic Geochemistry of Natural Waters, p. 497. Martinus Nijhoff/Dr W. Junk Publishers, Dordrecht, The Netherlands, 1985. 2. S. J. Tarapchak, J. Environ. Qua/., 1983, 12, 105. 3. W. Stumm and J. J. Morgan, Aquatic Chemistry, 2nd Ed., p, 780. Wiley, New York, 1981. 4. G. Anderson, in A. D. McLaren and G. H. Peterson, Soil Biochemistry, p. 67. Marcel Dekker, New York, 1967. 5. D. J. Cosgrove, Inositol Phosphates-Their Chemistry, Biochemistry and Physiology, p. 191. Elsevier, Amsterdam, 1980. 6. S. J. Eisenreich and D. E. Armstrong, Enoiron. Sci. Technol., 1977, 11, 497. I. S. E. He&s, H. E. Allen and K. H. Mancy, Science, 1975, 187,432. 8. R. A. Minear, J. E. Segars and J. W. Elwood, Analyst, 1988, 113, 645. 9. C. M. Clarkin, R. A. Minear, S. Kim and J. W. Elwood, Environ. Sci. Technol., 1992, %, 199. 10. J. B. Cotner and R. G. Wet&, Limnol. Oceanogr., 1992, 37, 232.
11. E. White and G. Payne, Can. J. Fish. Aquut. Sci., 1980, 37, 664.
12. E. Bentzen, W. D. Taylor and E. S. Millard, Limnol. Oceanogr., 1992, 37, 217. 13. D. Lean, in D. Povoledo and H. L. Golterman, Humic Substances. Their Structure and Function in the Biosphere; Proceedings of an International Meeting Held in Nieuwersluis, The Netherlands, 1972, p. 159. Pudoc,
Wageningen: Center for Agricultural Publishing and Documentation, 1975. 14. M. T. Downes and H. W. Paerl, J. Fish. Res. Bd. Can., 1978, 35, 1636.
Speciation of dissolved pho~ho~ 15. R. 1. Stevens and B. M. Stewart, Water Res., 1982, 16, 1507. 16. I. D. McKelvie, B. T. Hart, T. 1. Cardwell and R. W. Cattral1, AnalyJt 1989, 111, 1459. t7. Phannacia, Gel Fibation: Theory aad Practice, Pharmacia Fine Cbmiuds AB, Lund. Sweden, 1979. 18. J. H. Steward and M. E. Tate, 3. Chromatog., 1%9,4!$ 400. 19. A. M. Posner, Name, 1963, 198, 1161. 20. J.-C. Janson, J. Chromatogr., 1967, 28, 12. 21. P. A. Neddenneyer and L. B. Rogers, Awt. Gem., 1968,46,755. 22. J. C. Miller and J. N. Miller, St~t~t~s~r ~yzic~ Ckemistry, 2nd Ed., p. 227. Ellis Horwood, Chichester, 1988. 23. RiIZiEka and E. H. Hansen, IVow Injection Analysis, 2nd Ed.. 62, p. 498. Wiley, New York, 1988. 24. R. A. Minear, &&on. Sci. Tecknol., 1972, 6, 431.
TAL 4om-o
1993
2% P. R. Hesse, A Textbook of Soil Ckemicai Analysis, p. 520. John Murray, London, 1971. 26. I. D. McKelvie. Flow Injectioul Analysti of Dissolved Phosphorus SpecicJ in Na&ral and Wastewaters. PhD Thesis, La Trobe University, 1992. 27. J. H. Steward and M. E. Tate, J. Ckromatogr., 1971,6i& 75. 28. W. C. Weimer and D. E. Annstrong, Anal. Chim. Acra., 1977, w, 35. 29. M. T. Dowries, Water Res., 1978, 12, 743. 30. F. H. Rigler, Lim&. Oceungr., 1968, 13, 7. 31. W. Chamberlain and J. Shapiro, i&i& 1969, 14, 921. 32. R. M. Town and H. J. J. Powell, Am& Cirim. Acra, 1992, 2!%, 81. 33, P. R. Freeman and I. D. McKelvie, unpublished data. 34. D. J. Cosgrove, in W. W. Wells and F. Eisenberg, Cyclitols and Phosphoinosirides, p. 22. Academic Press, New York, 1978.
llo39-914Oj93 s6.00 + 0.00
CopyrightQ I993Fcrgamon Press Ltd
SPECIATION OF DISSOLVED PHOSPHORUS IN ENVIRONMENTAL SAMPLES BY GEL FILTRATION AND FLOW-INJECTION ANALYSIS I. D. MCKELVIE* and B. T.
HART
Water Studies Centre and Chemistry Department, Monash University, Caulfield East 3145, Victoria, Australia T. J.
CARDWELL and R. W. CA~TRALL
Centre for Scientific Instrumentation, La Trobc University, Bundoora 3083, Victoria, Australia (Received 16 February 1993. Revired 29
March
1993. Accepted 30
March
1993)
Summary-A study of the factors afkting separation and detection of dissolved organic and inorganic phosphorus species found in waters and sediients is reported. The system involved the use of gel titration and flow injection analysis (FIA). Orthophosphate and myo-inositol hexakisphosphate. as model solutes representative of low molecular weight P (LMWP) and high molecular weight P (HMWP). were separated on a Sephadex G25 cclumn incorporated into a flow-injection manifold which utilixed photo-oxidation and spectrophotometry for detection of dissolved reactive phosphorus (DRP) and dissolved organic phosphorus (DOP). The influence of eluent pH and ionic strength on adsorption and anionic exclusion of the model solutes is described, and the optimum eluent composition and sample size ate described. The method was used to determine LMWP and HMWP in natural and waste waters, and in sediment extracts. Potential limitations of this approach are discussed.
Phosphorus in natural waters and sediment is known to occur as orthophosphate and a variety of organic and condensed phosphorus species with a wide range of molecular weights.’ Some components of the dissolved organic phosphorus (DOP) fraction are thought to hydrolyse rapidly to orthophosphate when acidic molybate is used in the spectrophotometric analysis of orthophosphate as phosphomolybdenum blue, and for this reason the term “dissolved reactive phosphorus” (DRP) is used in reference to “orthophosphate”. Consequently, DRP concentrations may overestimate orthophosphateZ which is undesirable because orthophosphate is currently the best chemical measure of the dissolved phosphorus concentration biologically available for photosynthesis. In soils and sediments, phosphorus may occur as organic compounds such as sugar phosphates, phosphatidyl phosphates, inositol phosphates, phosphoamides, phosphoprotein, aminophosphonic acids, phosphorus-containing pesticides and organic condensed phosphates.3 Of this group, myo-inositol hexakisphosphate *Author for correspondence.
(also called phytic acid), is among the most stable;’ this compound occurs in free and ironcomplexed forms in soils and marine and lake sediments.5 It has been conjectured that this species and its hydrolysis products will be present in natural waters through groundwater input,’ and may comprise a significant proportion of dissolved organic phosphorus (DOP).47 However, only a few studies have actually demonstrated the existence of inositol phosphates in natural waters,8*9and to date little is known of the transport of these species from sediments and soils into stream and lake waters. Analysis of the DRP concentration in natural and wastewaters is commonly performed in an attempt to estimate the concentration of bioavailable phosphorus in the dissolved fraction. While orthophosphate has been shown in a number of studies to be the most bioavailable phosphorus species, lo*higher molecular weight phosphorus (HMWP) species display varying degrees of bioavailability.‘**‘* In order to determine the concentration of both “true” orthophosphate and HMWP in natural water samples, we attempted to separate these species according to differences in molecular weight using gel filtration followed by photo-oxidation
1981
I. D.
1982
MCKELVIE et al.
and subsequent phosphomolybdenum blue detection of the liberated orthophosphate using FIA. Separation of orthophosphate and HMWP in natural waters by gel filtration chromatography has been described by a number of authors”*‘1*‘~‘5using a variety of eluent conditions, large gel columns, slow flow rates and correspondingly long (30 mm-2 hr) elution times. The aim of the work described here was to develop a rapid technique allowing discrimination between these two molecular weight fractions. Small Sephadex G25 columns were investigated for the separation of model solutes representative of LMWP and HMWP, viz; orthophosphate and myo-inositol hexakisphosphate. Significant irreversible, or very slowly reversible, adsorption of phytic acid was found to occur when previously documented eluent conditions were used. In an attempt to overcome these problems, the influence of both ionic strength and pH on elution of orthophosphate and phytic acid was investigated.
EXPERIMENTAL
photolysed carrier/reagent solutions prior to the addition of chromogenic reagents. Reagents Alkaline persulphate reagent. A 0.14&U potassium persulphate (BDH, AnalaR) solution in 0.025M sodium tetraborate (May and Baker, Pronalys) was used. Acid molybdate reagent. Ammonium molybdate (BDH, AnalaR) (8.1 x 10w3it4) in 0.63M sulphuric acid (Ajax Chemicals). Tin(II) chloride reagent. A 8.9 x 10e4M solution of tin(I1) chloride (May and Baker, Pronalys) and 0.015M hydrazine sulphate (Ajax Chemicals, LR) in 0.5OM sulphuric acid were prepared. Model phosphorus compounds
Potassium dihydrogen orthophosphate (BDH, Pronalys) (mol. wt. of anion ca. 97.0); myo-inositolhexakis (dihydrogenphosphate), disodium salt dihydrate (i.e., phytic acid), 78% pure (Aldrich) [mol. wt. of phytate anion (- 8) ca. 837.81; both prepared as 100mg P/l. stock solutions.
Flow injection manifold
Gel jiltration conditions
LMWP and HMWP were determined postseparation using a flow injection manifold for determination of DRP + DOP similar to that described by McKelvie et a1.16 and shown in Fig. 1. An Applied Biosystems ABI 757 W-Vis chromatographic detector was used. For the high range standards, a detector sensitivity of 0.1 AUFS (absorbance units full scale) was selected, whereas 0.02 AUFS was used for the lower range. Accurel S6/2 (ENKA AG) microporous tubing was used for debubbling
Two Sephadex G25 columns were used: Column A. A ClO/lO column equipped with an AC10 adaptor was packed with pre-swollen Sephadex G25 Superfine (all from Pharmacia Fine Chemicals AB) to a bed height of 42 mm (bed volume = 3.3 ml), and used for eluent optimization and sediment extract separations. Column B. A Fast Desalting HRl O/IO high resolution column of bed height 100 mm (bed volume ca. 7.8 ml) (Pharmacia Fine Chemicals AB) was used for analysis of water samples.
Sephadex G25 column
uv
300x x 0.5 mm 600
_
Eluent AlkdinB persulphate Acid moiybdate Tin(M)
Peristaltic pump
Fig. 1. Flow injection manifold for gel filtration separation with post-column DRF + DOP detection. Pump flow rates shown as those used for the Sephadex HRlO/lO column. Flow rates were halved when column A was used.
Speciation of dissolved phosphorus
Columns were stored refrigerated in 0.02% (w/v) sodium azide when not in use. Injection volumes of either 100,350 or 600 ,ul were used depending on the size of column and concentration range of the samples. For the separation optimization studies using column A, 350-~1 injections were made throughout. Gel filtration separations of phytic acid and orthophosphate were investigated using eluents of either 0.034-0.137M NaCl (pH 5-9),‘1*14 0.025M sodium tetraborate (pH 9.1),15 or Tris-HCl buffers adjusted with O.lM NaCl to give total ionic strength (I) values of between 0.001 and 0.007M as required. All eluents were flltered through 0.45~pm Sartorius Type SM membrane fllters and allowed to degas for 15 min prior to measurement of pH and use. To investigate the feasibility of separating orthophosphate and HMWP by gel filtration, columns were calibrated using a range of solutes of varying molecular weights. Elution of these solutes was detected by measurement of W absorbance, or in the case of phytic acid and phosphate, by detection as DOP and DRP, respectively. Blue Dextran (Pharmacia Fine Chemicals AB), with a nominal molecular weight of 2 x lo6 Dalton was used to determine the void volume, V,, and nitrate ion (FW = 62.0) was used to estimate the total pore volume, Vi, of the gel co1umn.‘7 In NaCl and borate eluent solutions, direct W detection at 220 nm was used to determine nitrate. However, the Tris buffer used in later experiments had a large background absorbance, and a nitrate ion selective electrode (Activon) was used for detection of nitrate. Sample preparation Water samples were collected from several lotic and lentic environments in Victoria, a sewage treatment works and a leachate pond at a rural landfill tip. Samples were filtered on-site using a 0.45~pm Lida disposable syringe filter and stored at ~4” prior to analysis, which usually occurred within 48 hr. Filtered water samples were injected in triplicate (600 ~1) onto a Sephadex G25 HRlO/lO column (B) and eluted with a Tris/HCl eluent of I = 0.005M at pH 4.6-4.7 at a flow rate of 1.2 ml/mm. Lacustrine sediments (l-2 g) were extracted in 0.025M disodium tetraborate (ca. pH 9.2) and mixed end-over-end for 1 hr at room temperature. Diluted (1: 10) sediments extracts were analysed using the same Sephadex G25 Superfine column (A) used for the elution
0.8
I: : ;....... ‘hale.....+..............~..............~ ...... ........+a, e ........ 1............. .......
I)$_
.....
l.o;.
y”
1983
..........
\
.....
. ............
i 0.4_.
..............
0.*_
...........
i.4
..>..............i.
............
P’-F+-“w~;‘e ...
;
....aJTA ..I..............;...............>
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. 1.0
; 2.0
.
......
.j.. ............ ;
0.0.
. .......
j.. .....
y-i”
;
;
3.0
4.0
-?-+
\ 5.0
log(MW)
Fig. 2. Molecular weight calibration of different Sephadex G25 columns under differing elution conditions: (0) Column A, eluent: 0.2% NaCl @H 8.5) at 0.62 ml/min; injection volume 100 pl, (m) G25 bed height 103 mm, 10 mm id. eluent: 0.5% NaCl (pH 6.2) at 1.24 ml/min. Injection volume 350 pl, (A) Column B, eluent: 5 mM Tris (PH 4.5) at 1.24 ml/min. Injection volume 350 ~1.
optimization. Standard solutions of phytic acid and orthophosphate were prepared in the same matrix as the extracts. A Tris/HCl eluent of I = 0.005M and pH 4.65 at a flow rate of 0.65 ml/min was used, and triplicate 350~~1 injections were made. DRP and DOP+ DRP analyses were also performed on all water and sediment samples for comparative purposes. RESULT!3 AND DISCUSSION
ikiolecular weight calibration From molecular weight calibration data (Fig. 2), it is evident that an essentially linear relationship between the distribution coefficient, &, and log(molecular weight, MW) applies over the MW range which includes phytic acid and orthophosphate. Those which are significantly larger (aprotinin, myoglobin) fall outside this linear portion. Extrapolation of this linear portion of the calibration graph indicates that the operational exclusion limit is approximately lo3 Dalton, rather than the value of 5 x lo3 Dalton for globular proteins normally quoted.17 The observed k;l values of ca. 0.2 and 0.8 for phytic acid and orthophosphate respectively, are close to those previously reported for separation of these solutes on Sephadex G25 under similar elution conditions,‘8 and it is evident that phytic acid is eluting with a yd consistent with that of a non-aggregated, monomeric form. The calibration data, while indicating molecular weight-dependent elution behaviour, do not show resolution of the solutes. For the fast gel filtration separations on column (A), phytic
I. D. MCKELWE et al.
1984
acid and orthophosphate were incompletely resolved, but nevertheless quantifiable. However, separation of solutes with intermediate molecular weights was not feasible, and consequently analytical results are reported non-specifically as high molecular weight phosphorus (HMWP) and low molecular weight phosphorus (LMWP), rather than as DGP of a particular MW and orthophosphate. Eluen t selection Initial attempts to separate phytic acid and orthophosphate on even quite large Sephadex G25 columns (200 mm x 10 mm i.d.) using 0.2% (w/v) NaCl resulted in poor recoveries of phytic acid, and it was evident that some specific interaction, or slow reversible adsorption was occurring which was only slightly affected by pH. Despite poor recovery, phytic acid eluted with a I’,/ V, value which was consistent with its molecular weight, as shown in Fig. 2. It was observed that when either standard or sample was prepared in deionized water and injected into an eluent of higher ionic strength, notably better recovery of phytic acid was achieved compared with the situation where sample or standard was prepared in a matrix of the same ionic strength as the eluent. This is clearly seen in Fig. 3(a) and (b) where injections of mixtures of phytic acid and orthophosphate have been made in triplicate. Figure (3a) shows that when sample
04 Fig. 3. Triplicate injections of 300 pg P/I each of phytic acid and orthophosphate, showing the influence of sample zone ionic strength on elution of both species (a) eluent and sample zone: 0.2% NaCl (w/v) pH 9.7 with 5 min between injections, and (b) eluent: 0.2% NaCl (w/v) pH 9.7 with 4 min between injections, sample zone: deionized water. Elution conditions: column A, flow rate 0.62 ml/min, injection volume 350 pl. The numerals shown on the chromatograms are retention times in minutes. In both cases, the phytic acid peak elutes first.
zone and carrier had the same ionic strength, significant adsorption of phytic acid occurred (peaks at 3.44, 8.49 and 1344min). However, when the sample zone matrix was of much lower ionic strength, markedly improved recovery of phytic acid was noted [Fig. 3(b)], as indicated by the peaks at 3.19, 7.24 and 11.29 min. This effect is similar in some respects to the salt boundary elution effect previously described by Posner. ‘OPosner suggested that preparing a sample with a higher ionic strength would cause solute adsorption when the sample was added to a gel column. Elution with distilled water would then cause the progressive desorption of the adsorbed solute and this would move down the column as a concentrated zone at the distilled water-saline eluent interface. In the case illustrated by Fig. 3(b), the sample was dissolved in deionized water, and injected into a carrier/ eluent of 0.2% (w/v) NaCl. However, despite these conditions being the opposite of those described by Posner, the same type of elution persisted; i.e., phytic acid in a deionized water matrix showed much less adsorption than the same sample prepared in eluent. It is possible that the adsorptive process occurs at the head of the sample zone where there is considerable dispersion and hence mixing, with the saline eluent. Given that a large injection volume of 350 ~1 (10.6% of total column volume, V,) was used, it could be expected that the relatively undispersed mid-region of the sample zone would have a lower ionic strength than that of the eluent, and it is conceivable that it is this low ionic strength zone which causes the partial desorption of phytic acid which is observed. Although the mechanism of this process is unclear, it is quite evident from the relative sizes of the peaks, that better, albeit not complete recovery of phytic acid occurs when an ionic strength gradient exists across the sample zone, and for this reason the influence or ionic strength and pH on separation of orthophosphate and phytic acid in a deionized water matrix were studied systematically to obtain the optimum condition for separation and recovery of phytic acid. Mixed standards of phytic acid and orthophosphate in deionized water were injected onto the same column and eluted with carrier having a variety of pH and ionic strength conditions using Tris-HCl-NaCl solutions ranging from I = 0.001 to 0.071U. The results of these experiments are shown in Fig. 4 from
1985
Speciation of dissolved phosphorus
J”c4~z5.~6.~z7.~z9.4
I = 0.001
L\Vflo= 0.00 AVe/Vo = 0.00
AVcjVo = 0.00 AVe/Vo = 0.00
AVelvo = 0.00
~H~4x~4.7~~6.x~7~*.6
I =
0.003
AVe/‘.‘o = 0.38 I=o.oo5
AVe/Vo = 0.29 AVeJVo = 0.23 AVe/Vo = 0.00
AVe/Vo = 0.00
~=3~=4.5~6.5~=7.~=9.7
AVelVo = 0.46
AVeJVo = 0.40
AVejVo = 0.33 AVeJVo = 0.22 AVJvo
pH = 6.0
pH = 7.2
pH = 7.5
= 0.17 pH = 9.9
I = 0.01 _.JAAA AVePfo = 0.50 AVeJVo = 0.35 AVeJVo = 0.34 pH = 5.8
I =
pH = 7.2
AVe/Vo = 0.33
pH = 7.5
pH = 10.0
0.03 _ALfLJA AVe/Vo = 0.58
AVe/Vo = 0.55 AVeJVo = 0.52
pH =5.9
pH = 7.2
AVe/;go = 0.47
pH = 7.5
pH = 10.0
I = 0.05 _-JLJLJJ AVe/Vo = 0.59 0.05
AVeJVo = 0.58
pH = 6.2
AVe/Vo = 0.56
pH = 7.2
AVe/Vo = 0.46
pH = 7.5
pH = 9.7
I = 0.07 % <
0L
-JJJi\ 5 minutes
AVelvo = 0.57 AVJvo
= 0.61 AVe/Vo = 0.59
AVe/Vo = 0.50
Fig. 4. Variation in the elution of orthophosphate and phytic acid species as a function of total ionic strength and pH. Peak separation is expressed as AVJV,, where V, is the solute elution volume and V, is the void volume of the gel bed.
which a number of features of the chromatography are evident: (i) At low ionic strength (I = O.OOlM), phytic acid and orthophosphate co-elute under all pH conditions tested.
(ii) At slightly greater total ionic strength (I = O.O03M), orthophosphate shows partial resolution from phytic acid (AVJV,, = 0.23-0.38). However, at pH > 6.4, loss of separation is observed.
I. D. MCKELVIE et al.
1986
that of Blue Dextran, and even at high ionic strength (e.g., Z = O.O7M), with a large amount of adsorption occurring, there is only a small shift in the value of VJV,,. Despite the high charge on the phytic acid species, it appears that its elution is dictated by steric factors rather than charge considerations. This is consistent with the findings of Neddermeyer and Rogers” who suggested that larger charged ions would not be influenced by the Donnan exclusion effect to the same extent as smaller ions because they would be sterically excluded from the gel interior containing most of the charge. This may provide an explanation for the apparent absence of anion exclusion behaviour of phytic acid, despite its high charge. Neddermeyer and Rogers have also noted that solutes subject to the influence of anion exclusion display broadened front and sharp back edges, behaviour which is the converse of that normally observed in chromatography.*’ Examination of orthophosphate and phytic acid peaks reveals that while the orthophosphate peaks show broadened leading edges, phytic acid peaks do not, providing further evidence in support of the hypothesis that steric effects control the behaviour of phytic acid. Maximum recovery of phytic acid with respect to orthophosphate, based on peak height, was achieved for the specified column and flow conditions using eluents with Z = 0.005M and pH 4.5-6.5. Under optimal elution conditions (I = O.O05M, pH 6.5, cf, Fig. 4) the % recovery of phytic acid with respect to orthophosphate calculated using peak height was 97.4% (0, _ , = 0.2%, n = 3), but when calculated on the basis of peak area, was only 78.2% (c,_ , = 3.2%, n = 3). Given that a common phosphomolybdenum blue detection chemistry 2.0 was used, it is evident that significant adsorption pH range: 1.3 _7.5 of phytic acid had occurred even at this sol called optimum condition. Furthermore, when columns were cleaned with dilute sodium hydroxide solution (pH > 10) after the injection of several samples of phytic acid, a large desorp1 T I tion peak was observed; however this was not 1.2 d Q P O the case for orthophosphate. It was also appar0 ent from the uniformity of peak area data, 1.0 0 obtained over a wide range of ionic strength t and pH conditions, that little orthophosphate I 0.8 ! 0.08 0.06 0.04 0.00 0.02 adsorption occurred. It is unfortunate that even under salt-zone Total Ionic Strength elution conditions adsorption of phytic acid perFig. 5. Variation of VJV, for orthophosphate (0) and sisted. It would have been preferable to use only phytic acid (0) species as a function of total ionic strength one calibration substance, i.e., orthophosphate within the pH range 7.3-7.5.
(iii) At total ionic strength in excess of O.OOSM, significant adsorption of phytic acid was found to occur. Very slow desorption of some species (presumably phytic acid), indicated by a gradual return to baseline, was observed after elution of the orthophosphate peak (not shown by the data in Fig. 4). The co-elution of orthophosphate and phytic acid found to occur at the lowest ionic strength conditions tested (I = O.OOlM) was probably due to anionic exclusion of the orthophosphate. At Z = 0.003M some separation of the two species occurred at lower pH, but at pH > 6, the double- and triple-charged phosphate species are more prevalent, and the anion exclusion effect overcame the influence of increased ionic strength; a similar trend to this was observed for an eluent with Z = 0.005M. This anion exclusion effect appears to have been completely suppressed when the ionic strength was increased to O.OlM, and the phytic acid and orthophosphate peaks are as well separated as might be expected given the short column used. Predictably, however, there is significant adsorption of the phytic acid under these higher ionic strength conditions.20 Figure 5 shows the variation of VJV,, of both orthophosphate and phytic acid species with increasing ionic strength within the pH range 7.3-7.5. The orthophosphate shows a pronounced increase in VJV,, but a much smaller effect of ionic strength on phytic acid. This is perhaps surprising, given the highly charged nature of the phytic acid species (within the pH range used, the average charge on the phytic acid would be cu. - 8). The value of VJV,, for phytic acid is, however, always larger than
?
4
I
I
I
Speck&ion of dissolved phosphorus
1987
Table 1. Calculated limits of detection (lad) and sensitivity data for phytic acid and orthophosphate for the 42-mm bed height column (A)
Analyte Phytic acid Phosohate
High range (~OO-~~ 1(8 P/l.) Sensitivity Limit of detection (AU/M P/l.) w PP.) 44 12
1.338 x IO-’ 1.318 x IO-’
and a common peak-height : concentration calibration equation for all eluting species. This would only be possible in the absence of solute-gel interactions, given that all eluting species are converted from DOP to DRP before detection. There was, however, only a narrow range of pH and ionic strength conditions for which the separation and O/Orecovery of phytic acid (by peak height) was maximal. Even slight departure from the optimum ionic strength and pH conditions (I = O.OOSiV,pH 4.5-6.5) would cause the % recovery of phytic acid with respect to orthophosphate to change markedly. Such variations in the ionic strength could occur in the preparation of reagents, or may arise from some hydrodynamic artefact of the FIA manifold causing a variation in the degree of dispersion of the sample zone, and hence altering the ionic strength regime of the sample zone. Such artefacts may involve changes as simple as a fluctuation in carrier flow rate, or a variation in the sample volume. For these reasons, mixed standards of phytic acid and orthophosphate were used for quantitation of HMWP and LMWP, in preference to quantitation using orthophosphate alone and applying a response factor. It is recognized that this calibration approach may be deficient if an HMWP species elutes from the gel in a manner different to that of phytic acid. Linearity, sensitivity and limit of detection of mixed standard calibrations Mixed standards of phytic acid and orthophosphate were prepared over the ranges 50-1000 c(g P/l. and 10-100 pg P/l. Triplicate injections of 350 ~1 of these standards were made onto the same Sephadex G25 Superfine column that was used for all previous experiments under optimal separation conditions. At concentrations greater than approx 200 pg P/l., phytic acid and orthophosphate graphs were essentially co-linear, giving regression equations of A = (-2.63 x lo-‘) + (1.34 x lo-4)pg P/l, r* = 0.999,
Low range (10-100 l(g P/l.) Limit of detection Sensitivity ocg P/l.) (Auk? P/l.) 27 8.88 x 10-s 1.1 1.323 x lo-’
and A = (3.00 x 10-4) + (1.32 x 10-4)c(g P/l., r2 = 1.000, respectively. However, below ca. 100 pg P/l., the orthophosphate calibration is linear, but the phytic acid exhibits non-linear behaviour. At 50 pg P/l., there is a loss of approximately 50% of the phytic acid, while at < 10 p g P/l., phytic acid peaks were scarcely detectable, even though orthophosphate signals were still clearly discernible. Clearly, at concentrations of less than 100 pg P/l., the specific adsorption of phytic acid onto the gel poses a difficulty. Limits of detection (lod) have been calculated for these data using the linear regression method described by Miller and Milled and are listed with calibration sensitivity (slope) values in Table 1. Eflect of sample volume on peak response, resolution and O/O recovery The influence of sample volumes ranging from 100 to 600 ,ul on peak response, resolution, and % recovery (based on comparison with orthophosphate peak height) was investigated. A linear relationship between injection volume (S,) and peak height (Absorbance, A) was observed over the concentration range tested. For the dilute solutions analysed here, it can be shown that the relationship between the absorbances of undispersed (A”) and dispersed (A) sample, -$=
1 -exp(--kS,)
quoted by Ruzicka and Hansenz3 approximates to: A z k S, for a range of A values from 0.001 to 0.1. S1,2,the injection volume required to give a dispersion coefficient of 2, can be determined from 0.693 s l/2=k Using this relationship, and the k (slope) value from an A vs. S, plot, very high SI,2 values of
I. D. MCKELVIE
1988
ef cd.
0.6
0.6 0.4
o.o0
200
400
600
0
200
400
600
Injection Volume (pL)
Injection Volume (pL)
Fig. 6. Influence of sample volume on (a) resolution (for 500 pg P/l. each of phytic acid and orthophosphate), and (b) % recovery of 50 fig P/l. (-A-) and 500 pg P/l. (-A-) phytic acid with respect to orthophosphate. Column A, eluent flow rate 0.62 ml/min.
>20,000 ~1 at 50 pg P/l. and >4,000 ~1 at 500 pg P/l. were obtained. The very large values of & at 50 pg P/l. reflect the consideration dispersion that occurs in the gel column. Clearly, use of a smaller gel-filtration column would be preferable and more in agreement with conventional FIAchromatographic practice. Smaller columns would necessitate the use of small injection volumes if column overloading and attendent loss of resolution are to be avoided. However, the combination of small injection volumes and the high dispersion of the DOP + DRP detection manifold would result in poor sensitivity, and consequently the use of small columns and small injection volumes is precluded in this context. Figures 6(a) and (b) show the improvement in peak resolution and the corresponding decrease in % recovery of phytic acid (calculated from peak height data) as the injection volume is decreased to 100 ~1. The lower recovery of the 50 pg P/l. standard with respect to the 500 pg P/l. standard is probably due to a combination of preferential adsorption of phytic acid to the gel and dispersion. These results highlight the interrelationship between phytic acid adsorption and factors such as eluent-sample zone ionic strength factors like difference, and hydrodynamic sample injection volume, column size, and flow rate which influence dispersion.
trations of both HMWP and LMWP calculated from peak height may be mutually influenced by peak overlap. To determine the magnitude of this effect, several mixed standards all containing 300 pg PO,-P/l. and phytic acid concentrations varying between 50 and 400 pg phytic-P/l. were analysed. Figure 7 shows that the absorbance of the 300 pg PO,-P/l. standard increased linearly, by as much as 10.3%, as the concentration of phytic acid was increased from 50 to 400 c(g phytic-P/l. Similarly, the absorbance as. concentration plot for phytic acid displayed increasing deviation from linearity at higher concentrations because of overlap from the leading edge of the orthophosphate peak. Because this effect proved problematic in the analysis of some sediment extracts, a larger, high resolution column was employed for the analysis of water samples, as described in the following section. 0.05
g
0.03
1 a
0.02
0
100
200
300
IO
pg Phytic acid-P/L
Influence of phytic acid concentration on apparent concentration of orthophosphate for short columns
Phytic acid and orthophosphate peaks were only partly resolved on the short G25 Superfine column (A). Under these circumstances, the measured peak heights, and hence concen-
Fig. 7. Effect of differing phytic acid concentrations (0) on peak height of 300 cg P/l. orthophosphate (0) in short column gel filtration chromatography. Tris/HCl eluent at pH 4.75 and I = 0.005. Column A, injection volume 350 ~1. Eluent flow rate 0.62 ml/min. Lines of best fit for phytic acid and orthophosphate given by: y = (7.49 x IO-‘) + (7.77 x 1O-5)x + (1.04 x IO-‘)x2 r2 = 0.998 and y = (3.20 x 10-2) + (9.47 x 10e6)x r2 = 0.923, respectively.
Speciation of dissolved phosphorus
Analytical results-concentrations of HMWP and LMWP found in sediment extracts and waters Preliminary extractions of sediments with deionized water gave low yields of HMWP. Minea?” has noted that only about 40% of DOP was extracted from particulate material and Chiamydomonas cultures by distilled water, but that 85-100% was extracted when alkaline conditions (ca. pH 13) were used. Extraction of phosphorus from sediments was tested using Olsen’s reagent (OSM NaHCO, at pH 8.5);*’ however, the high bicarbonate concentration was found to interfere with the photo-oxidation of DOP, presumably through hydroxyl radical scavenging. Aluminium may also interfere in the photo-oxidation of DOP at concentrations > 100 pg A1/l.26 and for this reason acidic sediment extractions were avoided. HMWP and LMWP concentrations of the sediment extracts obtained by separation on the short (42 mm x 10 mm id) Sephadex G25 column with subsequent DOP analysis are shown with corresponding DOP and DRP data, expressed on a dry weight basis, in Fig. 8(a)-(c). The concentration of LMWP was significantly greater than the corresponding DRP data (P = 0.0058, paired t-value 3.91, d.f. = 7). Two possible explanations can be tendered, oiz; (i) Desorption of phytic acid, or other late eluting species, may enhance the LMWP peak. While the separation has been optimized to give maximum recovery of phytic acid, some phytic acid appears to undergo very slow desorption. Other phosphate-species, such as Vitamin B12 and 4nitrophenylphosphate, exhibited reversible adsorption and late elution when injected under these elution conditions. (ii) Organic phosphorus species of similar molecular weight range to orthophosphate (e.g., lower phosphate esters of myo-inositol) may be present and elute with the orthophosphate. Given the fairly poor resolution of the small column used, discrimination between orthophosphate and other low molecular weight phosphorus species would not be possible. From Fig. 8(a), it is evident that DOP values are significantly greater than those for HMWP (P = 7.0 x 10m4,paired t-value = 5.76, d.f. = 7). Between 65 and 97% of the DOP is represented by the HMWP of similar MW to myo-inositolhexakisphosphate, and this is consistent with suggestions regarding the stability of this phosphate ester and its probable role in the storage
1989
of phosphorus in sediments.’ Other researchers have reported similar recoveries of inositol phosphate esters from soils*’ and lake sediments.** The fact that the DOP values are all greater than the HMWP values [Fig. 8(b)] also lends some credence to any one of the explanations provided for the apparent discrepancy between LMWP and DRP. Moreover, it can be seen that when (DRP+DOP) and (HMWP+LMWP) data are compared [Fig. 8(c)], there is excellent agreement, as shown by the regression equation: (HMWP + LMWP) = 1.05 (DRP + DOP) - 1.34,
r = 0.987
(r significantly different from zero at P > 99%). The agreement between (HMWP + LMWP) and (DRP + DOP) suggests that there are no major recovery losses associated with the gel filtration separation that cannot be accounted for by calibration. It also supports the hypothesis that differences between DRP and LMWP can be explained by the co-elution of lower MW organic phosphorus species with orthophosphate. For water samples, LMWP and HMWP were separated on the larger, high resolution HRlO/lO column (B). Sample injections of 600 ~1 were necessary to compensate for the added dispersion associated with use of a large column, but this was compensated for by the complete resolution of phytic acid and orthophosphate, and the ability to use high eluent flow rates with this column. Flow rates of up to 6 ml/min are permissible, but in experiments reported here, flow rates in excess of 1.2 ml/min were avoided because the debubbler in the DOP photo-oxidation manifold did not function efficiently at these higher flow rates. Results of the analyses of the water samples are shown in Fig. 9(a)-(c). The importance of HMWP as a constituent of some waters is noted, as it comprised from 0 to 97% of the total HMWP + LMWP detected. There was no significant difference between the (HMWP + LMWP) gel filtration data and the (DRP + DOP) data for the same samples (P = 0.79, paired t = 0.27, d.f. = 9). However, HMWP was significantly greater than the DOP fraction (P = 0.03, paired t-value = 2.67, d.f. = 9) and LMWP is significantly less than the DRP fraction (P = 0.02, paired t-value = 2.85, d.f. = 9). These trends are opposite to those found for the extractable phosphorus in the
I. D. MCKELVIEet al.
1990
2.1176, R-8quw.d:
.9SOlX -
y I
20
3606
16 16 14
12 10
6 6 4 2 o+
d
64
_:
:-.
.
2
,
4
:
.
s
:
i
.
:
.
f&bO-PlkQ
:
12
.
:
.
1-4
:
.
1s
:
te
.
)
20
DOP significantly > HMWP (P = 7.0 x 1O4, paired t-value 5: 5.76, d.f, = 7) y r
.9364x + 1.6634. R-squared:
,681
6
04 d
W
.
I
I 4
i
I lo
I
li
LMWP significantly > DRP (P = 0.0058, paired t -value = 3.91, d.f. = 7) y
=
1.0446x -
lb
Cc)
RI(IDR%-PIkg
I
e
1.3407, R-squared:
3738
2-5
2’0 lb mg(DRP+DOP).P/kg
No significant difference between HMWP+LMWP
and DRP+DOP
30
(P = 0.14,
paired
t-value = 1.68, d.f. = 7)
Fig. 8. Comparison of (a) HMWP and DOP, (b) LMWP and DRP and (c) HMWP + LMWP and DRP+ DOP for sediments, expressed on a dry weight basis. The broken tines indicate the 95% confidence
intervals for the true mean of the y values.
sediment samples. One possible explanation for the higher DRP than LMWP data is that some of the DRP measured includes the products of rapidly hydrolysed dissolved organic phosphorus, a problem which has been reported
extensively.2*2g*M As DOP is determined by subtracting DRP from the total DRP + DOP, the effect of an inflated DRP value will result in significantly reduced DOP values. Chamberlain and Shapiro, for example, in a batch method,
Speciation of dissolved phosphorus
limited mixing and reaction between sample, molybdate and reductant to 6 set, but noted that some hydrolysis of DOP occurred even under these rapid reaction conditions.3’ For the flow injection DRP method used here, the time Y
(a)
I
+
between injection and emergence of the peak maximum was only 20-25 set, but it is feasible that some hydrolysis of DOP could occur even in this short period. Six of the samples analysed had LMWP 2.2045,
R-squwrd:
.SSBO
HMWP signif’i&nUy > DOP (P = 0.03. paired t-value = 2.67, d.f. = 9) Y
0
lb)
1.4033~
1991
,-20
DRP significantly
.936X
=
40
8.9436.
-
R-wurrod:
AI006
100
60
140
120
160
> LMWP (P = 0.02, paired l-value = 2.85, d.f. = 9) y
o
.7322x
+
17.0794,
R-squared:
.a092
160 160 140 120 100 60 60 40
0 0
w
20
40
No significant difference t-value = 0.27. d.f. = 9)
60
100 60 w(DRP+DOP)-P/L
between
120
140
160
160
HMWP+LMWP and DRP+DOP (P = 0.79. paired
Fig. 9. Comparison of (a) HMWP and DOP, (b) LMWP and DRP and (c) HMWP + LMWP and DRP + DOP for water samples. The broken lines indicate the 95% confidence intervals for the true mean of the y values.
1992
I. D. MCKELVIEer al.
values which were significantly lower than the DRP concentrations. However, there is no significant difference between (LMWP + HMWP) and (DRP + DOP) for most samples, and it is arguable that measurement of LMWP and HMWP provide better measures of nonhydrolysed organic phosphorus and orthophosphate than do DOP and DRP. In two diluted effluent samples the HMWP was undetectable; however, these samples were high in DRP and required dilution of 1: 50 prior to analysis in order to get them into the same concentration range as other samples. It is probable that this was sutIlcient to dilute the low concentration of HMWP to below the detection limit, or that the low concentrations of DOP (14 and 5 pg Pi/l. respectively) were comprised of late-eluting species. This study has highlighted some of the problems associated with the use of Sephadex G25 for the fractionation of dissolved phosphorus species in natural waters and sediment extracts, viz. partial adsorption of phytic acid species and late elution of other higher molecular weight species. This gel filtration approach to the separation of HMWP and LMWP is apparently the most rapid yet reported with an analysis rate of 12-15/hr, but it is not sufficiently sensitive for the analysis of dilute or pristine waters. It is envisaged that further research in this area could involve the use of complexing eluents to eliminate adsorption of HMWP species through complexation by metals adsorbed to the gel, along similar lines to that described by Town and Powell.32 In this study, only Sephadex G25 has been used, and it would be useful to repeat the ionic strength-pH studies on a wide range of gels, including the macro/microreticular Ultrogels, to determine optimum separation conditions. This would be labour-intensive by conventional gel filtration, but could be performed within a reasonable time using the gel filtration-FIA approach described. Separation of HMWP and LMWP in waters with a range of gels of different MW cut-off would provide further information on the concentration and speciation of dissolved organic phosphorus in natural waters. Detection of low concentrations of DOP compounds using the gel filtration-FIA system is clearly a problem. However, it was shown that phytic acid and phosphate could be concentrated onto an ion exchange precolumn prior to injection” and DOP analysis. This approach could be applied to the preconcentration of at
least the anionic HMWP species prior to gel filtration. While this study has identified that waters and sediment extracts contain HMWP in the same molecular weight range as phytic acid, and it is probably reasonable to assume that phytic acid is a major constituent of the HMWP, no information about the presence of other phosphate esters of myo-inositol can be obtained because of the inability of the gel to resolve the small differences in molecular weight involved. Low pressure anion exchange chromatography has been used extensively to separate myo-inositol phosphate esters,6J8*” and from the experience of this study, it should not be difficult to couple an anion exchange column with an FIA-DOP manifold to achieve rapid separations. Finally, the relevance of the HMWP and LMWP data in waters needs to be evaluated in terms of their bioavailability by comparison with appropriate algal bioassay techniques. Acknowledgenrenr-Support for this research by the Australian Research Council is gratefully acknowledged.
REFERENCES
1. E. M. Thurman, Organic Geochemistry of Natural Waters, p. 497. Martinus Nijhoff/Dr W. Junk Publishers, Dordrecht, The Netherlands, 1985. 2. S. J. Tarapchak, J. Environ. Qua/., 1983, 12, 105. 3. W. Stumm and J. J. Morgan, Aquatic Chemistry, 2nd Ed., p, 780. Wiley, New York, 1981. 4. G. Anderson, in A. D. McLaren and G. H. Peterson, Soil Biochemistry, p. 67. Marcel Dekker, New York, 1967. 5. D. J. Cosgrove, Inositol Phosphates-Their Chemistry, Biochemistry and Physiology, p. 191. Elsevier, Amsterdam, 1980. 6. S. J. Eisenreich and D. E. Armstrong, Enoiron. Sci. Technol., 1977, 11, 497. I. S. E. He&s, H. E. Allen and K. H. Mancy, Science, 1975, 187,432. 8. R. A. Minear, J. E. Segars and J. W. Elwood, Analyst, 1988, 113, 645. 9. C. M. Clarkin, R. A. Minear, S. Kim and J. W. Elwood, Environ. Sci. Technol., 1992, %, 199. 10. J. B. Cotner and R. G. Wet&, Limnol. Oceanogr., 1992, 37, 232.
11. E. White and G. Payne, Can. J. Fish. Aquut. Sci., 1980, 37, 664.
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