Journal of Environmental Quality 31:466-470 (2002)
© 2002 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
Article
SYMPOSIUM PAPERS
A Novel Technique for the Pre-Concentration and Extraction of Inositol Hexakisphosphate from Soil Extracts with Determination by Phosphorus-31 Nuclear Magnetic Resonance
Benjamin L. Turner* and
Ian D. McKelvie
Water Studies Centre and Chemistry Dep., Monash Univ., Clayton 3168, Victoria, Australia
* Corresponding author (bturner{at}nwisrl.ars.usda.gov)
Received for publication June 2, 2000.
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ABSTRACT
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Inositol hexakisphosphate (IP6) is often the dominant form of soil organic phosphorus (P), but is rarely investigated because of the analytical difficulties encountered in its extraction, separation, and detection in environmental samples. In particular, recent advances in the study of soil organic P with 31P nuclear magnetic resonance (NMR) have been of limited use for the study of IP6, because the technique does not discriminate between IP6 and other forms of P. This was addressed by developing a novel analytical procedure using the retentive properties of gel-filtration gels for IP6, which allows the combined selective extraction and pre-concentration of IP6 from soil extracts with determination by 31P NMR. While the technique is still in the developmental stage, the results demonstrate that the gel does not interfere with 31P NMR analysis and retains IP6 to concentrations well above those required to give clear spectral signals. The technique has considerable potential for application to the study of IP6 in soil extracts and water samples and, with development, could help to answer fundamental questions regarding the dynamics of organic P in the environment.
Abbreviations: IP6, inositol hexakisphosphate • 31P NMR, phosphorus-31 nuclear magnetic resonance
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INTRODUCTION
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THE MOST ABUNDANT CLASS of organic phosphorus (P) compounds in the environment is the inositol phosphates, a family of six congeners of hexahydroxy cyclohexane (inositol) that exist as inositol in various states of phosphorylation (bound to between 1 and 6 phosphate ions) (Fig. 1)
. Nine stereoisomers of inositol phosphates exist; the myo stereoisomer is by far the most common in nature, although neo-, scyllo-, and chiro-inositol phosphates have been reported in terrestrial and aquatic environments (Cosgrove, 1980). The dominant form of inositol phosphate in the environment is myo-inositol hexakisphosphate (IP6), which constitutes the major organic P compound in soils and aquatic sediments (Harrison, 1987; Suzumura and Kamatani, 1995). Despite its abundance, it remains poorly understood and little reliable information exists on the sources, pools, and dynamics of IP6 in the environment (Turner et al., 2002). The role of IP6 in supplying P to plants and algae is largely unknown and even its origins remain unclear in many cases (L'Annunziata, 1975). Research into IP6 has been limited by the lack of suitable analytical techniques for its determination in environmental samples, the main problems being poor recoveries of IP6 from soils by conventional extractants and from anion exchange columns during sample cleanup and separation (Anderson, 1964; Martin, 1970; Irving and Cosgrove, 1981). Furthermore, the concentrations of IP6 in environmental samples are too low for most analytical techniques, although new pre-concentration procedures have recently been developed that can address this (Nanny et al., 1995; Espinosa et al., 1999). These problems must be overcome before significant advances can be made in understanding IP6 dynamics (Turner et al., 2002).
This work describes a new approach to the use of 31P NMR for the determination of IP6 in soil extracts and water samples. Nuclear magnetic resonance is a powerful tool for the investigation of P forms in soil extracts (Condron et al., 1997), but it is limited for IP6, because of an inability to separate IP6 from orthophosphate and other phosphomonoester compounds. This is because phosphomonoester signals (including IP6) appear as a single envelope and frequently overlap with the orthophosphate signal, making quantification difficult (e.g., Hawkes et al., 1984; Condron et al., 1990). Further problems are encountered with interfering paramagnetics (such as Fe3+ and Mn2+), which cause line broadening and can require removal from the sample by chelating resins. Other problems are the low concentrations of organic P in soil extracts (relative to those required for 31P NMR), which means some form of pre-concentration is necessary. However, 31P NMR provides a simple method of determination compared to the time-consuming and complex separation and detection procedures used in previous studies of IP6 (e.g., Anderson, 1964; Martin, 1970; Irving and Cosgrove, 1981).
Inositol hexakisphosphate can be retained by gels used for molecular size separations such as Sephadex (Martin, 1970; McKelvie et al., 1993), although adsorption varies depending on solution parameters. For example, McKelvie et al. (1993) showed that optimum (but not complete) adsorption of IP6 to Sephadex G-25 gel occurred at ionic strengths >0.05 M and pH between 7.5 and 10, although Condron and Goh (1989) showed that there was negligible retention of soil organic P in NaOH extracts onto Sephadex G-100 gel columns. The adsorptive properties of these gels has presented a major problem for investigation of organic P in soil extracts and water samples (Martin, 1970; McKelvie et al., 1993), but provides a potential means of separation. We investigated the potential use of the retentive property of Sephadex G-25 gel for the selective extraction of IP6 from a solution, followed by analysis with 31P NMR. The proposed technique overcomes several of the major problems of determining IP6 in soil extracts with 31P NMR, namely the selectivity of extraction, pre-concentration, and preclusion of interference by other P compounds.
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METHODS
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Principle of the Method
Sephadex is manufactured by cross-linking dextran with epichlorohindrin; varying the degree of cross-linkage creates gels with different porosities. Sephadex gel has been shown to retain IP6, while excluding other organic P forms and orthophosphate (McKelvie et al., 1993). By using this property, IP6 can be removed from soil extracts in the presence of the gel, while impurities and other P compounds are removed by centrifugation, decantation, and washing prior to analysis with 31P NMR.
Sample Preparation
One-gram Sephadex G-25 (fine) gel powder (giving approximately 7 mL of gel) was mixed and, therefore, hydrated in 10 mL of ultrapure water containing 1 mg P as phytic acid (magnesium-potassium myo-IP6). A blank sample (no P) was included. The gel was allowed to swell and equilibrate by shaking end-over-end in 25-mL centrifuge tubes overnight at approximately 20°C. The mixtures were centrifuged for 5 min at low speed (<500 x g) to settle the gel and the supernatant was decanted and retained for total P analysis by sulfuric acid-persulfate digestion (Rowland and Haygarth, 1997). The remaining gels were then washed in ultrapure water and recentrifuged. The washed gels were resuspended in 5 mL ultrapure water to form a slurry, which was poured carefully into 10-mL NMR tubes. Aliquots of the initial supernatant solutions were also poured into NMR tubes, as was a sample of the phytic acid stock solution (100 mg P L-1). These samples were run for approximately 100 scans.
To test the selectivity of Sephadex gel for IP6, the procedure was repeated for a range of other organic and inorganic P compounds (Table 1). Samples were prepared as described above and the supernatant solutions were analyzed for total P. Recoveries are expressed as the percent recovery from solutions containing 1 mg P and are reported as means plus or minus the standard error of triplicate samples.
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Table 1. Recoveries of model phosphorus (P) compounds (%) from solutions containing 1 mg P after mixing with 1 mL Sephadex gel overnight at 20°C. Values are means of triplicate sam-ples ± standard error.
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To test whether Sephadex would retain IP6 to concentrations at which acceptable signal to noise ratios could be achieved, samples were prepared with 1 mL of gel (0.143 g powder) in 50 mL centrifuge tubes. For this experiment, Sephadex-IP6 mixtures were prepared according to the conditions for optimal retention of phytic acid by the gel (McKelvie et al., 1993). Forty milliliters of IP6 solutions containing 40 and 20 mg P as IP6 were mixed with Sephadex powder and 1 mL of 1.0 M Tris-HCl buffer, pH 8, containing 2 M NaCl (24 mM Tris, 49 mM NaCl final concentrations) and shaken overnight as described previously. These samples were run for approximately 700 scans.
Soil Extract
A soil extract was prepared for analysis by the Sephadex gel method. Two grams of soil (fine sandy clay loam, USDA Haplustult; total carbon 4.7%, pH in water 5.0, sand 38%, silt 49%, clay 13%, total P 685 mg kg-1) was shaken for 15 h overnight in 40 mL of 1 M NaOH, at a 1 to 20 soil to solution ratio. This was centrifuged for 1 h at 10 000 x g, filtered through an Advantec No. 2 filter paper (Advantec Toyo Kaisha Ltd., Tokyo), and 10 mL of solution was prepared for NMR analysis as for the IP6 samples described previously.
Phosphorus-31 Nuclear Magnetic Resonance Analysis
The "gel-slurry" samples were analyzed with 31P NMR, using a Bruker Advance DRX400 spectrometer (Bruker, Germany) (which uses a 31P operating frequency of 162 MHz), and data were collected with broadband (waltz) proton decoupling. The relaxation delay was 2.5 s, acquisition time was 0.84 s, with an approximate 40 000-Hz sweep width, 64 K data points for acquisition and 131 K for processing. The samples were run unlocked (no D2O). The number of scans was sufficient to obtain a recognizable signal. The 0 ppm position corresponded to the resonance of 85% orthophosphoric acid as the external reference and positive chemical shifts corresponded to increasing magnetic field strength (Costello et al., 1976).
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RESULTS
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No 31P NMR signal was present for the blank sample (ultrapure water + Sephadex gel), indicating that the presence of Sephadex would not interfere in the 31P NMR analysis (Fig. 2)
. The total P concentration in triplicate blank samples was 3.1 µg L-1 (SE = 1.76 µg L-1) and was, therefore, below the limit of detection for total P in water (Rowland and Haygarth, 1997). The Sephadex + IP6 sample showed a quadruplet signal in the range of +0.098 to +1.428 ppm, identical to the spectra of the IP6 standard solution (spectra not shown). The supernatant of the Sephadex + IP6 mixture showed no signal, indicating that the gel had completely retained IP6 from solution, which was confirmed by analysis of the supernatant for total P by sulfuric acid-persulphate digestion. Furthermore, other model P compounds were not retained by the Sephadex gel, as indicated by less than 95% recoveries of orthophosphate, pyrophosphate, DNA, ß-glycerophosphate, and glucose-1-phosphate (Table 1).
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Fig. 2. Phosphorus-31 nuclear magnetic resonance (NMR) spectra of a blank sample containing Sephadex G-25 (fine) gel and ultrapure water (top), a sample containing 1 mL of Sephadex gel and 40 mg P as phytic acid (center), and a sample of Sephadex gel that had been mixed with 10 mL of 0.5 M NaOH soil extract (bottom).
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The 20- and 40-mg phytic acid-P samples gave quadruplet peaks in similar areas between -0.06 and +1.58 ppm, respectively (see 40 mg sample, Fig. 2). This indicated that the gel could retain IP6 to concentrations well above those required to give clear spectral signals. The peak heights of these two samples were not in proportion, indicating that the limit for IP6 retention by the gel lies somewhere between the two concentrations (based on 1 mL Sephadex gel).
The soil extract sample showed a small signal at +5.063 ppm (Fig. 2), consistent with the position of IP6 in alkaline soil extracts (Newman and Tate, 1980), but even after more than 32 000 scans this peak was not clearly distinguishable from the background noise, indicating that the IP6 concentration was too low for effective analysis. Clearly, some form of pre-concentration would be required for application to real samples.
The results, therefore, showed that (i) the Sepahadex gel did not interfere with P determination by 31P NMR, despite the ‘solid’ nature of the matrix; (ii) phytic acid was completely retained by the Sephadex gel to concentrations above those required to give clear spectral signals; (iii) orthophosphate and other P compounds were not retained by the Sepahadex gel; and (iv) soil extracts would need pre-concentration prior to analysis with 31P NMR.
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DISCUSSION AND FUTURE DEVELOPMENT OF THE METHOD
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These preliminary results are promising, although substantial development is required. The study demonstrated that the basic principles of the method are sound, because Sephadex gel did not interfere with 31P NMR analysis and successfully retained IP6 from solution, while excluding other P compounds.
The quadruplet signal obtained for phytic acid has also been reported by other workers, although the chemical shift and number of peaks varies depending on the pH and the ionic strength of the sample, particularly the concentration of sodium (Costello et al., 1976). For example, at pH in the range of 4 to 10, phytic acid gives four resonance signals in the ratios of 1:2:2:1, which correspond to the positions of the orthophosphate ions on the inositol molecule.
Pre-concentration appears to be a fundamental prerequisite for this technique, but could be achieved relatively simply by preparing a small column containing 1 mL of Sephadex gel, through which a buffered stream of sample could be passed with a peristaltic pump. The gel would retain IP6, with the remaining sample containing other P compounds passing to waste. Presumably, measurable concentrations of IP6 from soil extracts could be obtained by passing sufficient volumes of extract through the gel column. The use of the common extracts for soil organic P, such as NaOH-EDTA (Cade-Menun and Preston, 1996), would require strong buffers to achieve the required pH for optimal retention of IP6 by the gel. However, this pre-concentration procedure would allow the determination of IP6 in waters given sufficient sample volume.
Quantification could be achieved by running a suitable range of IP6 standards, or more simply by including an internal reference standard containing a known concentration of P in a form that would not interfere with the peak of IP6 in a capillary tube inside the NMR tube. For example, Hinedi et al. (1988) used phosphonate as an internal reference standard (chemical shift around +20 ppm). In this case, the P concentration would be calculated from the ratio of peak area of the sample with that of the internal standard. An additional benefit would be that quantification would not require a pre-determined number of scans, but could be determined whatever the number of scans required to obtain a measurable signal.
Application of the technique to soil extracts might be further complicated by the retention by the gel of paramagnetics like Fe3+ and Mn2+, which interfere with the determination of IP6 by 31P NMR (Condron et al., 1997), although these compounds are reportedly not the cause of the poor resolution of solid-state 31P NMR signals of soils (Shand et al., 1999). A greater problem may be the nature of organic matter–IP6 associations in soils, which could protect IP6 from interacting with the gel. The prevalence of these associations in soils may necessitate the use of hypobromite oxidation prior to pre-concentration. This technique oxidizes all organic matter except inositol phosphates (Irving and Cosgrove, 1981; Nanny and Minear, 1997) and would free IP6 to solution, from where it could be extracted, pre-concentrated and analyzed by the Sephadex-NMR procedure.
The technique has considerable potential for the quantification of IP6 in soil extracts and a range of other environmental samples, including sediment extracts and waters, and might facilitate the determination of IP6 as a routine analytical technique. Interestingly, there is the possibility of extending the technique to 1H NMR for the identification (and possibly quantification) of the isomers of IP6, which has been demonstrated in solutions (Suzamura and Kamatani, 1993). This would give complete identification of the IP6 component.
An interesting application of the technique could be for the determination of ‘labile’ phosphomonoesters in soil extracts by 31P NMR. Currently, 31P NMR cannot distinguish individual phosphomonoesters, such as sugar phosphates, mononucleotides, and IP6, because the spectral signals overlap. The IP6 component dominates in most soils, yet other phosphomonoesters are probably more important in terms of short-term cycling and availability to plants. The Sephadex technique described here could be used to "clean up" soil extracts by removing IP6 and allow other phosphomonoesters to be quantified with 31P NMR.
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CONCLUSIONS
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The feasibility of using Sephadex gel for the selective extraction and pre-concentration of IP6 from solutions and detection with 31P NMR has been investigated. The technique is still at the developmental stage, but has considerable potential for quantifying IP6 in soil extracts and water samples. This would contribute considerably to our understanding of IP6 dynamics in the environment. Additional development is required to make the method quantitative and to investigate the potential problems involved in its application to soil extracts.
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ACKNOWLEDGMENTS
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Ben Turner thanks the Natural Environment Research Council, the Institute of Grassland and Environmental Research, and the British Grassland Society for funding a research visit to Monash University during 1999. The authors thank Dr. Jo Weigold for assistance with NMR analysis and Dr. Phil Haygarth and Dr. Leo Condron for helpful discussion. Ben Turner attended the 2nd European Symposium on NMR in Soil Science funded by a scholarship from the Wageningen NMR Centre.
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NOTES
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Benjamin L. Turner, current address: USDA-ARS, Northwest Irrigation and Soils Research Lab., 3793 N. 3600 E., Kimberly, ID 83341.
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REFERENCES
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ABSTRACT
INTRODUCTION
