Solution 31P Nuclear Magnetic Resonance Spectroscopy Research Articles

Dual isotopic (33P and 18O) tracing and solution 31P NMR spectroscopy to reveal organic phosphorus synthesis in organic soil horizons

Soil microorganisms can do both, mineralize and synthesize organic and condensed phosphate (P) species. Whereas P mineralization has been extensively studied, few studies have assessed the biological synthesis of organic P species, which can potentially accumulate in soil. The goal of this study was to investigate biotic and abiotic P transformations, particularly the synthesis of organic P species, upon water-soluble P addition in the organic (O) horizons of two beech forest sites with contrasting P availability.The two O horizons (low-P and high-P) were subjected to four different nutrient addition treatments (Control without addition, CN, P, and CNP additions) in an incubation experiment of up to 104 days. We combined isotopic tracing (33P-labelled P addition and 18O-enriched soil water) into sequentially extracted P pools with the characterization of organic P species (solution 31P nuclear magnetic resonance (NMR) spectroscopy) and soil respiration measurements.The P availability of the two O horizons shaped the microbial response to the nutrient additions. In the low-P O horizon, P addition stimulated microbial activity together with the increase of organic (phosphodiesters and phosphonates) and condensed (polyphosphates) P species, most likely from microbial origin. In the high-P O horizon, microbes were unaffected by the added P and abiotic processes controlled its fate. CN addition had no effect on P fate in the high-P O horizon but reduced the transformation of added P into organic P and increased soil-derived P in the resin P pool in the low-P O horizon. The 18O isotopic values in phosphate of the resin P pool suggest that the released P was biologically cycled.Our study confirms with a unique multi-analytical approach the microbial synthesis of phosphodiesters, phosphonates, and polyphosphates upon inorganic P addition under low P availability.

The molecular size continuum of soil organic phosphorus and its chemical associations

The chemical nature of most organic P (Porg) in soil remains ‘unresolved’ but is accounted for by a broad signal in the phosphomonoester region of solution 31P nuclear magnetic resonance (NMR) spectra. The molecular size range of this broad NMR signal and its molecular structure remain unclear. The aim of this study was to elucidate the chemical nature of Porg with increasing molecular size in soil extracts combining size exclusion chromatography (SEC) with solution 31P NMR spectroscopy. Gel-filtration SEC was carried out on NaOH-EDTA extracts of four soils (range 238–1135 mg Porg/kgsoil) to collect fractions with molecular sizes of < 5, 5–10, 10–20, 20–50, 50–70, and > 70 kDa. These were then analysed by NMR spectroscopy.Organic P was detected across the entire molecular size continuum from < 5 to > 70 kDa. Concentrations of Porg in the > 10 kDa fraction ranged from 107 to 427 mg P/kgsoil and exhibited on average three to four broad signals in the phosphomonoester region of NMR spectra. These broad signals were most prominent in the 10–20 and 20–50 kDa fractions, accounting for on average 77% and 74% of total phosphomonoesters, respectively. Our study demonstrates that the broad signals are present in all investigated molecular size fractions and comprise on average three to four components of varying NMR peak line width (20 to 250 Hz).The stereoisomers myo- and scyllo-inositol hexakisphosphates (IP6) were also present across multiple molecular size ranges but were predominant in the 5–10 kDa fraction. The proportion of IP associated with large molecular size fractions > 10 kDa was on average 23% (SD = 39%) of total IP across all soils. These findings suggest that stabilisation of IP in soil includes processes associated with the organic phase.

Combining diffusive gradients in thin‐films (DGT) and 31P NMR spectroscopy to determine phosphorus species in soil

AbstractThe diffusive gradients in thin‐films (DGT) technique shows in many publications a superior correlation to the amount of plant‐available phosphorus (P) in soil. However, this technique cannot give information on the plant‐available P species in soil. Therefore, we combined DGT with solution 31P nuclear magnetic resonance (NMR) spectroscopy. This was achieved by using a modified DGT device in which the diffusive layer had a larger pore size, the binding layer incorporated an adsorption material with a higher capacity, and the device had a larger exposure area. The spectroscopic investigation was undertaken after elution of the deployed DGT binding layer in a NaOH solution. Adsorption tests using solutions of known organic P compounds showed that a sufficient amount of these compounds could be adsorbed on the binding layer in order for them to be analyzed by solution 31P NMR spectroscopy. Furthermore, various intermediates of the hydrolysis of trimetaphosphate in soil could be also analyzed over time.Core Ideas We show a promising combination of DGT technique and solution 31P NMR spectroscopy. This technique combination can identify organic phosphorus species in solutions. Modified DGT device can analyze the hydrolysis of trimetaphosphate in soil.

Characterization of fluvo-aquic soil phosphorus affected by long-term fertilization using solution 31P NMR spectroscopy.

Reducing the applications of mineral phosphorus (P) fertilizers and supplementing them by organic fertilizers is becoming a necessary practice in the North China Plain due to overuse of mineral P fertilizers and improper disposal of organic wastes. Knowledge is needed about how the long-term substitution of mineral fertilizers by organic fertilizers affects soil P forms in order to understand soil P transformation and crop P uptake. In this study, we used solution 31P nuclear magnetic resonance (NMR) spectroscopy to characterize P forms in fluvo-aquic soil after 26 years of different fertilization management strategies, organic compost (OM), half compost in combination with half mineral fertilizer NPK (1/2 OM), mineral fertilizer NPK (NPK), mineral fertilizer NK (NK), and an unfertilized control (CK). Results showed that the P extraction efficiency using NaOH-EDTA varied from 13.0 to 27.7% for the soils of the treatments. 31P NMR spectra indicated that the majority of P was in the form of orthophosphate for all the treatments, which constituted 64.3-83.5% of the total extracted P. The application of P fertilizers significantly increased the concentrations of orthophosphate, monoesters and diesters regardless of the P fertilization method, although the proportions of monoesters and diesters were higher in CK. The proportions and concentrations of orthophosphate significantly decreased when all mineral fertilizers were replaced by compost. There was no significant difference in the proportions and concentrations of total organic P, corrected monoesters and diesters in NaOH-EDTA extracts of soils among NPK, 1/2OM and OM treatments. Decreasing mineral P fertilizers and partly replacing them by organic fertilizer in fluvo-aquic soil might increase soil test (Olsen) P and crop P uptake through the degradation of applied organic P forms.

The structural composition of soil phosphomonoesters as determined by solution 31P NMR spectroscopy and transverse relaxation (T2) experiments

In terrestrial ecosystems, a large proportion of the phosphorus (P) in soil is often found within soil organic matter. However, the majority of organic P in soil remains ‘unresolved’ and is largely observed as a ‘broad’ signal within the phosphomonoester region of a solution 31P nuclear magnetic resonance (NMR) spectrum on soil extracts. Our aim was to gain insight into the composition of four soils using the transverse relaxation (T2) time of the magnetisation in solution 31P NMR spectroscopy as a probe of their structure. We found the broad signal within the phosphomonoester region rapidly decayed compared to the sharp signals (i.e. myo- and scyllo-inositol hexakisphosphate) across all soils, which corresponded to the former having a shorter T2 time than the latter, and supports the existence of a broad signal due to supra-/macro-molecular structures. Furthermore, measures of the broad signal's line-width at half peak intensity based on T2 times were found to be less than that obtained from spectral deconvolution fitting. Therefore, our results strongly suggest that the broad signal is itself comprised of more than one component. The significance of this is that the chemical nature of a large proportion of soil organic P appears to be structurally complex.

Using solid 13C NMR coupled with solution 31P NMR spectroscopy to investigate molecular species and lability of organic carbon and phosphorus from aquatic plants in Tai Lake, China.

Forms and labilities of plant-derived organic matters (OMs) including carbon (C) and phosphorus (P) were fundamental for understanding their release, degradation and environmental behaviour in lake ecosystems. Thus, solid 13C and solution 31P nuclear magnetic resonance (NMR) spectroscopy were used to characterize biomass of six aquatic plants in Tai Lake, China. The results showed that carbohydrates (61.2% of the total C) were predominant C functional group in the solid 13C NMR spectra of plant biomass, which may indicate high lability and bioavailability of aquatic plants-derived organic matter in lakes. There was 72.6-103.7% of the total P in aquatic plant biomass extracted by NaOH-EDTA extracts. Solution 31P NMR analysis of these NaOH-EDTA extracts further identified several molecular species of P including orthophosphate (50.1%), orthophosphate monoesters (46.8%), DNA (1.6%) and pyrophosphate (1.4%). Orthophosphate monoesters included β-glycerophosphate (17.7%), hydrolysis products of RNA (11.7%), α-glycerophosphate (9.2%) and other unknown monoesters (2.1%). Additionally, phytate, the major form of organic P in many lake sediments, was detected in floating plant water poppy. These inorganic P (e.g. orthophosphate and pyrophosphate) and organic P (e.g. diester and its degradation products) identified in plant biomass were all labile and bioavailable P, which would play an important role in recycling of P in lakes. These results increased knowledge of chemical composition and bioavailability of OMs derived from aquatic plants in lakes.

Spectral sensitivity of solution 31P NMR spectroscopy is improved by narrowing the soil to solution ratio to 1:4 for pasture soils of low organic P content

Solution phosphorus (P) nuclear magnetic resonance (NMR) spectroscopy following extraction with sodium hydroxide–ethylenediaminetetraacetic acid (NaOH–EDTA) is the most powerful and widely used technique for characterising soil organic P. However, poor spectral sensitivity (related to the signal to noise ratio) can limit its applicability for soils of low organic P content, especially in subsurface layers. Sensitivity can potentially be improved by employing a much narrower (1:4) soil to solution ratio than usual (1:10 or 1:20), which increases the density of P in the NMR tube. Pasture soils were collected at two depths (0–4cm and 4–10cm) from across the high rainfall zone of eastern Australia, and were extracted with NaOH–EDTA at a 1:4 and 1:10 extraction ratio. The extracts were analysed for molybdate-reactive P (i.e. an estimate of inorganic P), and total extractable concentrations of aluminium (Al), iron (Fe), magnesium (Mg), manganese (Mn) and P using inductively coupled plasma optical emission spectroscopy (ICP-OES). Total concentrations of P in soil were determined by laboratory X-ray fluorescence. Extraction efficiency of total soil P was on average only slightly lower for the 1:4 extracts (41%) than for the 1:10 extracts (47%). Extraction efficiencies for paramagnetic (Fe, Mn) and non-paramagnetic (Al, Mg) metals were also similar at the two extraction ratios. Solution 31P NMR spectra of 1:4 extracts were considerably better in terms of spectral sensitivity than those of the 1:10 extracts, particularly for soils of low organic P content. This study demonstrates that in soils of low organic P content, NaOH–EDTA extraction using a 1:4 soil to solution ratio is a viable alternative to the more commonly used 1:10 soil to solution ratio.

The Organic P Composition of Vertisols as Determined by 31P NMR Spectroscopy

Solution 31P nuclear magnetic resonance (NMR) spectroscopy on sodium hydroxide–ethylenediaminetetraacetic acid (NaOH–EDTA) extracts can provide detailed characterization of soil organic P, but has not been previously applied widely to Vertisols. Vertisol soils were collected at two depths (0–10 cm and 10–30 cm) for chemical and spectroscopic analysis. Sodium hydroxide–EDTA extracted a relatively consistent proportion (17 to 37%; average 25%) of the total organic P content determined by the ignition–H2SO4 extraction technique. Orthophosphate monoesters were the dominant form of organic P detected by solution 31P NMR spectroscopy of the NaOH–EDTA extracts, the majority of which appeared to be present in large ‘humic’ molecules, based on the predominance of a broad peak in the NMR spectra; some smaller signals due to glycerophosphate, inositol phosphates, and RNA‐derived mononucleotides were also evident. The composition of organic P in the topsoil layer was very similar to that in the subsoil layer. Strong correlations were found between soil organic carbon (C) and nitrogen (N) and extractable organic P, which suggests that processes regulating the cycling of organic C and N are closely related to that for organic P in these Vertisols.

Assessing crop residue phosphorus speciation using chemical fractionation and solution 31P nuclear magnetic resonance spectroscopy

At physiological maturity, nutrients in crop residues can be released to the soil where they are incorporated into different labile and non-labile pools while the remainder is retained within the residue itself. The chemical speciation of phosphorus (P) in crop residues is an important determinant of the fate of this P. In this study, we used chemical fractionation and 31P nuclear magnetic resonance (NMR) spectroscopy, first separately and then together, to evaluate the P speciation of mature oat (Avena sativa) residue. Two water extracts (one employing shaking and the other sonication) and two acid extracts (0.2N perchloric acid and 10% trichloroacetic acid) of these residues contained similar concentrations of orthophosphate (molybdate-reactive P determined by colorimetry) as NaOH–EDTA extracts of whole plant material subsequently analysed by solution 31P NMR spectroscopy. However, solution 31P NMR analysis of the extracts and residues isolated during the water/acid extractions indicated that this similarity resulted from a fortuitous coincidence as the orthophosphate concentration in the water/acid extracts was increased by the hydrolysis of pyrophosphate and organic P forms while at the same time there was incomplete extraction of orthophosphate. Confirmation of this was the absence of pyrophosphate in both water and acid fractions (it was detected in the whole plant material) and the finding that speciation of organic P in the fractions differed from that in the whole plant material. Evidence for incomplete extraction of orthophosphate was the finding that most of the residual P in the crop residues following water/acid extractions was detected as orthophosphate using 31P NMR. Two methods for isolating and quantifying phospholipid P were also tested, based on solubility in ethanol:ether and ethanol:ether:chloroform. While these methods were selective and appeared to extract only phospholipid P, they did not extract all phospholipid P, as some was detected by NMR in the crop residue after extraction. These results highlight the need for careful interpretation of results from chemical fractionation, as separation can be compromised by incomplete recovery and side reactions. This study also highlights the benefits of employing a technique that can simultaneously detect multiple P species (solution 31P NMR) in combination with chemical fractionation.

Soil Phosphorus Forms along a Strong Nutrient Gradient in a Tropical Ombrotrophic Wetland

Phosphorus cycling influences productivity and diversity in tropical wetlands, yet little is known about the forms of P found in the accreting organic matter of these ecosystems. We used alkaline (NaOH-ethylenediamine tetraacetic acid [EDTA]) extraction and solution P-31 nuclear magnetic resonance (NMR) spectroscopy to characterize P in surface soils across a strong nutrient gradient within a tropical ombrotrophic peat dome. From the interior bog plain to the marginal Raphia taedigera swamp, total soil P increased from 14.6 to 70.9 g m(-3) and resin-extractable P from 0.1 to 30 mg kg(-1). Phosphatase activity declined across the same transect (364-46 mu mol methylumbelliferone kg(-1) min(-1)), indicating an increase in P availability toward the periphery of the wetland. Organic P identified by solution 31P NMR spectroscopy included phosphomonoesters (12-17%), phosphodiesters (10-14%), and phosphonates (up to 3.3% of total P). Inositol phosphates were not detected in these acidic peats. Inorganic P forms included orthophosphate (9-25% of total P), pyrophosphate (up to 3%), and long-chain polyphosphates; the latter occurred in concentrations (up to 24% of total soil P) considerably higher than previously found in wetland soils. The concentration of residual (unextractable) P was similar among sites (mean 280 mg kg(-1)), resulting in an increase in its proportion of the total soil P from 29% at the P-rich margins to 55% at the P-poor interior. This is the first information on the P composition of tropical wetland soils and provides a basis for further study of the cycling and contribution of P forms to the nutrition of plants and microorganisms.

Sample Pretreatment and Phosphorus Speciation in Wetland Soils

We assessed the influence of sample pretreatment on the amounts and forms of P extracted in NaOH–EDTA (ethylenediamine tetraacetic acid) from a series of contrasting wetland soils from the Florida Everglades. Samples of unconsolidated benthic floc and underlying soil (0–10 cm) were extracted either fresh (overnight refrigeration only), air dried (10 d at ∼30°C), or frozen at −80°C and lyophilized (∼48 h), before extraction and solution 31P nuclear magnetic resonance (NMR) spectroscopy. Significant differences in total P extraction following pretreatment were detected for one out of four benthic floc samples and three out of four soil samples, although the changes were inconsistent: in two cases the total P extraction increased, while in two others it decreased. Assessment of the P composition by solution 31P NMR spectroscopy revealed differences among treatments, although these were mostly within the range of error associated with replicate analyses; however, DNA was not detected in a fresh sample of calcareous benthic floc, despite representing an important component of the organic P extracted from dried samples. The apparent sample‐specific nature of the changes confirms the importance of carefully assessing pretreatment effects in studies of soil organic P in wetlands.

Refining 31P nuclear magnetic resonance spectroscopy for marine particulate samples: Storage conditions and extraction recovery

Solution 31P nuclear magnetic resonance (NMR) spectroscopy has recently been used to characterize phosphorus species within marine particles. However, the effects of sample collection, storage and preparation have not been thoroughly examined. In this study, samples of settling particulates collected from a 1200-m sediment trap located in Monterey Bay, California, were subjected to various storage options (i.e., no storage, refrigeration, freezing, and oven-drying and grinding) prior to extraction for solution 31P-NMR spectroscopy. Freezing, refrigerating and drying samples for periods of up to 6 months prior to extraction with 0.25 M NaOH + 0.05 M Na 2EDTA increased the concentration of extracted P by an average of 16% relative to samples extracted without storage. Pre-extraction storage also introduced some minor changes in P speciation, by increasing the percentage of orthophosphate by up to 15% and decreasing the percentage of pyrophosphate by up to 5%, relative to the abundances of these P species in samples extracted without storage. Drying caused the biggest changes in speciation, specifically decreasing more extensively the relative percentage of pyrophosphate compared to other treatments. Nevertheless, observed changes in speciation due to sample storage within a specific sample were small relative to differences observed among samples collected sequentially in the same area, or reported differences among samples collected at different locations. Samples were also analyzed by solid-state 31P-NMR spectroscopy before and after extraction, to examine extraction-related changes in P speciation. Comparison of solution with solid-state 31P NMR indicates that extraction with NaOH–EDTA removes the majority of organic esters, but only a variable portion of phosphonates (39–67%). In addition, there was preferential extraction of Ca-associated phosphate over Mg-, Fe- and Al-associated phosphate. Solution 31P NMR enables much higher resolution of P species within samples, particularly when it is important to speciate orthophosphate monoesters and diesters, or if polyphosphates are present. However, combining solid-state 31P NMR with solution 31P NMR spectroscopy for marine particles should be conducted when examining inorganic P speciation and the abundance of phosphonates.

Optimizing Phosphorus Characterization in Animal Manures by Solution Phosphorus‐31 Nuclear Magnetic Resonance Spectroscopy

A procedure involving alkaline extraction and solution 31P nuclear magnetic resonance (NMR) spectroscopy was developed and optimized for the characterization of P in animal manures (broiler, swine, beef cattle). Inclusion of ethylenediaminetetraacetic acid (EDTA) in the alkaline extraction solution recovered between 82 and 97% of the total P from the three manures, which represented a significant improvement on recovery in NaOH alone. Low concentrations of paramagnetic ions in all manure extracts meant that relatively long delay times (> 5 s) were required for quantitative analysis by solution 31P NMR spectroscopy. The manures contained inorganic orthophosphate, orthophosphate monoesters, orthophosphate diesters, and inorganic polyphosphates, but results were markedly influenced by the concentration of NaOH in the extractant, which affected both spectral resolution and the apparent P composition of the extracts. For example, extraction of swine manure and broiler litter with 0.5 M NaOH + 50 mM EDTA produced remarkable spectral resolution that allowed accurate quantification of the four signals from phytic acid, the major organic P compound in these manures. In contrast, more dilute NaOH concentrations produced considerable line broadening that obscured individual signals in the orthophosphate monoester region of the spectra. Spectral resolution of cattle manure extracts was relatively unaffected by NaOH concentration. Improvements in spectral resolution of more concentrated NaOH extracts were, however, compromised by the disappearance of phospholipids and inorganic polyphosphates, notably in swine and cattle manure extracts, which indicated either degradation or a change in solubility. The optimum extraction conditions will therefore vary depending on the manure type and the objectives of the study. Phytic acid can be accurately quantified in swine manure and broiler litter by extraction with 0.5 M NaOH + 50 mM EDTA, while a more dilute NaOH concentration should be used for complete P characterization or comparison among different manure types.

Optimizing Phosphorus Characterization in Animal Manures by Solution Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy

A procedure involving alkaline extraction and solution 31P nuclear magnetic resonance (NMR) spectroscopy was developed and optimized for the characterization of P in animal manures (broiler, swine, beef cattle). Inclusion of ethylenediaminetetraacetic acid (EDTA) in the alkaline extraction solution recovered between 82 and 97% of the total P from the three manures, which represented a significant improvement on recovery in NaOH alone. Low concentrations of paramagnetic ions in all manure extracts meant that relatively long delay times (>5 s) were required for quantitative analysis by solution 31P NMR spectroscopy. The manures contained inorganic orthophosphate, orthophosphate monoesters, orthophosphate diesters, and inorganic polyphosphates, but results were markedly influenced by the concentration of NaOH in the extractant, which affected both spectral resolution and the apparent P composition of the extracts. For example, extraction of swine manure and broiler litter with 0.5 M NaOH + 50 mM EDTA produced remarkable spectral resolution that allowed accurate quantification of the four signals from phytic acid, the major organic P compound in these manures. In contrast, more dilute NaOH concentrations produced considerable line broadening that obscured individual signals in the orthophosphate monoester region of the spectra. Spectral resolution of cattle manure extracts was relatively unaffected by NaOH concentration. Improvements in spectral resolution of more concentrated NaOH extracts were, however, compromised by the disappearance of phospholipids and inorganic polyphosphates, notably in swine and cattle manure extracts, which indicated either degradation or a change in solubility. The optimum extraction conditions will therefore vary depending on the manure type and the objectives of the study. Phytic acid can be accurately quantified in swine manure and broiler litter by extraction with 0.5 M NaOH + 50 mM EDTA, while a more dilute NaOH concentration should be used for complete P characterization or comparison among different manure types.

Organic Phosphorus Composition and Potential Bioavailability in Semi-Arid Arable Soils of the Western United States

The organic P composition of semi-arid arable soils is largely unknown, but such information is fundamental to understanding P dynamics in irrigated agriculture. We used solution 31P nuclear magnetic resonance (NMR) spectroscopy and phosphatase hydrolysis to characterize organic P in semi-arid arable soils from the western USA (organic C 2.0–30.7 g C kg−1 soil, clay 2–48%, pH 5.2–8.2, CaCO3 <1–480 g kg−1 soil). Total P concentrations ranged from 220 to 1210 mg P kg−1 soil, of which between 12 and 45% was extracted with NaOH–EDTA. Inorganic orthophosphate was the dominant P compound, but concentrations determined by solution 31P NMR spectroscopy were consistently greater than those determined by molybdate colorimetry. Concentrations of organic P were relatively small, and were dominated by orthophosphate monoesters (11–130 mg P kg−1 soil), with smaller concentrations of orthophosphate diesters (0–7 mg P kg−1 soil). Pyrophosphate was present in almost all soils at concentrations up to 14 mg P kg−1 soil. Bicarbonate-extractable organic P ranged from 1.7 to 22.8 mg P kg−1 soil, of which between 37 and 87% was hydrolyzed by phosphatase enzymes, suggesting its bioavailability. Soil organic P concentrations were positively correlated with mean annual precipitation, organic C, clay, and oxalate-extractable metals (Al, Fe, Mn), and negatively correlated with mean annual temperature and soil pH. However, CaCO3 concentrations were not significantly correlated with any soil property. These results indicate that equilibrium levels of organic P in semi-arid arable soils are controlled by a balance between the physical protection offered by the soil matrix and the suitability of the environment for biological productivity.

The phosphorus composition of temperate pasture soils determined by NaOH–EDTA extraction and solution 31P NMR spectroscopy

Information on the composition and dynamics of soil phosphorus (P) remains limited, but is integral to understanding soil biogeochemical cycles. We used solution 31P nuclear magnetic resonance (NMR) spectroscopy to characterise NaOH–EDTA extractable P in 29 permanent pasture soils from England and Wales (total carbon 29–80 g kg−1 soil, clay 219–681 g kg−1 soil, pH 4.4–6.8). Total P ranged between 376 and 1981 mg P kg−1 soil, of which between 45 and 88% was extracted with NaOH–EDTA. The extracts were dominated by orthophosphate monoesters (29–60% extracted P) and inorganic orthophosphate (21–55% extracted P), with smaller concentrations of orthophosphate diesters (2–10% extracted P), pyrophosphate (1–7% extracted P), phosphonates (0–3% extracted P), and traces of polyphosphates. Orthophosphate diesters were subclassified into phospholipids (1–7% extracted P) and DNA (1–6% extracted P). Signals slightly downfield of inorganic orthophosphate were tentatively assigned to aromatic orthophosphate diesters similar in structure to R-(−)-1,1′-binaphthyl-2,2′-diyl hydrogen phosphate. Such signals are rarely detected in soil extracts, but were present in relatively large concentrations in the samples analysed here (2–5% extracted P). Relationships between functional P groups and soil properties suggested that the various functional groups are involved in the soil P cycle to different extents. In particular, concentrations of orthophosphate monoesters appeared to be controlled by the potential for chemical stabilisation in soil, whereas DNA and pyrophosphate were strongly correlated with the microbial biomass, suggesting an active involvement in biological nutrient turnover.

Phosphorus‐31 Nuclear Magnetic Resonance Spectral Assignments of Phosphorus Compounds in Soil NaOH–EDTA Extracts

Soil P composition can be conveniently determined in alkaline extracts using solution 31P nuclear magnetic resonance (NMR) spectroscopy, but spectral assignments are based on fragmentary literature reports of model compounds in various extraction matrices. We report solution 31P NMR chemical shifts of model P compounds, including inorganic phosphates, orthophosphate monoesters and diesters, phosphonates, and organic polyphosphates, determined in a standardized soil P extractant (0.25 M NaOH and 0.05 M EDTA). Signals from nucleic acids (DNA −0.37 ppm, RNA 0.54 ppm) and phospholipids (phosphatidyl choline 0.78 ppm, phosphatidyl serine 1.57 ppm, phosphatidyl ethanolamine 1.75 ppm) could be differentiated in the orthophosphate diester region, and were identified in a sample of cultured soil bacteria. Inorganic and organic polyphosphates could be differentiated by the presence of a signal at −9 ppm from the α phosphate of organic polyphosphates. Some orthophosphate diesters, notably RNA and phosphatidyl choline, degraded rapidly to orthophosphate monoesters in NaOH–EDTA although DNA, other phospholipids, and orthophosphate monoesters were more stable. Changes in probe temperature had a marked influence on signal intensities and the relative magnitude of signals from orthophosphate monoesters and inorganic orthophosphate, and we suggest that solution 31P NMR spectroscopy of soil extracts be performed at 20°C.

Phosphorus-31 Nuclear Magnetic Resonance Spectral Assignments of Phosphorus Compounds in Soil NaOH–EDTA Extracts

Soil P composition can be conveniently determined in alkaline extracts using solution 31P nuclear magnetic resonance (NMR) spectroscopy, but spectral assignments are based on fragmentary literature reports of model compounds in various extraction matrices. We report solution 31P NMR chemical shifts of model P compounds, including inorganic phosphates, orthophosphate monoesters and diesters, phosphonates, and organic polyphosphates, determined in a standardized soil P extractant (0.25 M NaOH and 0.05 M EDTA). Signals from nucleic acids (DNA −0.37 ppm, RNA 0.54 ppm) and phospholipids (phosphatidyl choline 0.78 ppm, phosphatidyl serine 1.57 ppm, phosphatidyl ethanolamine 1.75 ppm) could be differentiated in the orthophosphate diester region, and were identified in a sample of cultured soil bacteria. Inorganic and organic polyphosphates could be differentiated by the presence of a signal at −9 ppm from the α phosphate of organic polyphosphates. Some orthophosphate diesters, notably RNA and phosphatidyl choline, degraded rapidly to orthophosphate monoesters in NaOH–EDTA although DNA, other phospholipids, and orthophosphate monoesters were more stable. Changes in probe temperature had a marked influence on signal intensities and the relative magnitude of signals from orthophosphate monoesters and inorganic orthophosphate, and we suggest that solution 31P NMR spectroscopy of soil extracts be performed at 20°C.

Soil and Litter Phosphorus‐31 Nuclear Magnetic Resonance Spectroscopy

Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy is an excellent tool with which to study soil organic P, allowing quantitative, comparative analysis of P forms. However, for 31P NMR to be tative, all peaks must be completely visible, and in their correct relative proportions. There must be no line broadening, and adequate delay times must be used to avoid saturation of peaks. The objective of this study was to examine the effects of extractants on delay times and peak saturation. Two samples (a forest litter and a mineral soil sample) and three extractants (0.25 M NaOH, NaOH plus Chelex (Bio-Rad Laboratories, Hercules, CA), and NaOH plus EDTA) were used to determine the differences in the concentration of P and cations solubilized by each extractant, and to measure spin-lattice (T1) relaxation times of P peaks in each extract. For both soil and litter, NaOH-Chelex extracted the lowest concentrations of P. For the litter sample, T1 values were short for all extractants due to the high Fe concentration remaining after extraction. For the soil sample, there were noticeable differences among the extractants. The NaOH-Chelex sample had less Fe and Mn remaining in solution after extraction than the other extractants, and the longest delay times used in the study, 6.4 s, were not long enough for quantitative analysis. Delay times of 1.5 to 2 s for the NaOH and NaOH-EDTA were adequate. Line broadening was highest in the NaOH extracts, which had the highest concentration of Fe. On the basis of these results, recommendations for future analyses of soil and litter samples by solution 31P NMR spectroscopy include: careful selection of an extractant; measurement of paramagnetic ions extracted with P; use of appropriate delay times and the minimum number of scans; and measurement of T1 values whenever possible.