High-Resolution Characterization of Organic Phosphorus in

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High-Resolution Characterization of Organic Phosphorus in Soil Extracts Using 2D 1H−31P NMR Correlation Spectroscopy Johan Vestergren,† Andrea G. Vincent,‡ Mats Jansson,‡ Per Persson,† Ulrik Ilstedt,§ Gerhard Gröbner,† Reiner Giesler,∥ and Jürgen Schleucher⊥,* †

Chemistry Department, Umeå University, S−901 87 Umeå, Sweden Department of Ecology and Environmental Sciences, Umeå University, S−901 87 Umeå, Sweden § Department of Forest Ecology & Management, SLU, S-90185 Umeå, Sweden ∥ Climate Impacts Research Centre, Department of Ecology and Environmental Sciences, Umeå University, S−981 07, Abisko, Sweden ⊥ Department of Medical Biochemistry and Biophysics, Umeå University, S−901 87 Umeå, Sweden ‡

S Supporting Information *

ABSTRACT: Organic phosphorus (P) compounds represent a major component of soil P in many soils and are key sources of P for microbes and plants. Solution NMR (nuclear magnetic resonance spectroscopy) is a powerful technique for characterizing organic P species. However, 31P NMR spectra are often complicated by overlapping peaks, which hampers identification and quantification of the numerous P species present in soils. Overlap is often exacerbated by the presence of paramagnetic metal ions, even if they are in complexes with EDTA following NaOH/EDTA extraction. By removing paramagnetic impurities using a new precipitation protocol, we achieved a dramatic improvement in spectral resolution. Furthermore, the obtained reduction in line widths enabled the use of multidimensional NMR methods to resolve overlapping 31P signals. Using the new protocol on samples from two boreal humus soils with different Fe contents, 2D 1H−31P correlation spectra allowed unambiguous identification of a large number of P species based on their 31P and 1H chemical shifts and their characteristic coupling patterns, which would not have been possible using previous protocols. This approach can be used to identify organic P species in samples from both terrestrial and aquatic environments increasing our understanding of organic P biogeochemistry.

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INTRODUCTION Phosphorus (P) is an essential element for life and its bioavailability often limits the productivity of terrestrial and aquatic ecosystems.1 Soil organic P compounds are often a major fraction of total soil P, and include numerous molecular species with varying bioavailability and mobility through ecosystems.2 Identification of organic P species is important to understand their origin and turnover in soils, and ultimately, ascertain their role in ecosystems and their effects on soil fertility. These questions need to be addressed urgently because of the unknown response of P biogeochemistry to global environmental changes, and the possibility that P fertilizer for agriculture may soon become scarce.3 One-dimensional (1D) solution 31P nuclear magnetic resonance spectroscopy (NMR) is currently the tool of choice for molecular-level characterization of organic P in soils,4 and has greatly advanced our understanding of organic P biogeochemistry. Solution-phase NMR requires extraction of organic P from soils, and NAOH/EDTA is commonly used as an extractant.4,5 Unfortunately, current 1D NMR methodology has several important drawbacks. First, spectra are often complicated by considerable signal overlap, especially in the monoester region, © 2012 American Chemical Society

because the observed signals usually have line-widths of tens of hertz and the 31P chemical shift dispersion is small. Second, the dependence of 31P chemical shifts on the sample matrix makes unambiguous spectral assignments and accurate quantification difficult. Finally, even when samples are spiked with reference materials, it is not possible to identify compounds if their 31P chemical shifts overlap completely.6 As a consequence, articles on 31P NMR of soils often only report the abundance of the monoester region as a whole. Whereas NaOH/EDTA extracts the greatest amount and diversity of P species compared to other methods,7 paramagnetic metal ions are also brought into solution.8 Even though the paramagnetic ions are scavenged as EDTA complexes, they still cause line broadening, and hence reduce resolution.4 2D NMR is well established in biochemistry as a technique for studying a wide range of P compounds.9−11 However, 2D NMR has, to the best of our knowledge, never been successfully Received: Revised: Accepted: Published: 3950

November 10, 2011 February 26, 2012 March 7, 2012 March 7, 2012 dx.doi.org/10.1021/es204016h | Environ. Sci. Technol. 2012, 46, 3950−3956

Environmental Science & Technology

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make a final volume of 2.5 mL in a 10 mm diameter NMR tube. Spectra were collected using a 36° pulse (12 μs pulse duration), an acquisition time of 211 ms, a relaxation delay of 1.8 s, and 10 240 scans. For treated samples prepared as described above, a 50° pulse was used (20 μs pulse duration), an acquisition time of 0.384 s, a relaxation delay of 2 s, and 24 576 scans. Spectra were processed using topspin 2.0 (Bruker) with a line broadening of 2 Hz, and 31P chemical shifts were expressed in parts per million (ppm) relative to an external standard of 85% orthophosphoric acid (at δ = 0.0). 1H chemical shifts were referenced to an external solution of 2,2-dimethyl-2-silapentane-5-sulfonate-d6 (DSS) in D2O. 2D 1H, 31P HSQC (heteronuclear single quantum coherence) spectra were recorded with a pulse sequence employing gradient coherence selection (according to Figure 13a in Sattler et al.13) on DRX 500 or DRX 600 spectrometers, each equipped with a 5 mm 1H, 13C, broadband probe optimized for 1 H observation and tuned to 31P. The sweep width and acquisition time in the 1H dimension were 12 ppm and 200 ms respectively, whereas in the 31P dimension they were 26 ppm and 20 ms, respectively. The duration of J-coupling evolution in the HSQC pulse sequence was set to 20 ms, and 31P decoupling was applied during 1H acquisition. The recycle time was 2.2 s and the experiment time for each 2D spectrum was about 40 h. Note that the 2D experiment starts with 1H magnetization, therefore 1H relaxation times have to be considered to ensure that spectra can be quantified. Detailed parameters are given in the Supporting Information. The 2D data matrix was processed by extending the 31P dimension to 480 points using linear prediction and zero-filling to 4k and 1k points in the 1H and 31P dimensions, respectively. Shifted squared sine-bell apodization was applied in both dimensions and was adjusted in the 1H dimension to optimize either sensitivity or resolution of the spectra. We performed 1D 31P and 2D 1H,31P NMR on a soil extract spiked with reference P compounds (1−4 mM, Supporting Information for list of compounds), to facilitate spectral assignments of organic P compounds in natural soil extracts.

applied to soil extracts. In 2D NMR spectra, each signal is identified by its coordinates in a two-dimensional plane, thus amplifying the resolving power of NMR. For example, it has been shown that phospholipids can be unambiguously identified in 2D 1H−31P NMR correlation spectra, based on their 31P chemical shifts in one dimension, and their 1H chemical shifts and signal fine structure in the second dimension.11 In addition, these spectra provide structural information that helps in identifying unknown compounds. The main objective of this study was to develop a 2D NMR procedure for complex environmental matrices such as soils. To achieve this we aimed to 1) develop a method to remove paramagnetic ions from solution without altering the P content of soil extracts; 2) ascertain a 2D experiment which provides optimal resolution and sensitivity in soil extracts and apply it to determine the organic P composition in soils; and, 3) produce a list of 2D 1H and 31P chemical shifts and signal fine structure of common soil organic P compounds to facilitate their identification in natural soils by 2D NMR.

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EXPERIMENTAL SECTION Sites and Soils. We used boreal forest humus soils from two sites close to Umeå, in Northern Sweden. The soils are typic haplocryods, the entire O horizon (5−15 cm) was sampled. Detailed information on the two sites, Ulterviken 2 and Sör-Grundbäck, is given in Wallander et al.12 Soils are denoted here as ‘soil-1’ (Ulterviken 2) and ‘soil-2’ (SörGrundbäck). After sampling and sieving (4 mm mesh size), the samples were dried (35 °C, 10 days) and then milled in a Retsch MM 400 ball mill (2 min, 2 kHz). A subsample of the milled soil was used for determination of acid-digestible (nitric acid) P and Fe by inductively coupled plasma optical emission spectrometry (ICP-OES, Perkin-Elmer). Soil-1 contained 16.5 and 0.99 g kg−1 of Fe and P, respectively, whereas soil-2 contained 2.1 and 0.84 g kg−1, respectively. Phosphorus was extracted by shaking 1.5 g of dry and milled soil in 30 mL of extraction solution containing 0.25 M NaOH and 50 mM Na2EDTA for 16 h.7 The samples were then centrifuged (30 min, 14000 g) and the supernatant was frozen at −80 °C and subsequently lyophilized. Each extraction yielded approximately 600 mg of lyophilized material. Sulfide Treatment to Remove Paramagnetic Ions. To remove paramagnetic ions from NaOH-EDTA soil extracts, 100 mg of lyophilized material was dissolved in a variable volume of D2O in an Eppendorf vial. To this solution, Na2S dissolved in D2O was added to a final concentration corresponding to 5−10 mol equiv of the iron (Fe) content of the sample. The high sulfide excess maintains reducing conditions throughout the precipitation. MDPA (methylene diphosphonic acid sodium salt, 5 μL of 100 mM solution in D2O, Sigma catalogue number M1886) was added as an internal standard and the volume of the extract/Na2S mixture was adjusted to 500 μL with D2O. After 18−20 h at room temperature, the sample was centrifuged (30 min, 7000 g) and the supernatant was transferred to a 5 mm diameter NMR tube, which was used to record 1D 31P and 2D 1H,31P spectra. NMR Experiments. Solution 1D 31P NMR spectra were recorded at 298 K using a Bruker DRX 500 spectrometer (Bruker, Germany), equipped with either a 10 mm broadband probe, tuned to 31P (untreated samples), or a 5 mm 1H, 13C, broadband probe optimized for 1H observation and tuned to 31 P (sulfide-treated samples). For 1D 31P NMR of untreated samples, 600 mg of lyophilized extract was dissolved in D2O to

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RESULTS AND DISCUSSION Figure 1 shows a 1D 31P NMR spectrum of the extract from soil-1 prepared without pretreatment with Na2S (bold line) and of the same soil extract after sulfide treatment (thin line). The spectrum for the nontreated soil extract shows only three broad peaks in the monoester region at 6.5, 5.4, and 5.0 ppm, with line widths of between 40 and over 100 Hz. These broad peaks do not provide sufficient information for the identification or quantification of individual P species. Signals in other regions (diesters, inorganic polyphosphate) had similarly large line widths. Compared to the spectrum for the untreated sample, the spectrum for the sulfide-treated sample (thin line) shows a dramatic improvement, with numerous sharp NMR resonances now clearly visible. The sulfide treatment reduced 31P line widths of orthophosphate and organic phosphates to 2−3 Hz, which is comparable to typical line widths for reference samples free of paramagnetic ions. Whereas the line widths of the untreated sample are larger than typical, the comparison was chosen to demonstrate that sulfide precipitation works even in this case. For soil-2 and 2 other podzols, line widths decreased from 5 to 8 to 2.5−3 Hz. The small line widths achieved using sulfide precipitation are a significant improvement over previous approaches for soil extracts, which typically result in line widths between 5 and over 20 Hz.6,14 The very similar 31P 3951

dx.doi.org/10.1021/es204016h | Environ. Sci. Technol. 2012, 46, 3950−3956


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more than 0.1 ppm.15 Because of this variation, peaks due to P species whose chemical shifts differ by 0.1 ppm or even more may switch positions in 1D NMR spectra making identification of these P species based on their chemical shifts difficult even if their signals are well resolved.16 Even spiking the sample with reference compounds may not allow all species in a sample to be identified, as compounds with identical chemical shifts have been observed.6 Finally, neither the 31P chemical shift nor spiking can be used to identify new P species. To obtain 2D 1H−31P correlation spectra, preliminary experiments (Figure S1 of the Supporting Information) showed that an HSQC pulse sequence employing a heteronuclear gradient echo in an amplitude-modulating pulse sequence (Figure 13a in Sattler et al.13) yielded spectra with the best resolution and excellent suppression of background signals. Part A of Figure 2 presents a 2D 1H−31P HSQC spectrum of a sulfide-treated extract of soil-1. The spectrum correlates 31P chemical shifts along the vertical axis with 1H chemical shifts along the horizontal axis. 2D NMR spectra are commonly displayed as contour plots, where the number of contours represents signal intensity similar to topographic maps. Correlations are possible when 1H and 31P nuclei are separated by no more than three bonds. Therefore, each P−O−C−H moiety gives rise to one cross peak in the 2D plane. Phosphodiesters, phosphomonoesters, and phosphonates are visible as well-resolved groups of signals in the 2D plane in separate 31P chemical shift ranges. Inorganic P signals are lacking, which allows observation of signals due to organic P compounds that would otherwise overlap with orthophosphate. Diesters appear as broad signals in both dimensions, which do not show improved resolution in the 2D relative to the 1D 31P spectrum. In the phosphonate region, a signal with two hydrogen signals was observed (signal X5). Two correlations can be visible for phosphonates because in a P−CH−CH moiety, the P nucleus interacts with both CH groups. The monoester region in the spectrum of soil-1 (part B of Figure 2) contains several cross peaks where the 1H chemical shifts allow the separation of species with overlapping 31P chemical shifts (dotted lines). For example, β-glycerophosphate (signal A) overlaps with uridine 3′ monophosphate (signal H), and the signals C and C′ of myo-inositol hexakisphosphate overlap with uridine 2′ monophosphate (signal G) and guanosine 2′ monophosphate (signal D) and an unidentified nucleotide (signal X2), respectively. These examples demonstrate that overlap may still occur in well-resolved 31P spectra. Therefore, unambiguous identification of P species from 1D 31P NMR spectra may prove impossible, even when samples are spiked with reference compounds. In contrast, 2D NMR enables the identification of P species based on resolved 1H chemical shifts, even if their 31 P chemical shifts overlap completely. Furthermore, 1H chemical shifts contain structural information, which helps to characterize unknown compounds. The 2D spectrum of soil-2 (part C of Figure 2) largely contains the same signals as for soil-1, but notable differences are also apparent. For example, a signal due to adenosine 5′ monophosphate (signal N) is only evident in the spectrum for soil-2 but not in soil-1. In soil-2, a phosphonate is observed (signal F) that we identify as the naturally occurring 2aminoethyl phosphonate H2N−CH2-CH2−PO3Na2 based on its 31P and 1H chemical shifts. Newman and Tate reported two signals in the phosphonate region with chemical shifts of 18.3 and 19.8 ppm and assigned them to a phosphonolipid and an alkyl phosphonic acid, respectively.17 Compared to these

Figure 1. 1D 31P NMR spectra of NaOH/EDTA extracts of soil-1, which is naturally high in Fe. Spectra without (bold line) and with (thin line) sulfide treatment are compared. The region displayed corresponds to phosphomonoesters and inorganic phosphate (6.5 ppm). Spectra are scaled for easy comparison.

line widths observed for sulfide-treated samples allow spectra to be readily compared. It is well-known that most paramagnetic heavy metals (e.g., Fe, Mn, Ni, Cu) form insoluble sulfides. Furthermore, sulfide reduces Fe3+ to Fe2+, lowering its affinity for phosphate groups. Hence, we expected sulfide to be an effective general reagent for removing paramagnetic metals from soil extracts. Upon overnight incubation of NaOH/EDTA soil extracts with Na2S, visible precipitation was observed, which was removed by centrifugation. It is essential to maintain reducing conditions during precipitation because iron sulfide is easily oxidized to iron hydroxide, which has a high sorption affinity for phosphates. Determination of Fe, Mn, and P in extracts of soil-1 by ICP-OES showed that sulfide treatment reduced the Fe concentration from 346 to 23 ppm, and Mn from 10 to 8 ppm. Recovery of P after sulfide precipitation was tested on four podzol samples and on six samples of Al- and Fe-rich dry tropical mineral soils, no loss of P was observed (recovery 94− 105%). In a control experiment, we found that the P monoester composition did not change upon sulfide treatment (Figure S4 of the Supporting Information). Removal of paramagnetic ions may increase phosphorus T1 relaxation times,7 so that the relaxation delay may have to be increased to obtain quantitative spectra. However, we have observed normal T1 relaxation times (approximately 1.5 s) upon sulfide treatment, and a possible reduced sensitivity due to longer relaxation delays is offset by the inherently higher sensitivity of sharper signals. Despite the narrow lines obtained following the sulfide treatment, unambiguous identification of all the P species will generally not possible by 1D 31P NMR due to the large number of species present and the small 31P chemical shift dispersion. For example, a 2 Hz line width corresponds to 0.01 ppm on a typical NMR instrument, and therefore, only signals whose chemical shifts differ by more than this value could be resolved. However, 31P chemical shifts of soil compounds often vary by 3952



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Figure 2. 2D 1H−31P HSQC spectra of sulfide-treated NaOH-EDTA soil extracts. (A) Overview of the spectrum for soil-1, showing distinct chemical shift ranges for phosphodiesters, phosphomonoesters and phosphonates. Note that MDPA had not been added to this particular sample. (B) Expansion of the spectrum for soil-1 showing the monoester region and processed to maximize resolution. (C) Overview of the spectrum for soil-2. See Table 1 for an explanation of peak labels; X, X2 etc. denote unidentified compounds. Solid lines indicate cross peaks belonging to the same compound. Dashed lines connect signals which overlap in the 31P dimension but are resolved in the 1H dimension.

signals, signals F and X5 have similar 31P chemical shifts (18.98

In addition to showing the correlations between the 31P and H chemical shifts, 2D spectra exhibit fine structure in the 1H dimension (part B of Figure 2), which is specific to each signal. The fine structures of four compounds are shown in more 1

and 20.7 ppm), but they both lack a cross peak to the glycerol moiety that would be expected for a phosphonolipid. 3953



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coupling partners, resulting in a quintet signal. For αglycerophosphate (trace B), the hydrogens in the CH2 group are not equivalent (diastereotopic) and therefore have different chemical shifts. Thus, they couple to each other, in addition to the central hydrogen, giving rise to two double−doublets. Trace C shows signals of myo-inositol hexakisphosphate and uridine 2′ phosphate, with their respective fine structures. A singlet is observed for myo-inositol hexakisphosphate because its 1H−1H couplings are small due to the compound’s conformation.18 For the nucleotides uridine 2′ phosphate and guanosine 2′ phosphate (traces C, D), the 1H coupled to P has two 1H coupling partners, giving rise to triplets. In summary, the multiplet patterns contain information on the molecular structure in the vicinity of the P nuclei, which allows more definitive identification of known compounds and provides information on the structure of hitherto unknown compounds. 2D spectra of reference P compounds common in soils and measured in soil NaOH/EDTA extracts are given in the supplementary Figure S2 of the Supporting Information, and their 31P and 1H chemical shifts and multiplet patterns are summarized in Table 1. The 31P chemical shifts observed in this study are in close agreement with previous work.19 Note that there are small chemical shift differences between reference compounds (Table 1) and compounds observed in natural soil extracts (Figure 2). Nevertheless, most compounds are unambiguously identified in the 2D spectra based on the combination of 31P and 1H chemical shifts and the 1H fine structure. The spectra displayed in Figure 2 reveal several unknown P species (marked X−X6), which are concealed in conventional 31P NMR. In conventional 31P NMR, spiking with reference compounds is a common way to identify P species.6 However, it has been pointed out that some compounds cannot be identified by spiking because their chemical shifts are too similar,6 and that agreement of chemical shifts does not prove the identity of a compound.20 As mentioned above, P species with overlapping 31P chemical shifts are often resolved in the 1 H dimension of 2D spectra, therefore spiking in conjunction with 2D NMR may be the most powerful tool to identify P species. By combining the advantages of 1D and 2D NMR, several strategies can be used for the quantification of organic P species in soils. First, if 2D NMR shows that all P species are resolved in a 1D 31P spectrum, the 2D spectrum can be used to identify the P species, and the 1D spectrum for quantification. Alternatively, if 31P signals overlap in the 1D 31P spectrum, cross peaks in the 2D spectrum can be integrated to quantify P species. However, because 1H−31P coupling constants vary among species, different P species may exhibit different sensitivities in 2D experiments so that cross peak integrals of different compounds may not be directly comparable. Therefore, absolute quantification of P species (e.g., in μmol P) requires determination of compound-specific sensitivity factors11 by integrating the 2D spectra of reference samples of known composition. Finally, if the interest is in determining differences in the abundance of individual compounds between soils, 2D integrals can be directly used without the need to determine compound-specific sensitivity factors. For example, integration of signals B and A (Figure 2) shows that the concentration of α-glycerophosphate is similar in soil-1 and soil-2, whereas β-glycerophosphate is approximately five times less abundant in soil-2 compared to soil-1. Such relative changes often allow deducing trends in P composition and to derive valuable biogeochemical conclusions.

detail in parts A−D of Figure 3, which displays 1H traces extracted from part B of Figure 2. Each trace shows the signal of

Figure 3. 1H traces extracted from the spectrum displayed in part B of Figure 2 to reveal 1H−1H coupling patterns, which reflect the arrangement of 1H nuclei around the POCH moiety. The hydrogen atoms giving rise to the signals are indicated in bold in the molecular structures, whereas their coupling partners are shown inside circles.

the hydrogen nucleus − marked in bold in the respective structure − that couples to a particular P nucleus. The fine structures are independent of sample conditions and can be used to empirically identify compounds even if their chemical shifts are affected by for example changes in pH. For example, signals C and G overlap in the 31P dimension, but their fine structures (part C of Figure 3) enables them to be clearly identified as myo-inositol hexakisphosphate and uridine 2′ phosphate. The fine structures in the 1H traces are multiplets, which arise from 1H−1H couplings between the H nuclei marked in bold in the structures and neighboring hydrogens (circled in the structures) in the respective molecule. The β-glycerophosphate trace (part A of Figure 3) shows the signal of the central hydrogen in the glycerol moiety, which has four hydrogen 3954



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Table 1. List of 31P and 1H Chemical Shifts, Multiplet Structures and Their Respective Assignments for Organic P Reference Compounds in Soil Extract Solution, Obtained from 1D 31P and 2D 1H−31P NMR (Figure S2 of the Supporting Information); J Denotes 1H−1H Coupling Constants Measured in Hz; s: Singlet; d: Doublet; dd: Double−Doublet; t: Triplet; q: Quintet; AB: AB Spin System; m: Multiplet compound A B

β glycerophosphate α glycerophosphate

31

P shift (ppm) 4.95 5.27

C C′ C″ C‴ D E F

myo-inositol hexakisphosphate

guanosine 2′ monophosphate guanosine 3′ monophosphate 2-aminoethyl phosphonate

4.65 (C4,6) 4.55 (C5) 5.03 (C1,3) 5.97 (C2) 4.33 4.90 18.98

G H I J K L

uridine 2′ monophosphate uridine 3′ monophosphate choline phosphate ethanolamine phosphate α-D-glucose-1- phosphate guanosine 5′ monophosphate

4.62 4.85 4.25 4.91 3.39 4.78

M

DNA

N

adenosine 5′ monophosphate

−0.5 −0.0 4.77

O

phosphatidyl choline

0.69

P Q R

phosphatidyl ethanolamine adenosine 2′, 5′ diphosphate

1.79 4.54 (2′) 4.81 (5′)

S T

MDPA (reference) scyllo-inositol hexakisphosphate

17.39 4.14

1

H shift (ppm)

fine structure

4.07 3.68,

q dd

3.75

dd

4.52 4.27 4.48 4.48 4.89 4.58 1.59, 3.28 4.54 4.38 4.08 3.69 5.34 3.84, 3.89 3.8 − 4.2, 4.62 − 4.98 3.85, 3.90 4.23, 3.79 3.81 4.93 3.88, 3.94 1.80 4.47

s s s s t d m m t t broad broad s d d

d d t weak AB t d d s s

J 6.0 7.5, 12 6.0, 12

6.4 8.0

6.6 6.5

13 13

12 12 6 15 6.3 15 15

that the precursor RNA was contained in cells of soil organisms. This is further supported by the fact that large amounts of glycerophosphate were detected in the soil samples, which is a breakdown product of phospholipids of cellular membranes. These results suggest that a substantial amount of the detected organic P was originally present in soil organisms and was released and broken down to the observed species during sample preparation. Whereas 2D 1H−31P correlation experiments have been tested on reference organic P compounds found in soils, such as inositol phosphates,23 they have not previously been applied to soil extracts. The results of this study demonstrate that removal of paramagnetic ions is essential in 2D NMR experiments of soil P compounds when significant amounts of paramagnetic ions are present. That paramagnetic ions must be removed agrees with theory because the sensitivity of 2D NMR experiments declines with increasing line widths of the observed compounds.13 We have observed very narrow 31P signals in extracts of dry tropical soils, and 2D experiments succeed on these samples without sulfide treatment. 2D 1 H−31P NMR allows many more P species to be resolved than traditional 1D 31P NMR, and the combination of 31P and 1 H chemical shifts and 1H fine structure enables unambiguous identification. Although the time taken to record each 2D

Extraction of soils with NaOH/EDTA is known to hydrolyze several forms of phosphodiesters. This is considered an unavoidable drawback of the method, but it has been pointed out that it does not exclude deriving the original P composition when hydrolysis products can be traced back.21 Therefore, when a hydrolysis product is observed, it must be determined what fraction of the compound was originally present in the soil, versus formed during extraction.19 Whereas the longer sample preparation time for sulfide treatment increases hydrolysis (Figure S3 of the Supporting Information), the 2D methodology is very well suited to trace observed compounds back to their precursors. For example, nucleotides are normal cellular metabolites but are also formed during hydrolysis of RNA. In metabolites, the phosphate group is usually connected to the 5′ position, whereas the 2′ and 3′ isomers are formed by RNA hydrolysis.22 These isomeric forms are difficult to distinguish based on their 31P chemical shifts, but they can be easily identified based on their 1H chemical shifts and multiplet structures. Besides a small amount of 5′ AMP (signal N), several 2′ and 3′ phosphates were observed in the 2D spectra for the soil samples (Figure 2), namely signals D, G, H, and probably X2, X3, and X4 (based on their fine structures). Thus, the majority of nucleotides detected were products of RNA hydrolysis. Because free RNA is rather unstable, this suggests 3955



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Extractants, metals, and phosphorus relaxation times. J. Environ. Qual. 2002, 31 (2), 457−465. (9) Perlman, M. E.; Davis, D. G.; Gabel, S. A.; London, R. E. Uridine diphospho sugars and related hexose phosphates in the liver of hexosamine-treated rats − identification using 31P-(1H) two-dimensional NMR with HOHAHA relay. Biochemistry 1990, 29 (18), 4318− 4325. (10) Cromsigt, J. A. M. T. C.; van Buuren, B. N. M.; Schleucher, J.; Wijmenga, S. S. Techniques for resonance assignment and structure determination for RNA. Methods Enzymol. 2001, 338, 371−399. (11) Petzold, K.; Olofsson, A.; Arnqvist, A.; Gröbner, G.; Schleucher, J. Semi-constant-time P,H-COSY NMR: Analysis of complex mixtures of phospholipids originating from Helicobacter pylori. J. Am. Chem. Soc. 2009, 131 (40), 14150−14151. (12) Wallander, H.; Mörth, C. M.; Giesler, R. Increasing abundance of soil fungi is a driver for N-15 enrichment in soil profiles along a chronosequence undergoing isostatic rebound in northern Sweden. Oecologia 2009, 160 (1), 87−96. (13) Sattler, M.; Schleucher, J.; Griesinger, C. Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog. Nucl. Magn. Reson. Spectrosc. 1999, 34, 93−158. (14) Ding, S. M.; Xu, D.; Li, B.; Fan, C. X.; Zhang, C. S. Improvement of 31P NMR spectral resolution by 8-hydroxyquinoline precipitation of paramagnetic Fe and Mn in environmental samples. Environ. Sci. Technol. 2010, 44 (7), 2555−2561. (15) Turner, B. L.; Mahieu, N.; Condron, L. M. The phosphorus composition of temperate pasture soils determined by NaOH-EDTA extraction and solution P-31 NMR spectroscopy. Org. Geochem. 2003, 34 (8), 1199−1210. (16) Doolette, A. L.; Smernik, R. J. Soil organic phosphorus speciation using spectroscopic techniques. In: Phosphorus in Action: Biological Processes in Soil Phosphorus Cycling; Bünemann, E. K., Oberson, A., Frossard, E., Eds.; Springer: Berlin, Heidelberg, Germany, 2011, pp 6−17. (17) Newman, R. H.; Tate, K. R. Soil phosphorus characterization by P-31 nuclear magnetic resonance. Commun. Soil Sci. Plant Anal. 1980, 11 (9), 835−842. (18) Barrientos, L. G.; Murthy, P. P. N Conformational studies of myo-inositol phosphates. Carbohydr. Res. 1996, 296, 39−54. (19) Turner, B. L.; Mahieu, N.; Condron, L. M. Phosphorus-31 nuclear magnetic resonance spectral assignments of phosphorus compounds in soil NaOH−EDTA extracts. Soil Sci. Soc. Am. J. 2003, 67 (2), 497−510. (20) Bünemann, E. K.; Smernik, R. J.; Doolette, A. L.; Marschner, P.; Stonor, A.; Wakelin, S. A.; McNeill, A. M. Forms of phosphorus in bacteria and fungi isolated from two Australian soils. Soil Biol. Biochem. 2008, 40 (7), 1908−1915. (21) Turner, B. L.; Cade-Menun, B. J.; Condron, L. M.; Newman, S. Extraction of soil organic phosphorus. Talanta 2005, 66 (2), 294−306. (22) Lipkin, D.; Talbert, P.; Cohn, M. The mechanism of the alkaline hydrolysis of ribonucleic acids. J. Am. Chem. Soc. 1954, 76 (11), 2871− 2872. (23) Murthy, P. P. N. Identification of Inositol phosphates by nuclear magnetic resonance spectroscopy: Unravelling structural diversity. In: Inositol phosphates: linking agriculture and the environment; Turner, B. L., Richardson, A. E., Mullaney, E. J., Eds.; CAB International: Wallingford, UK, 2007 pp 7−22.

spectrum in this study was rather long, it could be reduced by an order of magnitude by using cryogenically cooled probes, allowing the routine use of 2D spectra. For identification of unknown compounds, the 2D HSQC experiment can be extended to a third dimension. For example, in a 3D HSQCTOCSY (total correlation spectroscopy) experiment, the third dimension would enable the chemical shifts of all hydrogens in the chemical moiety linked to P to be determined. This study represents a significant advancement in the speciation of soil organic P, which is a crucial first step toward improving understanding of the biogeochemistry of organic P. The proposed method helps to resolve signals due to orthophosphate monoesters, which are the most prevalent but difficult to assign fractions of organic P in soils. Although this methodology was tested on boreal humus soils, it would also be useful for other soils and matrices with naturally high amounts of paramagnetic ions, such as tropical soils and natural waters.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information on the experimental setup and spectra of reference compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +46 90 7865388, fax: +46 90 7869795, e-mail: jurgen. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Swedish research councils VR (GG, JS, RG) and FORMAS (JS), CMF (GG, UI), and the Kempe (AV, JS, GG) and Wallenberg (JS) foundations.

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REFERENCES

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High-Resolution Characterization of Organic Phosphorus in

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