Optimized procedure for the determination of P species in soil by liquid-state 31P-NMR spectroscopy
© Li et al.; licensee Springer. 2015
Received: 13 October 2014
Accepted: 12 December 2014
Published: 18 March 2015
Liquid-state 31P-NMR spectroscopy becomes progressively an important role for studying phosphorus (P) dynamics in soil. Soils of different origin and organic matter content were used to optimize sample preparation and re-dissolution procedures to improve characterization of P species in soil by 31P-NMR spectroscopy. The efficiency of P extraction from an untreated fresh soil was compared to that from freeze-dried and air-dried soil samples.
A freeze-drying pretreatment not only provided the greatest extraction yields of total and organic P from both farmland and forest soils but also enhanced the intensity of signals for inorganic and organic P species in 31P-NMR spectra, except for polyphosphates. Re-dissolution of freeze-dried soil extracts in relatively dilute alkaline solution and addition of a small aliquot of concentrated HCl to the NMR tube prior to analysis improved the quality of NMR spectra. Finally, the visibility of relatively weak P signals, such as for phosphorus diesters, phosphonates, polyphosphate, phospholipids, and DNA were reproducibly enhanced when 31P-NMR spectra were generated after at least 15 h of acquisition time.
The optimized procedure presented here ensured the greatest detectability of inorganic and organic P species by liquid-state P-NMR spectroscopy in soil extracts.
KeywordsLiquid-state 31P-NMR spectroscopy Soil pretreatment before extraction Re-dissolution of P extracts Inorganic and organic P species
Solution-state 31P-NMR spectroscopy represents a useful tool to follow the cycle of phosphorus (P) in the environment . However, due to the relatively low concentration of P compounds present in environmental compartments (soils and sediments), natural organic matter (NOM), and microbial biomass, the procedures for sample preparation for the detection of P content by NMR require a careful setup of experimental conditions, including the removal of paramagnetic species [25,27,11]. In fact, an optimization of sample pretreatment and NMR experimental parameters become critical to improve the final quality of 31P NMR spectra and minimize variations in the determination of P compounds [7,18,21].
Several works were devoted to improve procedures in order to obtain meaningful and reproducible liquid-state 31P-NMR spectra. In particular, the extracting solution may affect the quantitative solubilization of P species [3,20]. Cade-Menun and Preston  found that an aqueous solution containing both NaOH and EDTA extracted the greatest diversity of P forms and the largest percentage of total phosphorus in soil. Recently, it has been shown that a 0.25 M NaOH and 0.05 M EDTA extracting solution was most efficient in solubilizing a maximum range of organic P forms from sediments [26,28], while minimizing the co-extraction of Fe (III) and Mn (II) metals, which may significantly affect the resolution and intensity of 31P NMR spectra [14, 1]. Addition of HCl or Chelex to extracts was also found to be useful in reducing interferences [13,24]. Recently, the capacity of bicarbonate dithionate (BD), EDTA, Chelex-100, and 8-hydroxyquinoline (HQ) were compared in order to prevent solubilization of Fe and Mn from environmental samples . It was found that HQ was the most efficient treatment, although it was also observed that excessive HQ in the extracting solution produced an insoluble residue during subsequent freeze-drying of extracts.
Extracted matter is normally concentrated prior to solution-state 31P-NMR analysis by either rotary evaporation or freeze-drying . However, the latter procedure was reported to lead to considerable degradation of polyphosphate and glucose 6-phosphate and significant reduction of NMR signals of phospholipids extracted from a calcareous sediment . The disappearance of phopholipids and pyrophosphate in extracts from a sediment from Taihu Lake in China was also noted after freeze-drying of samples . On the other hand, while rotary evaporation of aqueous extracts is a time-consuming enrichment procedure as compared to freeze-drying, it also fails to sufficiently enrich P content in samples with very low P concentration. This becomes evident for soil-water leachates or dissolved organic matter, for which a 1,000- to 2,000-fold enrichment is usually required [4,19,8]. In these cases, sample freeze-drying followed by re-dissolution in the proper solution for NMR analysis may be the most useful method.
The aim of this work was to set up a reproducible procedure to extract and concentrate organic P species solubilized from soil samples of different organic matter content. Sample extraction and re-dissolution and spectral acquisition time were assayed to optimize detection of P species by 31P-NMR spectroscopy.
Materials and chemical properties
A surface (0 to 20 cm) layer of an alluvial clayey loam agricultural soil was collected at the Experimental Farm of the University of Naples Federico II at Castel Volturno (CE). This soil was used without (sample A) and with (sample B) addition of 125 q ha−1 of farm compost as P source. A volcanic silty loam soil sample (0 to 20 cm) was collected from the forested area around the Royal Palace of Portici (NA) (sample C). The overlaying litter material was collected from the same forest soil (sample D). The compost used here to amend the agricultural soil and extract P (sample E) was derived from a pilot composting plant build at the Experimental Farm of the University of Naples Federico II at Castel Volturno (CE). The pH of the samples was determined by a pH meter (HANNA Instruments, Padova, Italy) in a soil:water suspension of 1:2.5 ratio. The C, H, and N content of samples was determined by an elemental analyzer (Eager 200, Fisons, Ipswich, UK), while the content of the organic matter (OM) was achieved by the Walkley-Black method. Organic P was calculated in soils and compost by difference in P content before and after ignition at 550°C and followed by extraction with 1 M HCl . Total P in both original sample materials and their extracts (TPE) was obtained by first digesting samples in concentrated H2SO4 and HClO4 for 2 h and the measuring total dissolved P in digested solutions by a UV-vis spectrometer (Lambda 25, Perkin Elmer, Waltham, MA, USA) by the molybdate colorimetry method .
Sample treatments and preparation for NMR acquisition
Soils, litter, and compost samples used in this study were cleaned from plant remains, passed through a 1-mm sieve, and divided into three aliquots. One aliquot (fresh) was subjected to direct extraction of P species, the second aliquot was first freeze-dried, and a third aliquot was left to air-dry (20°C to 25°C) for 2 weeks before extraction.
About 4 g of each aliquot of soil and compost was mixed with 0.25 M NaOH and 0.05 M EDTA solution at a 1:8 soil:solution ratio and shaken at 20°C for 16 h. The mixture was centrifuged at 12,000 g (20°C) for 30 min, and the clear supernatant solution was collected into a 50-mL centrifuge tube. An amount (1.1 mL for each 10 mL extract) of a 3% aqueous 8-hydroxyquinoline (HQ) solution was added to the tube to remove Fe and Mn metals . The solution pH was adjusted to 9.0 ± 0.1, and, after at least 30 min, the solution was centrifuged. Thirty millimeters of the supernatant was separated, frozen at −80°C, and freeze-dried. The resulting powder was finely ground before further analysis.
The freeze-dried powder obtained with samples extraction was divided into two aliquots of about 1 g each. One aliquot was re-dissolved in 2 mL of a 1 M NaOH solution, while a second aliquot was re-dissolved in 2 mL of a 10 M NaOH solution. After about 2 h, the samples were centrifuged at 12,000 g for 30 min. Then, 930 μL of the supernatant were transferred into a 5-mm NMR tube, together with a deuterated solution of metylendiphosphonic acid-P,P′-disodium salt (MDP) (Epsilon Chimie, Guipavas, France) as internal standard (δ = 16.62 ppm), for a final 2.65 mM concentration. Moreover, in order to flocculate possible suspended particles, which may be present in the redissolved extracts and interfere with NMR analyses, 100 μL of concentrated HCl was further added to the NMR tube prior to acquisition of 31P-NMR spectra. All samples were prepared in triplicates for NMR analyses.
The P containing solutions were analyzed by a 400-MHz Bruker Avance spectrometer (Bruker AXS, Inc., Madison, WI, USA), equipped with a 5-mm Bruker broadband inverse (BBI) probe, operating at 31P resonating frequency of 161.81 MHz, applying 6 s initial delay and a 45° pulse length ranging between 8.5 and 9.5 μs (−2 dB power attenuation). The 31P-NMR spectra of sample solutions were acquired from 5, 10, and 15 h of acquisition time. The 5 h of acquisition time comprised 3,000 transients and 5,461 time domain points, while the 31P spectra for the 15-h acquisition time consisted in 9,000 transients, 16,384 time domain points, and a spectral width of 250 ppm (40,650 Hz). Except for the samples used to determine the best acquisition time, the rest of the 31P-NMR spectra of this study were acquired with 15 h of acquisition time. An inverse gated pulse sequence, with 80-μs length Waltz16 decoupling scheme, with around 15.6 dB as power level, was employed to decouple phosphorous from proton nuclei . All spectra were baseline-corrected and processed by MestReC software (v. 126.96.36.199). The free induction decays (FID) for solution-state 31P-NMR spectra were transformed by applying a fourfold zero filling and a line broadening of 6 Hz. Signals were assigned according to literature [6,15,22,12]. The relative proportions of P species were estimated by integration of 31P-NMR spectral peaks and expressed in respect to the concentration of total P in extracts (TPE).
Results and discussion
Some properties of the sample materials used in this study
(mg g −1 )
(mg g −1 )
(mg g −1 )
(mg g −1 )
(mg g −1 )
Farmland soil added with compost
Phosphorus in extracts
Total P (TP E ) and organic P (OP E ) in soil extracts (mg kg −1 ) and relative distribution of P
18 to 24
5.3 to 3.3
1.6 to −0.3
−1.1 to −1.6
−19 to −21
Farmland soil added with compost
Air-drying decreased considerably the signals for P monoesters, phospholipids (PL), DNA, and polyphosphates in farmland soil, either with or without compost addition, while these species were not as extensively decreased in the fresh soil (Table 2). Advanced microbial degradation of organic P may be the cause of the reduced values in air-dried samples.
Influence of re-dissolution
The powder materials resulting from freeze-drying soil extracts required to be re-dissolved prior to analysis by liquid-state 31P-NMR spectroscopy. We compared re-dissolution of extracts in either a 1 M or a 10 M NaOH solution.
It was also noted that a displacement of signals chemical shifts in the 10 M NaOH solution (Figure 2). The chemical shift of pyrophosphate was approximately 0.5 ppm downfield as compared to the sample re-dissolved in 1 M NaOH, while those of polyphosphates and DNA were 0.6 ppm downfield. Signals of orthophosphate monoesters were found in the 5.3 to 3.3 ppm range in 1 M NaOH, while they were reduced in a smaller 5.1 to 3.5 ppm interval in 10 M NaOH.
It was also observed that complete sample re-dissolution in alkaline solutions may form suspended particles, which may reduce spectral quality and undermine TP and OP detectability. To alleviate this problem, samples re-dissolved in 1 M NaOH were added with 100 μL HCl before undergoing NMR analyses. This procedure was found to decrease the amount of particles suspended in the alkaline solution and successful in providing more intense signals in 31P-NMR spectra (Figure 2).
NMR acquisition time
Solution-state 31P-NMR spectra obtained in this work revealed that soil and compost samples which were freeze-dried before NMR analysis ensured a larger detectability of total and organic P than samples undergone NMR experiments either after air-drying or directly without pretreatment. Thus, a freeze-drying pretreatment represents the method of choice for natural samples with low P concentration and whose NMR detectability of P signals is poor.
We also showed that re-dissolution of extracts from natural samples into 1 M NaOH solution makes P signals more visible by 31P-NMR spectroscopy than when a stronger alkaline solution is used. Furthermore, when suspended particles may interfere with spectral quality, addition of 100 μL of concentrated HCl to the NMR tube containing the alkaline re-dissolved extracts, the intensity of NMR signals are improved. Finally, we showed that at least 15 h of NMR acquisition time were needed in order to reach the sufficient intensity of P signals and enable distinction among sample treatments. We believe that the procedure optimized here to obtain 31P-NMR spectra for natural samples such as soil and humus-rich materials (litter and compost) may become useful to foster studies on total and organic P dynamics in the environment.
This work was supported by the EU-7FP project BIOFECTOR (grant agreement no. 312117). The scientific work at CERMANU of the first author was sponsored by the National Natural Science Fund Projects of China (No. U1133604), China Scholarship Council (CSC).
- Ahlgren J, De Brabandere H, Reitzel K, Rydin E, Gogoll A, Waldebäck M (2007) Sediment phosphorus extractants for phosphorus-31 nuclear magnetic resonance analysis. J Environ Qual 36:892–898View ArticlePubMedGoogle Scholar
- Aspila KI, Agemian H, Chau ASY (1976) A semi-automated method for the determination of inorganic, organic and total phosphate in sediments. Analyst 101:187–197View ArticlePubMedGoogle Scholar
- Cade-Menun BJ, Liu CW, Nunlist R, McColl JG (2002) Soil and litter phosphorus-31 nuclear magnetic resonance spectroscopy: extractants, metals, and phosphorus relaxation times. J Environ Qual 31:457–465View ArticlePubMedGoogle Scholar
- Cade-Menun BJ, Navaratnam JA, Walbridge MR (2006) Characterizing dissolved and particulate phosphorus in water with 31P nuclear magnetic resonance spectroscopy. Environ Sci Technol 40:7874–7880View ArticlePubMedGoogle Scholar
- Cade-Menun BJ, Preston CM (1996) A comparison of soil extraction procedures for 31P-NMR spectroscopy. Soil Sci 63:770–785View ArticleGoogle Scholar
- Cade-Menun BJ (2005) Characterizing phosphorus in environmental and agricultural samples by 31P nuclear magnetic resonance spectroscopy. Talanta 66:359–371View ArticlePubMedGoogle Scholar
- Cheesman AW, Turner BL, Inglett PW, Reddy KR (2010) Phosphorus transformations during decomposition of wetland macrophytes. Environ Sci Technol 44:9265–9271View ArticlePubMedGoogle Scholar
- Condron LM, Frossard E, Newman RH, Tekeley P, Morel J-L (1997) Use of 31P NMR in the study soils and the environment. In: Nanny MA, Minear RA, Leenheer JA (ed) Nuclear magnetic resonance spectroscopy in environment chemistry. Oxford University Press, New York. USAGoogle Scholar
- Ding SM, Xu D, Li B, Fan CX, Zhang CS (2010) Improvement of 31P NMR spectral resolution by 8-hydroxyquinoline precipitation of paramagnetic Fe and Mn in environmental samples. Environ Sci Technol 44:2555–2561View ArticlePubMedGoogle Scholar
- Ding SM, Bai XL, Fan CX, Zhang L (2010) Caution needed in pretreatment of sediments for refining phosphorus-31 nuclear magnetic resonance analysis: results from a comprehensive assessment of pretreatment with ethylenediaminetetraacetic acid. J Environ Qual 39:1668–1678View ArticlePubMedGoogle Scholar
- Dong L, Yang Z, Liu X, Liu G (2012) Investigation into organic phosphorus species in sediments of Baiyangdian Lake in China measured by fractionation and 31P NMR. Environ Monit Assess 184:5829–5839View ArticlePubMedGoogle Scholar
- Doolette AL, Smernik RJ, Dougherty WJ (2011) Overestimation of the importance of phytate in NaOH-EDTA soil extracts as assessed by 31P NMR analysis. Org Geochem 42:955–964View ArticleGoogle Scholar
- Makarov MI, Haumaier L, Zech W, Malysheva TI (2004) Organic phosphorus compounds in particle-size fractions of mountain soils in the northwestern Caucasus. Geoderma 118:101–114View ArticleGoogle Scholar
- Makarov MI, Haumaier L, Zech W, Marfenina OE, Lysak LV (2005) Can 31P NMR spectroscopy be used to indicate the origins of soil organic phosphates? Soil Biol Biochem 37:15–25View ArticleGoogle Scholar
- Makarov MI, Haumaier L, Zech W (2002) Nature of soil organic phosphorus: an assessment of peak assignments in the diester region of 31P NMR spectra. Soil Biol Biochem 34:1467–1477View ArticleGoogle Scholar
- Mazzei P, Piccolo A (2012) Quantitative evaluation of noncovalent interactions between glyphosate and dissolved humic substances by NMR spectroscopy. Environ Sci Technol 46(11):5939–5946View ArticlePubMedGoogle Scholar
- Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27:31–36View ArticleGoogle Scholar
- Reitzel K, Ahlgren J, DeBrabandere H, Waldebäck M, Gogoll A, Tranvik L, Rydin E (2007) Degradation rates of organic phosphorus in lake sediment. Biogeochemistry 82:15–28View ArticleGoogle Scholar
- Toor GS, Condron LM, Di HJ, Cameron KC, Cade-Menun BJ (2003) Characterization of organic phosphorus in leachate from a grassland soil. Soil Biol Biochem 35:1317–1323View ArticleGoogle Scholar
- Turner BL, Cade-Menun BJ, Condron LM, Newman S (2005) Extraction of soil organic phosphorus. Talanta 66:294–306View ArticlePubMedGoogle Scholar
- Turner BL, Condron LM, Richardson SJ, Peltzer DA, Allison VJ (2007) Soil organic phosphorus transformations during pedogenesis. Ecosystems 10:1166–1181View ArticleGoogle Scholar
- Turner BL, Mahieu N, Condron LM (2003) Phosphorus-31 nuclear magnetic resonance spectral assignments of phosphorus compounds in soil NaOH-EDTA extracts. Soil Sci Soc Am J 67:497–510View ArticleGoogle Scholar
- Turner BL, Newman S (2005) Phosphorus cycling in wetland soils: the importance of phosphate diesters. J Environ Qual 34:727–733View ArticleGoogle Scholar
- Turner BL (2004) Optimizing phosphorus characterization in animal manures by solution phosphorus-31 nuclear magnetic resonance spectroscopy. J Environ Qual 33:757–766View ArticlePubMedGoogle Scholar
- Turner BL (2008) Soil organic phosphorus in tropical forests: an assessment of the NaOH-EDTA extraction procedure for quantitative analysis by solution 31P NMR spectroscopy. Eur J Soil Sci 59:453–466View ArticleGoogle Scholar
- Xu D, Ding SM, Li B, Jia F, He X, Zhang CS (2012) Characterization and optimization of the preparation procedure for solution P-31 NMR analysis of organic phosphorus in sediments. J Soils Sediments 12:909–920View ArticleGoogle Scholar
- Zhang R, Wu F, He Z, Zheng J, Song B, Jin L (2009) Phosphorus composition in sediments from seven different trophic lakes, China: a phosphorus-31 NMR study. J Environ Qual 38:353–359View ArticlePubMedGoogle Scholar
- Zhang WQ, Shan BQ, Zhang H, Tang W (2013) Assessment of preparation methods for organic phosphorus analysis in phosphorus-polluted Fe/Al-rich Haihe river sediments using solution 31P-NMR. PLoS One 8:e76525View ArticlePubMed CentralPubMedGoogle Scholar
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