Envlron. Scl. Technol, 1004, 28, 1521-1527
Use of Lanthanide Shift Reagents with ‘‘P FT-NMR Spectroscopy To Analyze Concentrated Lake-Water Samples Mark A. Nanny and Roger A. Mlnear’
Institute for Environmental Studies, University of Illlnois at Urbana-Champaign, 1101 West Peabody, Urbana, Illinois 6 1801
Several lanthanide shift reagents (LSR) were synthesized from praseodymium, europium, dysprosium, and terbium using ethylenediaminetetraacetic acid, trans-l,a-diarninocyclohexane-N,N,N‘,N’-tetraacetic acid, and diethylenetriaminepentaacetic acid for the purpose of separating overlapping 31P FT-NMR spectral signals of dissolved organic phosphorus compounds present in concentrated lake-water samples. The extent of change in signal position and line width of several known phosphorus compounds dissolved in pure water was examined as a function of solution pH and the molar ratio of LSR to phosphorus. Praseodymium ethylenediaminetetraacetate (PrEDTA) was determined to be the most effective LSR. Concentrated lake-water samples mixed with PrEDTA were examined with FT-NMR. The use of PrEDTA provided separation of overlapping signals, indicated that phosphorus compounds were becoming isolated from the solution environment during sample concentration, enhanced the sensitivity of certain signals by possibly acting as a T1 relaxation agent, and did not precipitate in the presence of a high concentration of humic and fulvic acids.
Introduction Phosphorus-31 Fourier transform-nuclear magnetic resonance spectroscopy (31P FT-NMR) has been used to identify and characterize phosphorus species in a variety of environmental samples. 31PFT-NMR spectra of soil samples (1-4), freshwater sediments (4,marine sediments (6),wastewater treatment plant-activated sludge (7), and lake water (8,9)indicate the presence of orthophosphate, monoester and diester phosphates, phosphonates, pyrophosphates, and polyphosphates. 31PFT-NMR spectroscopy has provided valuable insight regarding the identity and behavior of phosphorus compounds in these systems. But even so, identification of individual phosphorus species is impeded because 31PFT-NMR signals are usually close together or overlap. Characterization and identification of phosphorus species would be greatly enhanced if the 31PFT-NMR signals could be “separated”, i.e., increase the spectral region in which the signals appear so that the signals are no longer close together or overlapping. Lanthanide shift reagents have been used extensively for this purpose since 1969for lH and 13CNMR spectra (10). Lanthanide shift reagents interact with other molecules by associating with a Lewis base present on that molecule, forming a direct contact with an alcohol, carboxylate, amine, or phosphate group. The harder the base, the greater the affinity for the interaction (11). Because of direct contact, a change in the electronic structure around the associated atom occurs. Atoms that are not directly associated with the LSR also have their electronic environment perturbed by the lanthanide atom; the degree
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of perturbation is indirectly related to the distance between the atom and the LSR. The angle between the LSR and the atom also influencesthe perturbation (11). These types of “through-space” interactions are termed pseudo-contact interactions. The degree of change in the chemical shift that occurs is directly related to the amount of interaction between the atom and the LSR. There have been a few studies using 31P FT-NMR to examining phosphorus compounds with lanthanide shift reagents in organic solvents. Trimethyl phosphate, triethyl phosphate, and the phosphonate (EtO)zP(O)(Et) have been studied using Eu(N03)~ and Pr(N03)3 (12). Trimethyl phosphate, triethyl phosphite, C ~ H S P ( O C H ~ ) ~ , (CdW$“CH2CH3, (CeHs12PCH2CH3,( C H d W V W H 3 , and (CH&H20)3PS were examined with praseodymium, europium, and ytterbium complexesof 2,2,6,6-tetramethyl3,5-heptanedione (dpm), and 1,1,1,2,2,3,3-heptafluoro-77-dimethyl-4,6-octanedione(fod) (13). The compounds RzNCzH4P(C6Hs)z,where R = H or CH3, were examined with Eu(dpm)a (14),and Berkova et al. (15) examined 17 phosphonates using Eu(fod)s and Pr(fod)a. Because many lanthanide shift reagents are insoluble in water (111,such as Eu(fod)g and Eu(dpm)3, or have the possibility of causing precipitation at high pH values upon the formation of the hydroxide species (161, numerous studies have been direct toward finding lanthanide shift reagents that are soluble in aqueous solutions. Elgavish and Reubens have examined the use of ethylenediaminetetraacetic acid (EDTA) with Pr, Eu, and Gd (17-19) in aqueous solvents. Other lanthanide shift reagent (LSR) complexes that have been examined for aqueous solvents are (5‘)-carboxymethyloxysuccinic acid with Eu and Yb (20) and propylenediaminetetraacetatewith Eu (21). To date, there has been little effort to develop and examine the use of a lanthanide shift reagent for phosphorus compounds in heterogeneous aqueous solvents, Le., samples that contain numerous dissolved species ranging from inorganic salts, dissolved organic carbon, and mixtures of phosphorus compounds. The goal of this research was to develop and examine lanthanide shift reagents for such samples containing phosphorus compounds, primarily concentrated lake-water samples, and then use these lanthanide shift reagents in the 31PFT-NMR spectroscopic identification and characterization of dissolved organic phosphorus compounds in lake water. Several parameters had to be met for a lanthanide shift reagent to work effectively with environmental samples containing phosphorus species. These parameters were as follows: (1)The LSR must be soluble in an aqueous solution. (2) The LSR must be able to interact with phosphorus containing species such that it provides adequate changes in the signal position with a minimum of signal broadening. (3)The insoluble LSR-phosphorus complexes must not form. (4)If a LSR is to be used in a heterogeneous solution, the LSR must not form insoluble complexes with dissolved organic carbon species, Le., dissolved humic and fulvic acids. (5) Since the 31P FTEnviron. Scl. Technol., Vol. 28, No. 8, 1994 1521
NMR signal is usually a function of sample pH, it would be beneficial for the LSR to be stable and effective over a wide pH range. In light of these requirements and the previously mentioned studies, the following compounds were examined as potential lanthanide shift reagents: (1)ethylenediaminetetraacetic acid (EDTA) with praseodymium (Pr), europium (Eu), dysprosium (Dy), and Terbium (Tb); (2) trans-1,2-diaminocyclohexane-N,N,N’,”-tetraaceticacid (CDTA) with Pr and Eu; and (3) diethylenetriaminepentaacetic acid (DEPA) with Pr. The efficacy of each LSR was examined with several phosphorus compounds in pure water. Each LSR compound was evaluated on its ability to induce shifts in the signal position while minimizing line broadening. The dissolved organic phosphorus in concentrated lake-water samples was then examined using 31PFT-NMR and the best performing LSR.
Experimental Section Lanthanide Shift Reagent Synthesis and Characterization. A 3.5-g sample of the appropriate lanthanide chloride salt (Johnson Matthey, 99.9% purity) was dissolved in 25 mL of water and added dropwise to a stirring aqueous solution of the chelating ligand at -80 “C. The chelating ligand, EDTA (Sigma),CDTA (Sigma),or DEPA (Aldrich), was present in 10% molar excess to ensure complete complexation of the lanthanide atoms. The solution was allowedto sit overnight, and if no precipitation occurred, ethanol or some other aqueous soluble organic solvent was added to precipitate the lanthanide complexes. Usually the lanthanide complexes were quite soluble, and an organic solvent had to be added. After drying the precipitate overnight in a vacuum desiccator, an IR spectrum was obtained to ensure that the solid material was the organic complex rather than the lanthanide chloride salt. The LSR was then dissolved to form a 0.05 M aqueous solution, and a 10-fold molar excess of the chelating ligand was added. Addition of excess ligand was necessary to prevent precipitation of the LSR with inositol hexaphosphate and the concentrated lake-water samples. PrEDTA, which was the eventual LSR of choice, was recrystallized in pure water, and an elemental analysis was obtained. An elemental analysis of 20.34% C, 4.91 % H, and 4.64 % N indicated that the PrEDTA complex has nine HzO molecules associated with it. Model Phosphorus Compounds. The efficacy of the LSR was measured by examining the extent of signal shifting and line broadening as a function of the LSR/P molar ratio and pH (4.0,7.0,and 11.0). The phosphorus compounds used were inositol hexaphosphate (IHP) (Sigma),adenosine monophosphate (Eastman Kodak Co.), choline phosphate (Sigma),DNA (degraded free acid from Herring sperm, Sigma), KHzP04 (J. T. Baker), and phosphonoacetic acid (Aldrich). IHP (22), adenosine monophosphate (23), and DNA (24-26) were chosen because of evidence that these compounds exist in natural waters. Phosphonoacetic acid was used because 31PFTNMR spectroscopy has detected phosphonates in marine sediments (6) and fresh water (9). Adjustments in pH were made with additions of 0.1 M NaOH or HC1solutions. A 200-mg P/L solution of each phosphorus compound was used with the following LSR/P molar ratios: 0.00, 0.25, 0.50,0.75,1.00,1.50,and 2.00. The value, Achemical shift/ A(LSR/P molar ratio) was calculated for the region of 1522
Environ. Sci. Technol., Voi. 28, No. 8 , 1994
linear behavior using a simple least-square fit. Signal width was measured at half the height of the signal. All 31PFT-NMR were collected with a GN 300 narrow bore or a GN 300 wide bore nuclear magnetic resonance spectrometer (General Electric) at the University of Illinois, School of Chemical Sciences Molecular Spectroscopy Laboratory. All 31P FT-NMR were obtained at 121.648 Hz. Magnet shimming and signal phasing were done automatically by computer. NMR parameters were as follows: pulse width = 24 ps, delay time = 2.00 s, number of acquisitions = 200, and spectral width = 10 000 Hz, and all samples were proton decoupled. All free induction decays underwent exponential multiplication, using a linebroadening value of 5 Hz, to improve sensitivity. All chemical shift values were relative to 85% H3P04, and line widths were measured at half-height. Samples were placed in 10-mm glass NMR tubes with coaxial inserts (Wilmad Corp.) containing deuterium oxide (Sigma), which was used to lock the magnet. Sample Collection and Concentration. Lake water was collected from Crystal Lake, a small urban impoundment in Champaign County, Illinois. Lake-water samples were concentrated volumetrically several thousand-fold and fractionated according to molecular size using tangential flow ultrafiltration (UF) and reverse osmosis (RO) membranes (27). Four membranes of decreasing pore size were placed in series: 0.2 pm (to remove nondissolved material), 30 000 Da, 1000Da, and a reverse osmosis rated at 95 % NaCl rejection. The retentate of each membrane was continuously recycled while the filtrate passed into the next membrane. Further sample concentration was achieved with Amicon stirred batch concentrators employing UF membranes of 500 or 1000Da, depending upon the sample. This provided total soluble phosphorus concentrations ranging from 5 to 60 mg of P/L. Total soluble phosphorus (TSP)and soluble reactive phosphorus (SRP) concentrations were measured using ascorbic acidmolybdate reagent (28). Concentrated Lake-Water Samples. Treatment of the concentrated lake-water samples was similar to that of the model phosphorus compounds. Because of the high dissolved organic carbon (DOC) concentration in the concentrated samples (several hundred to a few thousand mg of C/L, depending upon the sample and the degree of concentration), the variability of the TSP concentration from sample to sample, and the probable interaction of the DOC with the LSR, there was no attempt to accurately control the LSR/P molar ratio; just as long as the LSR was in a high enough excess to adequately interact with the phosphorus. To each 3.0-mL sample, 0.4 mL of the 0.05 M LSR solution was added. The pH was adjusted to desired values with 0.1 M NaOH and 0.1 M HCl. 31P FT-NMR parameters were the same as with the model phosphorus compounds, except that the number of acquisitions was 30 000-40 000, and a line-broadening value of 35 Hz was used.
Results Model Phosphorus Compounds. The efficacy of each LSR was measured by examining individual known phosphorus compounds with each lanthanide complex and measuring the shift of the signal position and the amount of signal broadening that occurred (Table 1). The change in the chemical shift position is linear in the LSR/P molar
Table 1. AChemical Shift/A(LSR/P Molar Ratio) Values and Percent Increase in Signal Width upon Addition of LSR as Function of LSR Used and Sample pH for Various Phosphorus Compounds in Aqueous Solutions choline adenosine inositol phosphonoacetic phosphate monophosphate hexaphosphate orthophosphate DNA acid 0.81,33% 0.85,7% barely detectable 1.02,21% 0.71,19% PrEDTA, pH = 4 1.01,0% 0.48,22% 1.19.237% PrEDTA, pH = 7 1.57,222% 0.34,42% 0.28,5% barely detectable 2.49, low SINb 0.41; 8% PrEDTA, pH = 11 0.79,455% 0.34,119% 0.54,251% PrCDTA, pH = 4 0.21,197 % 0.28,5% 0.24,198% PrDEPA, pH = 4 0.09,139% 0.16,200% 4 3 7 , 3 0 1% EuEDTA, pH = 4 -0.55,300% barely detectable 0.01,75% EuEDTA, pH = 11 0.03,371% -0.44, low SINb -1.09,491% EuCDTA, pH 11 -1.43,712% 1.00, low SIN* 1.11,415% DyEDTA, pH = 4 1.16,275% TbEDTA, pH = 4 signal not detected signal not detected signal not detected a Rows without data indicate that measurements were not made. Low S/N indicates that the signal to noise ratio was too low (S/N < 2) for the signal width to be measured.
ratio range of 0.00-1.00. At LSR/P molar ratio values above 1.00, the chemical shift position remains fairly constant. Upon the initial addition of the LSR to the sample, there was a dramatic increase in the signal width. Further additions of LSR did not cause any appreciable increase in the signal width. Table 1provides the percent increase in the signal width upon the initial addition of the LSR. The lower and upper chemical shift limits for the shift of the monoester phosphate signals were 0.0-5.0 PPm. The CDTA and the DEPA complexes allowed excessive signal broadening to occur, and although the DyEDTA complexdid provide adequate change in the signal position, the amount of signal broadening that occurred was unacceptable. Due to the poor behavior of these and some of the other lanthanide shift reagents, they were not tested with all the model phosphorus compounds. PrEDTA, at a pH of 4,was determined to be the most effective LSR for phosphorus compounds in aqueous solvents based upon the change of the signal position with respect to the LSR/P molar ratio (Achemical shift/A(LSR/P molar ratio)) and the increase in signal width upon addition of the LSR. Concentrated Lake-Water Samples. Figure 1 illustrates that PrEDTA does separate overlapping signals in 31P FT-NMR spectra of concentrated lake-water samples. The 31PFT-NMR spectra of the concentrated lake water samples, before the addition of PrEDTA, usually consist of a large broad signal centered at a chemical shift position of 1.0-1.5 ppm. Diester and monoester phosphates appear in the region encompassed by this broad signal. Depending upon the season, a small second signal is detected in the chemical shift region of 4.0-5.0 ppm. These signals indicate the presence of monoester phosphates. Addition of PrEDTA to a RO retentate sample causes the large broad signal at 1.0-1.5 ppm to split into two broad signals, one appearing at 4.0-5.0 ppm and one remaining at 1.0-1.5 ppm. Figure 2 demonstrates that PrEDTA is probably enhancing signal sensitivity for certain compounds. Addition of PrEDTA changes the spectrum of a 30 000-Da retentate sample from a single broad signal at 1.5 ppm, with a shoulder near 0.5-0.0 ppm, to a spectrum containing three signals: the original signal at 1.5 ppm, a shifted signal at 5.5 ppm, and a third signal at 0.0 ppm. Based upon these chemical shift positions, the signal at 0.0 ppm is probably due to a diester phosphate, and the signals at 1.5 and 5.5 ppm are due to monoester phosphates. Since PrEDTA only causes signal shifts to higher chemical shift positions, the new signal at 0.0 ppm is most likely caused
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by PrEDTA enhancing the sensitivity of the signal that appears as a shoulder in the 0.5-0.0 ppm region of the original spectrum, rather than actually shifting a signal from the large 1.5 ppm signal to a lower chemical shift position. The spectra in Figure 2 also have signals in the phosphonate region of 20.0-30.0 ppm. Because of their low S/N, changes in their chemical shift position upon addition of PrEDTA are unmeasurable. Signals in this region have been detected by 31PFT-NMR in other 30 000 retentate samples (9). Figure 3 demonstrates that a much higher Pr/P molar ratio is required for concentrated lake-water samples as compared to model phosphorus compounds in pure water. Changes in the signal position of the examined phosphorus species in pure water were evident at a Pr/P ratio of 0.25Environ. Scl. Technol., Vol. 28, No. 8, 1994 1523
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0.50, while a concentrated 30 000-Da sample required a Pr/P ratio of at least 8 to produce the desired signal shifting. This indicates that other components in the concentrated sample matrix are competing with the phosphorus compounds for interaction with the LSR. The results in Figure 4 show that, unlike the results with model phosphorus compounds in pure water, PrEDTA is most effective at higher pH values for concentrated lake-water samples. A 30 000-Da retentate sample was examined at pH values of 5.6 and 10.5, both before and after the addition of PrEDTA. At a pH of 5.6, little change is seen in the 31P FT-NMR spectrum after the addition of PrEDTA. The peak of the large broad signal only shifts from 1.8to 2.2 ppm, and the spectral sensitivity is enhanced from a signal to noise ratio of 6.5-9.0. Increasing the pH to 10.5, without PrEDTA present, causes two new small signals to appear at 4.5 and 3.4 ppm, in addition to the original signal at 1.4 ppm. The appearance of the two smaller signals most likely results from signals shifting to higher chemical shift values as the pH is increased. It is well-documented (8, 29) that as the phosphate moiety becomes deprotonated the chemical shift position of its 31PFT-NMR signal increases. Based upon sample pH and signal position, previous research (8) indicates that the signal at 3.4 ppm is due to orthophosphate and that the signal at 4.5 ppm is due to a monoester phosphate. The addition of PrEDTA to the pH = 10.5 sample causes a dramatic separation of signals, causing the spectrum to appear as two very sharp and separate signals; one at 4.2 ppm and the original at 1.5 ppm. 1524
Environ. Sci. Technol., Vol. 28, No. 8, 1994
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Discussion Model Phosphorus Compounds. Since the amount of change in the chemical shift position and the extent of signal broadening are a function of the bonding between the LSR and the phosphate group, a LSR was desired that would bind strongly enough with the phosphate group so that sufficient signal shifting would occur,but only minimal signal broadening would be present. Because the model phosphorus compounds were all dissolved in only pure water, the degree of bonding was a function of the lanthanide atom, the chelating ligand, and the sample pH. Terbium and dysprosium complexes interacted with the phosphate group such that a fair amount of signal shifting was achieved, but extreme line broadening occurred. Europium, on the other hand, when complexeswith CDTA and EDTA (at a pH = 11)provided little signal shifting.
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A t pH = 4,the signal shifting ability of EuEDTA increased modestly and, as expected for europium shift reagents, caused signal shifts to occur toward lower chemical shift values, Le., upfield. Examining the praseodymium complexes (PrEDTA, PrCDTA, and PrDEPA) at pH = 4 demonstrates that EDTA is the ligand of choice, for very little signal broadening occurred and satisfactory signal shifting was attained. The performance of PrEDTA as a function of pH illustrates that pH = 4 is optimal. At higher pH values, excessive line broadening occurs with PrEDTA. This is likely the result of the interaction between the phosphate moiety and the lanthanide complex increasing as the phosphate moiety becomes deprotonated and increases its electronegativity. It is fortunate that pH = 4is the optimal pH because the range of the chemical shift region for monoester phosphate signal shifts appears to be limited to 0.0-5.0 ppm. That is, the signal is bound by an upper limit as to how far it can be shifted by the LSR. At lower pH values, most 31PFT-NMR signals of monoester phosphate compounds appear in the upfield portion of the monoester phosphate region, Le., at lower chemical shift values. Increasing the pH causes the phosphate groups to become deprotonated, and in turn, the chemical shift of their 31PFT-NMR signals increases, Le., the signals move downfield and appear at higher chemical shift values. Since PrEDTA causes the 3lP FTNMR signals of monoester phosphates to shift downfield or toward higher chemical shift values, the greatest amount of change that is available is when the signals are at low chemical shift values rather than at chemical shift values close to the upper limit of the monoester phosphate region.
Concentrated Lake-Water Samples. Figure 1shows the separation of two signals in the 31PFT-NMR spectrum of a RO retentate sample. One of these two signals is in the same chemical shift position as the original single signal before the addition of PrEDTA. This indicates that the phosphorus compounds comprising this signal are isolated from the PrEDTA. It is possible that the phosphate species comprising this signal at 1.5 ppm are such that they do not interact with the PrEDTA; e.g., DNA at pH = 11 has a Achemical shift/A(LSR/P molar ratio) equal to 0.28, thus the DNA signal hardly shifts from its position in the presence of PrEDTA. It is also possible that the 1.5ppm signal after the addition of PrEDTA results from organic phosphorus compounds that have become incorporated into structures that formed during the concentration procedure, isolating the organic phosphorus from the solution environment. This aggregate incorporation hypothesis is supported by other 31PFT-NMR data (8, 9) and by HPLC data (30). The structures which form during the concentration process are probably either humic and fulvic aggregates,condensed silicates, or possibly a mixture of both. Phosphorus compounds can bind to humic or fulvic macromolecules or to silica polymers via several mechanisms: hydrogen bonding, metal ion bridging, and hydrophilic bonding with an amphile portion of the molecule. As the sample concentration proceeds, the humic acid and molecules or the silicate polymers begin to aggregate, incorporating the sorbed phosphorus compounds into the interior of a macromolecular or colloidal structure and thus isolating it from the solution environment. The 31P FT-NMR signal that does change its Environ. Sci. Technol., Vol. 28, No. 8, 1994 1525
position upon addition of PrEDTA could be either dissolved phosphorus species or phosphorus compounds that are sorbed to the aggregate surface. Even though they are still sorbed, they have the ability to interact with the solution environment. Similar behavior has been seen with 31PFT-NMR of phospholipid bilayer vesicles in the presence of Pr ions (31). Before the addition of Pr, only one signal is detected; the combined signal of both the inner and surface phospholipid phosphate groups. After the addition of Pr, two signals are seen; one signal still in the original position due to the inner phosphate groups and a second signal at a different position due to the surface phosphate groups interacting with Pr. Figure 2 illustrates that the addition of PrEDTA to a concentrated lake-water sample selectively enhances signals. An explanation is that PrEDTA could be acting as a TI relaxation agent for the phosphorus nuclei. It is not surprising that PrEDTA could act in this manner because several transition metal complexes are used as T1 relaxation agents. The possibility that the new signal at 0.0 ppm is actually shifted upfield from the large original signal envelopeat 1.5ppm is unlikely because Pr complexes induce downfield shift rather than upfield shifts. Instead, the 0.0 ppm signal probably results from a diester phosphate for which the T1 relaxation time has decreased and, in turn, becomes visible in the 31PFT-NMR spectrum. Figure 3 demonstrates that the ratio of Pr to P required for a signal shift is greater in a concentrated lake-water sample than for phosphorus compounds dissolved in pure water. It is hypothesized that the excess of PrEDTA is required because, in addition to interacting with organic phosphorus species, the PrEDTA also interacts with the dissolved organic carbon compounds present in the sample matrix. PrEDTA interacting with the concentrated sample matrix is not unexpected, for there is a wide variety of “hard” bases present, such as carboxylic acids, carbonyl groups,hydroxyl groups, and other polar functional groups, with which PrEDTA can interact. Since PrEDTA does not precipitate in the presence of the concentrated sample matrix, PrEDTA would likely be a viable candidate as a LSR for I3C FT-NMR spectroscopy of humic and fulvic compounds. Figure 4 illustrates that for concentrated lake-water samples, PrEDTA is most effective at high pH values. It is hypothesized that the greater influence of PrEDTA upon the 3lP FT-NMR spectra at higher pH is due to the increased electrostatic attraction between the deprotonated oxygen atoms of the phosphate group and the PrEDTA. In a concentrated lake-water sample matrix, the greater electrostatic attraction of the phosphorus moiety is required in order for it to effectively compete for PrEDTA interaction given the large number of other “hard” bases present. The lack of movement of some signals to higher chemical shift positions upon increasing pH in the original sample indicates that some of the phosphorus is isolated from the solution environment. As mentioned previously, the 31P FT-NMR signal position of phosphorus compounds is a function of pH; as the pH is increased, the chemical shift position of the signal also increases. The hypothesis that phosphates is being isolated into the interior of aggregates during sample concentration is further supported by the splitting of the 3lP FT-NMR signal into two signals upon addition of PrEDTA (one at 4.2 ppm and the other at the original position of 1.5 ppm). The lack of movement in 1528
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the signal at 1.5 ppm indicates that a major portion of the phosphorus is isolated and, thus, prevented from interacting with PrEDTA, while the signal shifted to 4.2 ppm results from phosphorus which is either dissolved in solution or sorbed to the aggregate surface and interacting with PrEDTA. Conclusion PrEDTA was found to be an effective LSR for 31PFTNMR spectroscopy of aqueous samples, primarily concentrated lake-water samples. Not only did PrEDTA provide separation of overlapping signals, but it also reinforced the hypothesis that phosphorus compounds are becoming incorporated into aggregate structures during sample concentration. These phosphorus compounds can be either incorporated into the aggregate interior, sorbed to the aggregate surface, or dissolved in solution. PrEDTA may also have the potential to enhance 31PFT-NMR spectral sensitivity by acting as a T1 relaxation agent. The fact that PrEDTA does not precipitate in the presence of a highly concentrated sample matrix indicates that I2Cenriched PrEDTA could be a tool to aid in 13C FT-NMR analysis of humic acid and fulvic compounds. Acknowledgments This work was supported through the grant 31Phosphorus Fourier Transform Nuclear Magnetic Resonance Analysis of Organic Phosphorus in the Hydrosphere from the United States Geological Survey (Grant 1434-92-G2260). Literature Cited Tate, K. R.; Newman, R. H. Soil Biol. Biochem. 1982, 14, 191-196. Hawkes, A. E.; Powlson, D. S.;Randall, E. W.; Tate, K. R. J. Soil Sci. 1984, 35, 35-45. Condron, L. M.; Goh, K. M.; Newman, R. H. J. Soil Sci. 1985,36, 199-207. Condron, L. M.; Frossard, E.; Tiessen, H.; Newman, R. H.; Stewart, J. W. B. J . Soil. Sci. 1990, 41, 41-50. Oluyedun, 0. A.; Ajayi, S. 0.; van Loon, G. W. Sci. Total Environ. 1991, 106, 243-252. Ingal, E. D.; Schroeder, P. A.; Berner, R. A. Geochim. Cosmochim. Acta 1990,54, 2617-2620. Jing, S. R.; Benefield, L. D.; Hill, W. E. Water Res. 1992, 26, 213-223. Nanny, M. A.; Minear, R. A. In Environmental Chemistry of Lakes and Reservoirs; Baker, L., Ed.; Advances in Chemistry Series;American ChemicalSociety: Washington, DC, 1994. Nanny,M. A.; Minear,R. A. Natl. Meet.-Am. Chem. SOC., Diu. Enuiron. Chem. 1993, 33, 183-185. Hinkley, C. C. J. Am. Chem. SOC.1969, 91, 5160. Morrill, T. C. In Lanthanide Shift Reagents in Stereochemical Analysis; Morrill, T. C., Ed.; VCH Publishers: New York, 1986; pp 1-18. Sanders, J. K. M.; Williams, D. H. Tetrahedron Lett. 1971, 30, 2813-2816. Mandel, F. S.; Cox, R. H.; Taylor, R. C. J. Magn. Reson. 1974,14, 235-240. Taylor, R. C.; Walters, D. B. Tetrahedron Lett. 1972, I, 63-66. Berkova, G. A.; Zakharov, V. I.; Smirnov, S. A.; Morkovin, N. V.; Ionin, B. I. 2%. Obschch. Khim. 1977, 47, 1431-2. Sinha, S. P. Europium; Springer-Verglag: Berlin, 1967. Elgavish, G. A,; Reuben, J. J. Am. Chem. SOC.1976, 98, 4755-4759. Reuben, J. J. Am. Chem. SOC.1976,98, 3726-3728.
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Received for review December 9, 1993. Revised manuscript received April 4, 1994. Accepted April 11, 1994.'
*Abstract published in Advance ACS Abstracts, May 15, 1994.
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