Environ. Sci. Technol. 1992, 26, 199-204
An HPLC Postcolumn Reaction System for Phosphorus-Specific Detection in the Complete Separation of Inositol Phosphate Congeners in Aqueous Samples Catherine M. Clarkln,t*tRoger A. Minear,*,+Seungdo Kim,+ and Jerry W. Elwood5
Institute for Environmental Studies, University of Illinois, 1101 West Peabody Drive, Urbana, Illinois 6 1801
A method for the separation of all the inositol phosphate congeners has been developed and demonstrated. Phosphorus-specific detection is achieved by postcolumn illumination by high-intensity ultraviolet radiation with resultant formation of reduced phosphomolybdate by flow injection of an ascorbic acid/molybdate reagent and spectrophotometric detection. Separations are achieved with a complex gradient between 0.1 and 0.5 N NaCl and constant concentration of Na4EDTA (0.5 mM). Submilligram per liter levels of phosphorus can be detected with 50-250-pL injections. Preliminary results with field samples are presented along with a proposed sample concentration procedure necessary to obtain sufficientphosphorus levels.
Introduction In spite of the importance of phosphorus to aquatic ecosystems, where it is frequently the limiting nutrient, information regarding the chemical speciation remains minimal. Because of the extremely low concentrations of total soluble phosphorus (TSP) and soluble reactive phosphorus (SRP), frequently presumed to be principally orthophosphate, use of many analytical techniques for speciating phosphorus is precluded without prior concentration. The work reported herein is part of an effort to develop analytical techniques and concentration procedures that in combination will aid in obtaining better information on soluble phosphorus speciation in aquatic systems. One group of compounds that may have potential significance in the aquatic ecosystem is the family of inositol phosphates. The soil and plant linkage to the aquatic environment are the basis for suspecting the presence of inositol phosphates in natural waters. The inositol phosphates, especially the hexaphosphate congener, have been of interest to food and soil chemists (1-3). Inositol hexaphosphate has been shown to represent from 2 to 59% of base-extractable soil organic phosphorus (3). This knowledge and interest in the importance of phosphorus as a growth-limiting nutrient in aquatic ecosystems have led to assays of the inositol phosphates in freshwater sediments ( 4 , 5 ) and subsequently in the water column itself (6-9). However, only circumstantial evidence has been presented in support of inositol phosphate presence in the aquatic dissolved organic phosphorus compartment. No information has been presented for soil or groundwaters or rainfall/throughfall samples. Analytical methods for determination of inositol phosphates have been summarized by numerous authors (10-13). Attempts at applying HPLC as an analytical method for separation, detection, and quantification of inositol phosphates have been presented (14-18) but +Universityof Illinois. Present address: ENVIRON Corp., 4350 North Fairfax Drive, Suite 300, Arlington, VA 22203. 5 Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6036. 0013-936X/92/0926-0199$03.00/0
without actual separation of the various congeners. Recently, ion-pair reversed-phase HPLC has been used for cereal grain assays (11, 19-25). However, the organic reagent content presents analytical interferences for phosphorus-specific detection, and other detection approaches are not of sufficient sensitivity for nonradiolabeled compounds for aquatic environmental work. Similarly, more efficient ion-exchange HPLC separation has been recently reported (26,27), but again detection was either by radiometry for 32Por tritiated components or by relatively insensitive and/or nonspecific methods. Earlier work by Minear et al. (28,29) has demonstrated the separation capability of ion-exchange HPLC for inositol phosphates using an aqueous eluent with a low organic content. Use of manual analysis of collected fractions substantiated the separation capabilities and clearly demonstrated the need for a real-time phosphorus-specific detection method that could function in-line. This paper reports on the development of a postcolumn reaction system coupled with a flow injection analyzer that is designed to accomplish this objective. Complete separation of all six inositol phosphates has been achieved using an anion-exchange column. Other compounds can also be separated with the technique, and preliminary results on highly concentrated field samples demonstrate the utility of the method.
Experimental Section Apparatus. A schematic diagram of the HPLC system is shown in Figure 1. The reagent pumps were Millipore Waters Model 510 and gradient was controlled by a Millipore Waters Model 680 automated gradient controller. The injector was a Rhenodyne high-pressure rotary manual loop injector with 250-pL-volume loop. An anion-exchange Aminex A-27 column, 250 X 4 mm, from Biorad Laboratories was used for separation. A guard column, also from Biorad, 40 X 4 mm, was in-line with all samples. The columns were stored in 0.1 M NaC1/0.5 mM tetrasodium ethylenediaminetetraacetate (Na,EDTA) between uses. A postcolumn reactor coupled with a flow injection analyzer allowed for color development and subsequent spectrophotometric detection. The reactor was a UV oxidizer (see Figure 2). The photoreactor platform was wrapped with 100 or 200 f t of Teflon microbore tubing through which eluent from the column would flow. The UV lamp was an Ace-Hanovia photochemical lamp with a power of 450 W. The apparatus was shielded by a metal chimney and cooled with an exhaust fan at the top. Exposure of organic phosphate compounds to UV light as they passed through the photoreactor converted compounds to orthophosphate plus an organic product. The flow injection analyzer converted orthophosphate to a reduced phosphomolybdate product, which was detected in the visible region at 668 nm. Constraints of the HPLC detector range prevented use of the more sensitive absorption at 885 nm. The ascorbic acid/molybdate reagent of the Murphy and Riley method (30) for orthophosphate assay was pumped via a SSI Model 222 HPLC pump. The
0 1991 American Chemical Society
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1 6
1 20
40
60
80
0
Tempratwe ( "C)
Fbun 3. Effect of bath temperature on response (total flow 0.00 mllmin. tubing diameter 0.3 mm to 0.012 In.).
Fan
"
Flguro 2. Commerclal platform reactor (Ace Glass. 7892)wim mlcrobore Teflon tubing wrapped ebwt posts.
reaction tmk place in an additional 100 f t of Teflon microbore tubing immersed in a Napco Model 210A water bath. The phosphomolybdate derivative was detected using a Linear Model UVIS 200 detector. Peaks were recorded simultaneously on a strip chart recorder and a Nelson analytical data acquisition system. The Nelson system consisted of a Nelson analytical 760 Series interface and an IBM personal computer with Nelson software. Chemicals. Standards were made to concentrations of 50-100 mg of P / L in distiUed/deionized water or in HPLC grade water from American Burdick and Jackson. The inositol phosphate (IP) compounds studied were inositol monophosphate (IMP), diphosphate (1,4-IDP), triphosphate (1,3,4ITriP), tetraphosphate (1,4,5,6-ITetP), pentaphosphate (IPP),and hexaphosphate conger (IHP). All compounds were members of the myo-inositol series. 2-IMP and IHP were obtained from Sigma; the other IP congeners were obtained from Calhiochem. Standards were stored in the refrigerator, except IDP, ITriP, and ITetP. which were keut frozen. Stock solutions were diluted needed. Orthophosphate standards were made using KHzPOl from J. T. Baker. Other compounds examined were adenosine 5'-monophosphate (AMP, Eastman, Kodak Co.), ,6-elvcerouhosDhate (6-GP. Fisher Scientific Co.. 4% a). and D N A ' ( S i a Chemical'Co., degraded, herring spemj: Concentrations of phosphate in each standard were conf i e d using the Murphy and Riley method (30)for total soluble (TSP) and soluble reactive phosphorus (SRP). Procedures. For optimization of color development in the bath, the A-27 HPLC column and the oxidizer unit were removed from the system and replaced by -1 m of I _
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Environ. Scl. Technd.. Vol. 26,No. 1. I992
0.022 in.-diameter Teflon tubing. The substrate used was orthophosphate and the solvent wm water. The reagent used in the flow injection analyzer was the same as used for TSP and SRP analysis. To determine the amount of conversion to Orthophosphate in the UV oxidizer unit, the system was run as shown in Figure 1, but without the column in-line. For HPLC runs, a gradient ranging from 0.1 M NaC1/0.5 mM NQEDTA to 0.5 M NaCl/0.5 mM Na,EDTA was used. For HPLC runs flow rate was constant at 0.5 mL/min with molybdate reagent flow rate at 0.1 mL/min. NQEDTA prevents on-column hydrolysis of organic phosphates.
Results and Discussion Optimization of Color Development. In preliminary studies (30, we found a leveling off of the response with reaction temperature at 60 "C. This result was c o n f i i e d as shown in Figure 3. However, when reaction temperature was elevated beyond 60 'C, the signal response showed a rapid increase after 75 O C . By operating at 55 O C , in the middle of the plateau region, small variations in temperature should not have a significant effect on response. Another important factor in achieving optimum color developments was residence time in the bath This could be varied by changing the size of tubing or the flow rate through the bath reador. Tubing was 30.5 m in length and either 0.81 or 0.30 mm in diameter. With a solvent flow of 0.5 mL/min and a molybdate reagent flow of 0.1 mL/min, residence times in the bath with these tubings were 26.4 and 3.7 min, respectively. Due to excessive end-mixing leading to peak broadening with the 0.81mm-diameter tubing, the 0.30-mm-diameter tubing is recommended for the reaction in the water bath. Since peaks will become broader when oxidizer and column are added to the system, it is important that the bath reador alone does not cause excessive broadening. Peak width for the O.&mm-diameter tubing was 1.6 times that for the 0.3-mm-diameter tubing. For qualitative work, peak width would be the more important factor rather than completeneas of reaction, i.e., response factor. If necessary, increasing the length of the 0.3-mm-diameter tubing in the bath may lead to a more complete reaction with a minimum of peak broadening. Use of crocheted tubing has been reported as a means to reduce the mixing problems (32,33)that produce peak
7.0
1 t
I Ratio of Reagent (%)
I rn
11.1%
154%
20%
3.0
'
0
0.2
I
I
1
I
I
0.3
0.4
0.5
0.6
0.7
33%
0.8
Flow Rate (rnL/rnin)
Flgure 4. Effect of flow rate on response (54 OC, tubing diameter 0.30 mm).
Flgure 6 . Standard curve for each reagent ratio (55 OC, 50-pL sample).
h
6.00
4
4.00
%
a"
3'00 2.00 1.00
At = 3 minutes
1
I/ 1
At = 2.5 minutes
_I_ic/j_
0.00
0
10
20
30
40
50
60
Percent Reagent (%)
Figure 5. Effect of reagent ratio on response (54 OC, tubing diameter 0.30 mm, flow 0.6 mL/min) for 1 mg of PO4 P/L.
broadening. However, when it was used to wrap the Teflon tubing around the reactor frame, we experienced frequent leaking problems with the tubing. Flow rates of both solvent and molybdate reagent were varied, maintaining a constant reagent to solvent ratio of 20%. Lower total flow rates gave longer residence times in the bath and thus increased response. See Figure 4. The 0.5 mL/min flow rate for solvent was dictated by a pressure drop with the column in-line. It was fortuitous that this gave an adequate balance between peak area enhancement and peak-broadening effects. When the ratio of solvent flow to reagent flow was changed but the overall flow rate was kept constant, residence time in the bath was kept constant, dilution effects were eliminated, and the optimum reagent ratio could be determined. With 1mg of P / L orthophosphate, reagent ratios from 11 to 50% yielded a relatively constant peak area (Figure 5 ) , in agreement with preliminary results [Minear et al. (31)]. At higher concentrations of phosphorus, higher reagent ratios give better response and extend the dynamic range, as shown in Figure 6. A 20% ratio is recommended unless large concentrations of orthophosphate are present. With a solvent flow of 0.5 mL/min, this means a reagent flow of 0.1 mL/min. With repeated injections of orthophosphate, allowing specific loop-clearing times, time for adequate separation could be determined. Without column or oxidizer in-line, clearing times of 1.5 min and greater gave good separation; greater than 2.5 min gave complete separation, as shown in Figure 7. Oxidizer Performance. To optimize conversion of organic phosphates to orthophosphate, the most important
r\ L
At = 1 minute
J 0
10
Time (minutes)
Figure 7. Peak separations obtained for successive Injections at indicated intervals. Column was not in-line.
factor to consider is time of exposure to UV radiation. This could be varied by changing the flow rate through oxidizer tubing or by changing tubing diameter, in which case other factors may effect conversion,such as penetration of light through the entire diameter of the tubing. Optimal flow rate was determined by varying both solvent and reagent flow rates while keeping a constant reagent to solvent ratio of 20%. Under these conditions, both oxidizer and bath residence times varied. Lower flow rates resulted in larger peak areas, but these were accompanied by significant peak broadening. A solvent flow rate of 0.5 mL/min was determined to be acceptable once again. Teflon tubings of 0.30, 0.68, and 0.81 mm in diameter and 30.5 m in length were tested to determine peak response and peak-broadening effects. The two larger diameter tubings showed excessive peak broadening due to end-mixing, as demonstrated in Figure 8. This figure shows the result of duplicate 250-pL injections of 3 mg of P / L IHP using a loop-clearingtime of 3 min. This demonstrates the increase in ability to distinguish peaks of similar retention times with increasingly smaller diameter tubing. The lower residence time with the smaller tubing does not result in loss of efficiency because breakdown of most compounds is near 100% when sample concentrations are approximately 1mg of P/L. For compounds with Environ. Sci. Technol., Vol. 26,No. 1, 1992
201
A 0.81 mm diameter
L
0.68 mm diameter
Concentration (rngPlL)
Figure 9. Percent conversion to orthophosphate as a functlon of concentration for selected compounds. 60
I
50
0 Time (minutes)
Flgure 8. Peak-broadening effects of larger tubing (clearing time 3 min, 3 mg of IHP P/L, 20-pL sample).
a
IMP DNA
Table I. Comparison of Selected Compound Responses in Water and Aqueous Gradient Solvents Used for Separations compd
concn, mg of P/L
PO4 IHP IMP DNA
1 1 1 1
H,O 3.4 1.6
2.5 1.7
IMP
o H
response, fiV-9 x lo6 0.1 M NaC1" 0.5 M NaC1" 3.4 3.4 3.6 3.7
w AMP
IPP 0
3.4 3.1 3.7 3.5 Cancentrallo" (rnQPIL)
Also contains 0.5 mM NarEDTA.
less than 100% breakdown, doubling the tubing length to 200 ft increased conversion. Residence time in the 0.3 mm X 61 m tubing was 8.9 min with little deterioration in peak separation. Quantitative Relationships. Initial studies using a 30.5-m Teflon coil in the oxidizer found less than 100% conversion of inositol hexaphosphate to orthophosphate. At the operational flow rate of 0.5 mL/min, and a concentration of 1.1mg of P/L, only 63% of IHP was converted to orthophosphate. Doubling the tubing length resulted in 100% conversion; 0.81 mg of P/L DNA showed an increased conversion from 61 to 88% with the same increase in tubing length. These experiments were done using water as the solvent. With NaC1/Na4EDTA as solvent and the 61-m tubing in the oxidizer, both IHP and DNA showed 100% conversion to orthophosphate. IMP, IDP, ITetP, IPP, AMP, and 0-GP gave greater than 100% of the expected orthophosphate response. Table I illustrates the NaC1/Na4EDTA solvent effect on the apparent photodegradation efficiency for the organophosphate compounds relative to that in water. This enhancement of molybdate-based response could possibly result from the partially degraded organic material in solution effecting the reduction of phosphomolybdate or directly absorbing light as 668 nm. There is also a dependence upon the compound concentration. Figure 9 shows change in percent conversion with change in concentration of substrate for IHP, IMP, and DNA. Other studies performed for other purposes qualitativelyindicate that, for IHP, IPP, IMP, BGP, AMP, 202
Environ. Sci. Technol., Vol. 26, No. 1, 1992
Flgure 10. Response vs concentration for several compounds (55 2 5 0 - ~ Lsamples).
OC,
and DNA, percent conversion to orthophosphate drops off significantly at higher concentrations. Typically, response factors are nonlinear for most compounds above a few milligrams of phosphorus per liter, but as shown in Figure 10, the dynamic range extends beyond 100 mg of P/L. While nonlinearity is not desirable for analytical methods, computer control of data acquisition makes this problem manageable. These experiments were done using water as the solvent. Enhancement of response is expected when the salt solvent is used. Because of the high cost of the intermediate IPS and the extremely limited quantities available (100 pg of total compound for each intermediate), standard curves were not obtained for these compounds. Signals were obtained for concentrations of all the IP congeners at 0.01-0.05 mg of P/L, which translates into a detection limit for this method in the 10 ng of P range. HPLC Separations. Gradients ranging from 0.1 to 0.5 M NaC1/0.5 mM Na4EDTA were tested that would achieve good separation of IMP and PO4 and of IPP and IHP with adequate time for separation of intermediate IPS at intermediate times. An early gradient using a 30-min isocratic step with 0.1 M NaCl followed by a 60-min linear gradient to 0.5 M NaCl yielded excellent separation of orthophosphate and IMP (elution at 50 and 40 min, respectively). IHP eluted at approximately 95 min but there was no separation between IHP and IPP. Given that the initial isocratic step resolved the early-eluting species, a variety of gradients were tried that included intermediate
I
Water Samples
I
Tangential Flow Microporous Filtration (0.2 pn)
5d Cation Exchange (Na form)
Ultrafiltration*
(MW cutoff - 1000 Dalton)
Reverse Osmosis**
Permeate (Waste)
Time(min)
Figure 11. Gradient used for Inositol congener separatlon.
11-
(76mmdiamter Mw cutoff 500
Pressure Filter Batch Concentrator (500 Da)
H
Pressure Filter Batch Concentrator (5M) Da)
IMP IDP
~
Exchange Column*-*
I
(43 mm diamter MW cutoff - 500 Dalton)
IHP Pressure Filter
i
Final volume 5 or 6 ml
Figure 12. Separation of inositol phosphate congeners: IMP, inositol monophosphate; IDP, inosltol dlphosphate; ITP, inositol triphosphate; ITRP, Inositol tetraphosphate; IPP, inositol pentaphosphate; IHP, inositol hexaphosphate. AMP
i
0
BGP
ea
IHP
ha0
io
,.e
,Lo
Figure 13. HPLC separation: (A) DNA; (B) IMP, AMP, PGP, IPP, IHP, and DNA.
isocratic steps and variable rates of change. After much experimentation, an effective separation was achieved. This gradient is shown in Figure 11. Although lengthy and complicated in form, this gradient gives good separation of all IPS (Figure 12). Conceivably, other modifications could improve separations, especially if separation of the various isomers for the individual IPS is desired or if other than the myo-inositol series were to be investigated. In anticipation of the presence of other compounds in natural water samples, the elution behavior of a few selected compounds was examined. There is the potential for some interference since it can be seen in Figure 13 that DNA and 0-GP elute concurrently with IHP and PO4, respectively. However, AMP is well separated from all other compounds. While some minor variability was experienced in the individual compound elution times, they were typically reproducible within f4%, The variation in elution time for a particular compound could result from limitations in gradient reproducibility
*: Tangential Flow UlnafiiUabon(1000 Dalton Cut-off) **: Reverse Osmosis (40% or 80% NaCl RejecUon membrane) ***: Inuoduced when RO is used
Figure 14. Flow diagram for concentration procedures.
or variables such as inconsistent pumping resulting from such problems as wear of plunger seals. It is to be noted that travel time between the end of the column and the detector is approximately 16.5 min. Attempts at separating IHP and DNA were unsuccessful. Since these are considered to be environmentally important compounds, other methods of separation or detection will be developed. One possibility is to separate IHP from DNA using a 1000-Da ultrafiltration membrane and then perform HPLC on both retentate and filtrate. Another possible approach is to detect DNA using fluorometry methods and quantitate it independently. To distinguish orthophosphate from concurrently eluting P-GP, orthophosphate can be evaluated via a dark run, Le., with the UV lamp off. Application to Field Samples. Preliminary data have been obtained from field samples. Streamwater samples, from Walker Branch Watershed located on the Oak Ridge Department of Energy Reservation in eastern Tennessee, have been treated as shown in Figure 14. About 25% of the initial TSP is retrieved by this technique with resultant concentration factors of 10000-20 000. Two samples concentrated by this procedure were subjected to HPLC separation. The earlier sample shown in Figure 15 was processed with a simple intermediate gradient (not that of Figure 11). A peak eluting at the same retention time as IHP was observed. A sample processed 10 months later and using the gradient of Figure 11 (Figure 16) showed considerably different behavior, including the presence of an early-eluting (essentially unretarded by the column) peak of organic phosphorus. That it was not SRP was confirmed by repeated injection without UV illumination. The peak labeled DNA-like elution behavior was spread out in the same fashion as DNA standards, although the presence of IHP cannot be entirely ruled out either. Environ. Sci. Technol., Vol. 26, No. 1, 1992 203
IHP, 83-86-3; AMP, 61-19-8; phosphate, 14265-44-2;P-glycerophosphate, 17181-54-3; water, 7732-18-5.
Literature Cited
Tune (mutcs)
Flgure 15. HPLC analyses: (A) standard solutions (orthophosphate, field sample taken on May 20, 1988 (soluble reactive IMP, and IHP); (6) P, 2.81 mg of P/L; soluble unreactive P, 1.92 mg of P/L). u h m ( P O S z W Wharged or catbn
form Of ~Wri2PMJphoNJ)
A , L ,
DNA
omphasphstc
LO
,OD
21
>‘a
i,l
Figure 16. HPLC analysis for field sample taken on May 20, 1989 (soluble reactive P, 1.07 mg of PIL; soluble unreactive P, 1.62 mg of P/L).
While much remains to be resolved with natural samples containing unknown P species in general, what has been demonstrated is the capability of the method to separate organic P water samples when employed in conjunction with high-volume concentration techniques. It should be pointed out that attempts to utilize the oxidizer system with ion-pairing techniques on C18 (reversed-phase) columns were unsuccessful. Reagent incompatibility led to the formation of precipitates in the system lines with subsequent system failure.
Conclusions With this method, inositol phosphates and other organophosphate compounds are well separated and can be detected in the 10-ng range. This is an improvement over previous methods of detection discussed in earlier papers (29, 31) in that it is specific and sensitive. The length of time required to separate compounds is a major disadvantage, but it is possible that the gradient could be shortened somewhat depending on the compounds being separated. It is clear that when applied in conjunction with the high-volume concentration scheme described, this procedure can provide greater assessment of soluble organic phosphorus in natural waters. The method should also have application in soil and food chemistry, where inositol phosphates and other organic phosphorus compounds are present in much higher concentrations. Registry No. IMP, 105182-27-2; 1,4-1DP, 47055-78-7; 1,3,4ITriP, 98102-63-7; 1,4,5,6-ITetP, 121010-58-0; IPP, 20298-95-7;
204
Environ. Sci. Technol., Vol. 26, No. 1, 1992
Cheryan, M. In CRC Crit. Rev. Food Sei. N u t r . 1980,13, 297. Cosgrove,D. J. Soil Biochemistry; McLaren, A. D., Petersen, G. H., Eds.; Marcel Dekker: New York, 1967; pp 216-228. Dalal, R. C. Adv. Agron. 1977, 29, 83-117. Sommers, L. E.; Harris, R. F.; Williams, J. D. H.; Armstrong, D. E.; Syers, J. K. Soil Sei. SOC.Am. R o c . 1972,36, 51-54. Weimer, W. C.; Armstrong, D. E. Anal. Chim. Acta 1977, 94, 35-47. Minear, R. A.; Walanski, K. A. Report No. 86, University of Illinois, Water Research Center, Urbana, IL, 1974. Herbes, S. E.; Allen, H. E.; Mancy, K. H. Science 1975,187, 432-434. Eisenreich, S. E.; Armstrong, D. E. Environ. Sei. Technol. 1977, 11, 497-501. Minear, R. A. Technical Report No. 64, University of Tennessee, Water Resources Research Center, Knoxville, TN, 1978. Iberkeas, D. Cereal Foods World 1983, 28, 352-357. Cilliers, J. J. L.; van Niekerk, P. J. J. Agric. Food Chem. 1986, 35, 680-683. Hong, J. K.; Yamane, I. Soil Plant Nutr. 1980,26,497-505. Irving, G. C. L.; Cosgrove, D. J. Commun. Soil Sei. Plant Anal. 1981,12,495-509. Hixson, S. W. M.S. Thesis, The University of Tennessee, Knoxville, 1977. Tangendjaja, B.; Buckle, K. A.; Wootton, M. J. Chromatogr. 1980,197, 274-277. Camire, A. L.; Clydesdale, F. M. J . Food Sei. 1982, 47, 575-578. Graf, E.; Dintzis, F. R. Anal. Biochem. 1982,119,413-317. Knuckles, B. E.; Kuzimicky, D. D.; Betschart, A. A. J. Food Sci. 1982, 47, 1257-1258. Lee, K.; Abendroth, J. E. J.Food Sei. 1983,48,1344-1351. Sandberg, A. S.; Ahderinne, R. J . Food Sei. 1986, 51, 547-550. Sulpice, J.-C.; Phillippe, G.; Joumet, E.; Rendu, F.; Renard, D.; Poggioli, J.; Giraud, F. Anal. Biochem. 1989,179,90-97. Hsu, F.-F.;Sherman, W. R. J . Chromatogr. 1989, 479, 437-440. Lehrfeld, J. Cereal Chem. 1989, 66, 510-515. Shayman, J. A.; Barcelon, F. S. J. Chromatogr. 1990,528, 143-154. Patthy, M.; Aranyi, P. J. Chromatogr. 1990,523, 201-216. Prestwich, S. A.; Bolton, T. B. Biochem. SOC.Trans. 1990, 18,623-624. Taylor, G. S.; Garcia, J. G. N.; Dukes, R.; English, D. Anal. Biochem. 1990,188, 118-122. Segars, J. E.; Minear, R. A.; Elwood, J. E.; Mulholland, P. J. ORNL/TM-9737, Oak Ridge National Laboratory, Oak Ridge, TN, 1986. Minear, R. A.; Segars, J. E.; Elwood, J. W.; Mulholland, P. J. Analvst Vol. 113. 1988. 113. 645-649. Murph;, J.; Riley, J: P. Anal. Chim. Acta 1962,27,31-36. Minear, R. A.; Davidovitz, Z.; Dehghani, M. Natl. Meet. -Am. Chem. Soc., Diu. Environ. Chem. 1987, 27(1), 135-138. Uihlein, M.; Schwab, E. Chromatographia 1982,15, 140. Brinkman, U. A. Th. Chromatographia 1987, 24, 190.
Received for review June 12,1991. Accepted July 29,1991. This work is currently under support through a grant (Organic Phosphorus Compounds in the Hydrosphere: Characteristics, Identity and Dynamics) from the U S . Geological Survey (Grant 87-INT-M-0141) with matching f u n d s from the university of Illinois Campus Research Board. Earlier development work was supported by Contract DE-AC05-840R21400 with M a r t i n Marietta Systems with a subsequent subcontract (Contract 88-DOE-SBCIM-0770) to t h e University of Illinois.
