Hali/Califor vitrains from coal from infrared, broadline NMR, and data from dehydrogenation experiments. He concluded that this ratio should be in the range 1.6 to 1.8 for coals having carbon contents between 82.5 and 92.5%. For coal extracts, the lH and 13C NMR results give values between 1.7 and 1.9. It is interesting to reflect upon one of the earliest parameters used in coal research as an indication of carbon aromaticity, the atomic C/H ratio. The parameter has been used in the past to estimate minimum fa values for whole coals and other material derived from coal (26). The plot of fa vs. C/H in Figure 1clearly indicates that the elemental constitution of the materials listed in Table I gives only a crude estimate of f a .
ACKNOWLEDGMENT The authors gratefully acknowledge gifts of samples from Koppers Company, FMC Corporation, University of North Dakota, and the Exploratory Engineering and Product Technology Groups at the Pittsburgh Energy Research Center ar,d are indebted to Josef Dadok for use of the Mellon Institute spectrometer. The Mellon Institute spectrometer facility is supported by PHS Grant RR 00292.
LITERATURE CITED (1) J. K. Brown and W. R . Ladner, Fuel, 38, 87 (1960). (2) R . A. Friedel and H. L. Retcofsky, "Proceedlngs of the 5th Carbon Conference", Vol. 11, Pergamon Press, New York, N.Y., 1963, p 149. (3) 0.Takeya, M. Itoh, A. Suzuki, and S . Yokoyama, Bull. Chem. Soc. Jpn., 36, 1222 (1963). (4) G. Takeya, M. itoh, A. Suzuki, and S. Yokoyama, J. Fuel SOC.Jpn., 43,837 (1964). ( 5 ) H. L. Retcofsky and R. A. Friedei in "Spectrometry of Fuels", R. A. Friedei, Ed., Plenum Press, New York, N.Y., 1970, pp 70-89. (6) C. W. DeWalt, Jr., and M. S. Morgan, frepr., Div. FuelChem., Am. Chem. Soc., 1962, p 33.
Y. Maekawa, S. Ueda, Y. Hasegawa, Y. Nakata, S. Yokoyama, and Y. Yoshida, frepr., Div. FuelChem., Am. Chem. SOC., 1975, p 1. D. R. Clutter, L. Petrakis, and R. K. Jensen, Prepr., Div. Petrol. Chem.,Am. Chem. Soc., 1972, p C19. C. R. Clutter, L. Petrakis, R. L. Stenger, Jr., and R. K. Jensen, Anal. Chern., 44, 1395 (1972). I. G. C. Dryden, Fuel, 41, 301 (1962). J. B. Stothers, "Carbon-13 NMR Spectroscopy", Academic Press, New York, N.Y. 1972, pp 33-34. C. G. Levy and G. L. Nelson, "Carbon-13 Nuclear Magnetlc Resonance for Organic Chemists", Wiiey-Interscience, New York, N.Y., 1972, pp 2931. K. D. Bartle, T. G. Martin, and D. F. Williams, Chem. lnd. (London), 313 (1975). H. L. Retcofsky and R. A. Friedel, Fuel, 55, 363 (1976). C. A. Johnson, M. C. Chervenak. E. S.Johanson, and R. H. Wolk in "Coal Processing Technology", prepared by the Editors of Chem. Eng. frogr., American Institute of Chemical Engineers, New York, N.Y., 1974, pp 22-23. V. L. Brandt and 9. K. Schmid, Chem. Eng. frogr. 65 (12), 55 (1969). R . C. Merrill, L. J. Scotti, L. Ford, and D. J. Domina in "Coal Processing Technology", Vol2, prepared by the Editors of Chern. Eng. Progr., American Institute of Chemical Engineers, New York, N.Y., 1975, pp 88-93. P. M. Yavorsky, S. Akhtar, J. J. Lacey, M. Weintraub, and A. A. Reznik, Chem. Eng. frog., 71, 79 (1975). R. G. Ruberto and D. M. Jeweil, Papers presented in part at the National Science Foundation Workshop, "Analytical Needs of the Future as Applied to Coal Liquefaction," Greenup, Ky., August 21-23, 1974, and at the Symposium "Supplemental Fuels from Coal: New Analytical Needs", 26th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 3-7, 1975. J. Dadok, R. F. Sprecher, A. A. Bothner-by, and T. Link, Paper presented at the 1lth Experimental NMR Conference, Pittsburgh, Pa., 1970. J. Dadok and R. F. Sprecher, J. Magn. Reson., 13, 243 (1974). H. L. Retcofsky and R. A. Friedel in "Coal Science," R. F. Gould, Ed., Adv. Chem. Ser. No. 55, American Chemical Society, Washington, D.C., 1966, pp 503-515. Ref. 5,pp 99-1 19. K. D. Bartle, T. G. Martin, and D. F. Williams, Fuel, 54, 226 (1975). H. L. Retcofsky, App. Spectrosc., in press. D. W. Van Kreveien, "Coal," Elsevler, Amsterdam, 1961, p 446.
RECEIVEDfor review October 15,1976. Accepted January 4, 1977.
Determination of 2-Aminoethyl Dihydrogen Phosphate by Phosphorus-3 1 Fourier Transform Nuclear Magnetic Resonance Spectrometry Ian K. O'Neill" and Martln A. Pringuer Laboratory of the Government Chemist, Cornwall House, Stamford Street, London SE 1 9NQ, England
Fourier transform NMR spectrometry has been used to obtaln an accurate assay of 2-amlnoethyl dlhydrogen phosphate dlssolved in 50% aqueous sucrose solution. Slmple precautlons eilmlnated those quantltation errors arising from the spectrometer but It was necessary to study spln-lattice relaxation ( T I )to obtain an accurate assay technlque. The use of K3Cr(CN)Bas relaxlng agent overcame unusual and pronounced TI effects caused by the presence of sucrose. Quick specific analyses were obtained with a relative standard deviatlon of less than 3%.
lH NMR spectrometry has been used extensively for the structural and quantitative analysis of pharmaceutical substances (1,2). Although 31PNMR spectrometry has provided similar detail for phosphorus-containing materials ( 3 ) ,the lower inherent sensitivity of this nucleus requires application of the pulse-Fourier transform mode in many situations. While this has allowed observation of low concentrations of phosphorus compounds ( 4 ) ,accurate quantitative FTNMR 588
ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
spectrometry is difficult to achieve ( 5 ) . Nevertheless, the features of high specificity and minimal sample preparation make quantitative FT NMR a particularly desirable general objective. 2-Aminoethyl dihydrogen phosphate, I, is a phospholipid constituent H~NCHZCH~OPO~H
(1)
that is included in some proprietary tonics at 5% w/v and for which we needed an assay method. A chromatographic separation and ninhydrin determination (6) requires extensive preparative and calibration effort. 1H NMR spectrometry could not be used because of the presence of 500h w/v sucrose, but it was found that 31PFT NMR had sufficient sensitivity and specificity. The aim of this work is to secure accurate 31P FT NMR quantitation.
EXPERIMENTAL Apparatus. 31PFT NMR spectra were taken with a JEOL PFTfacilities operating 1OOP spectrometer equipped with 25.03 MHz
30
26
20
15
10
5
0
6 ppm
Figure 1. 31P FT NMR spectra of (a)2-aminoethyl dihydrogen phosphate (I) in aqueous proprietary solution;(b) sodium pyrophosphate; (c)as (a) with added sodium pyrophosphate in the presence of 0.03 M K3Cr(CN)8,
showing resolution of P043- impurity. Spectra referred to c-(Napod4 a t reduced field (1.45 Tesla). In each analysis, ten pulses (9 ps = 60° flip angle, repetition time 7 s) were applied and each apodized free induction decay was automatically transformed and co-added in the as reference. Spectral frequency domain using the P ~ 0 7 ~singlet width and frequency filter were both 2500 Hz, and data were collected in 4095 points with an acquisition time of 0.81 s. The phase-corrected absorption mode was integrated. Spin-lattice relaxation was measured by the 18O0-7-9O0 technique (7). Samples were examined a t a probe temperature of 23 OC in 10-mm NMR tubes and chemical shifts referenced to a capillary tube containing saturated aqueous tetrasodium cyclotetraphosphate. Reagents. 2-Aminoethyl dihydrogen phosphate (Fluka, 99+%) mp 239-241 " C , (8) and KHzP04 (Fisons, 99.5%) were used as received. Na4P207-10H20 (Hopkin and Williams, AnalaR) was checked by "P FT NMR spectrometry and found to contain less than 0.2% PO4 j-. A 10% w/v aqueous solution of potassium hexacyanochromate, K.$r(CN)B, (ROC/RIC) was used as the relaxing agent. Two batches of proprietary solution of the phospholipid I were examined, both being declared as containing 5% w/v I and 50% w/v sucrose in water, this being equivalent to 4.17 w/w I in the solution. Sample Preparation. Reagents and the proprietary solution were weighed directly into NMR sample tubes with brief warming to assist dissolution as necessary. Three mixtures of KH2P04 and NadPlOi. lOHzO were accurately prepared and dissolved in 4 ml water to give approximately 15% w/v solutions and then 1.5 ml of relaxing agent was added before quantitation. Accurately known amounts of approximately 140 mg each of both I and NadPzOTlOHzO were dissolved in 3 ml water and then 0.3 ml of relaxing agent was added for analysis. For each gram of proprietary solution of I, 0.1 ml of relaxing agent and an accurately weighed portion of approximately 60 mg NadPlOj. 1 0 H ~ were 0 added for assay.
RESULTS AND DISCUSSION Sodium pyrophosphate was chosen as internal standard for assay of I because it has a sharp 31Presonance placed conveniently relative to that of I. Addition of pyrophosphate to the proprietary solutions of I effected a convenient 3 ppm downfield shift for I so as to reveal orthophosphate (see Figure 1). This impurity was present in the proprietary solutions and the chemical shift change possibly arose from complexation effects. There are four principal factors to be overcome in obtaining FT NMR quantitation ( 5 ) and of these, the Nuclear Overhauser effect (9) was absent in this work since decoupling was unnecessary. Changes in signal intensity with separation from the irradiation frequency, and through frequency filter
I
0 06
0
01
Mol. 1-' of K3Cr (CNIgaddad
Flgure 2. The effect of the addition of K3Cr(CN)s on T , of I (A)and P2074- (X) in aqueous solution and of I (0) and P2074- ( 0 )in the
presence of 50% w/v sucrose Table I. :IlP FT NMR Assay of Standard Solutions Containing Added Relaxing Agent Mole ratio, P2074-/H2P04-
Mole ratio, P2O7*-/I
1.240 2.172
... ... ...
3.711
*..
... *..
0.3013 0.2624 0.3042
Found
Re1 % error
1.236 2.174 3.682 0.3055 0.2655 0.3097
-0.3 +0.1 -0.8
+1.4 +1.2 +1.8
effects (IO)were both minimized by having the resonances of I and internal standard separated by only 500 Hz and observing both signals within 1000 Hz of the irradiation frequency. However, a large difference in the spin-lattice relaxation times, T I ,of I and pyrophosphate was revealed by measuring the ratio of the integrals for these substances over a range of pulse repetition times. A constant ratio could only be obtained with repetition times greater than 30 s, thereby indicating a need to reduce 2'1 values and hence reduce the apparent large difference. It is known that traces of paramagnetic substances can drastically reduce 7'1 values and so there is an a priori need to consider T I of compounds of interest in actual samples t o be assayed. An aqueous K3Cr(CN)6 solution was added as relaxing agent (11)both to swamp the effect of any unknown trace paramagnetics and also to effect the required reduction of T I .There was a striking difference in the consequent T I values for the sample of I and pyrophosphate prepared from pure materials and the proprietary solutions. It can be seen in Figure 2 that the addition of relaxing agent to the solutions containing 50% sucrose produced little change in the pyrophosphate T1 but a fourfold reduction in the T 1of I. The ANALYTICAL CHEMISTRY, VOL. 49, NO. 4, APRIL 1977
589
Table 11. 31PFT NMR Assay of Proprietary Solutions of 1" Containing Added Relaxing Agent Duplicate assays, % wlw found 4.15, 4.11 4.07, 4.10 4.21, 4.15 4.06, 4.10 4.26, 4.28 4.21,4.11 4.16, 3.99 4.25,4.18
Av % w/w
found Batch A f = 4.12 cr = 0.49
Batch B f = 4.18 cr = 0.095
declared I equivalent to 4.17 w/w. presence of sucrose has reduced the TI of pyrophosphate to one-tenth of its value in aqueous solution although producing only a slight reduction in the T I of I. A possible cause of this unexpected effect in the T I of pyrophosphate was competitive complexation of the pyrophosphate ion between sucrose and relaxing agent. This abnormal effect emphasizes the need to evaluate T I in actual assay samples. Addition of sufficient relaxing agent reduced T1 of I and pyrophosphate to less than 1s. A pulse repetition time of 7 s was chosen to provide more than enough time, i.e., greater than 7 T I ,to preclude saturation effects. Accuracy of quantitation was tested by comparing pyrophosphate and orthophosphate in admixture over a threefold concentration ratio. Accuracy in assaying I was tested by comparing pyrophosphate and pure I. The results in Table I show that the technique had a relative error of less than 2%; these results were obtained by integration since relative peak heights were found to be adversely affected by changes in resolution and concentration. Four portions of each proprietary sample were assayed in duplicate for I. The results (Table 11) accord with the declared concentrations and show that the method has a standard deviation of less than 2.1% in the presence of the large concentration of sucrose. Thus, the addition of relaxing
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agent was effective in overcoming gross T1 differences of those phenomena whatever the origin had been.
CONCLUSION The results indicate that 31PFT NMR can yield quantitative results providing sufficient care is taken to counter differential relaxation effects which can vary unpredictably in different sample matrices. The technique can provide a quick, specific assay with little sample preparation. This should be applicable to a variety of phosphorus-containing samples; we have already obtained useful results for polyphosphate mixtures (12) and polyphosphates in meat (13). ACKNOWLEDGMENT We thank Margaret Pratt of the Medicines Inspectorate for technical assistance, and R. A. Smith of Albright and Wilson Ltd for a sample of tetrasodium cyclotetraphosphate. LITERATURE CITED (1) A. F. Casy, "PMR Spectroscopy in Medicinal and Biological Chemistry", Academic Press, New York, 1971. (2) F. Kasler, "Quantitative Analysis by NMR Spectroscopy", Academic Press, New York, 1973. (3) M. M. Crutchfield, C. H. Dungan, J. H. Letcher, V. Mark, and J. R. van Wazer, "P3' Nuclear Magnetic Resonance" in "Topics in Phosphorous Chemistry", Vol. 5, M. Grayson and E. J. Griffith, Ed., Wiley-Interscience, New York, 1967. (4) T. W. Gurley and W. A. Ritchey, Anal. Chem., 47, 1444 (1975). (5) I. K. O'Neill and M. A. Pringuer, Org. Magn. Res., 6, 398 (1974). (6) S.Moore and W. H. Stein, J. Bo/.Cbem., 211, 907 (1954). (7) R. L. Vold, J. S.Waugh, M. P. Klein, and D. E. Phelps. J. Cbern. Pbys., 48, 3831 (1968). (8) "Dictionary of Organic Compounds", Eyre and Spottiswoode, London, 1965, Vol. 5, p 130. (9) P. L. Yeagle, W. C. Hutton, and R. B. Martin, J. Am. Chem. SOC., 97,7175 (1975). (IO) B. Thiault and M. Messerman, Org. Magr. Res., 7, 575 (1975). (1 1) i. D. Campbell, C. M. Dobson, R. J. P. Williams, and A. V. Xavier. J. Magn. Res., 11, 172 (1973). (12) I . K. O'Neili, M. A. Pringuer, H. J. Prosser, and C. P. Richards, unpublished work. (13) I. K. O'Neill and C. P. Richards, presented at the Third International NMR Conference, St. Andrews, Scotland, July 1975.
RECEIVEDfor review October 12,1976. Accepted November 12, 1976.
