Anal. Chem. 1988, 60, 1393-1397
dition to the least squares standard deviation), these results indicate that the differences between the FIA and batch data points are within statistical error. The FIA can produce data significantly faster than a manual procedure (a D value is obtained in 20 min or less), and because the method is almost totally automated, it can be run with minimal supervision and carries out operations in a more reproducible manner. These factors enabIe multiple D values to be acquired with the FIA easier, faster, and with better analysis statistics than with the batch system. It can also be advantageous to have a system such as the FIA that features on-line extraction, phase separation, and detection. This FIA should also be useful for the study of extraction systems that feature either unstable ligands or extracting complexes because there is much less time between the extraction and detection. The solution flow rates and even the extraction coil length on the FIA can be readily adjusted. These simple adjustments should facilitate the acquisition of kinetic measurements for extraction systems. The major limitation of this type of measurement on the FIA would be encountered when very slow flow rates or a very long extraction coil is required because of slow extraction kinetics (i.e., 5 min or more of contact time is required). Solvent Extraction Chemistry. Least-squares fits were employed for the pH, ligand, and synergist dependency plots (shown in Figures 2,3, and 4, respectively) for both the batch and FIA data. These data are summarized in Table I. The least-squares slopes were 2.04, 1.90, and 1.12 (obtained by averaging the FIA and batch data), respectively, for the pH, ligand, and synergist dependencies. The correlation coefficients of these plots were 0.994 or better. With use of the equation log D = log C + npH + n log [HL].,, + n log [SI,,, (derived elsewhere (1)) and the data obtained, the resulting extracting complex is readily derived as U02(BMPPT)2(TOPO),-where C is a constant and [HL] and [SI are the ligand and synergist concentrations respectively. For this study, [HL],,, and [S],,, are equal to the original concentra-
1393
tions of ligand and synergist because their solubilities in the aqueous phase are very small and [L-I,, is also very small (1). Deviations from the whole number slopes can be attributed to variations in activity coefficients (as described in ref 16). A similar solid-state structure has been reported with dimethyl sulfoxide as the synergist (16).
ACKNOWLEDGMENT We thank the Los Alamos National Laboratory for funding for this project. We also thank Raul Morales and Bob Ryan for helpful discussions on topics relevant to this paper. Registry No. TOPO, 78-50-2; HBMPPT, 62574-32-7; U, 7440-61-1.
LITERATURE CITED Lo, T. C.; Baird, M. H. I.; Hanson, C. Handbook of Solvent Extraction; Wiley: New York, 1983; Chapter 2. Ruzicka, J.; Hansen, E. Now Injection Analysis; Wiley: New York, 1981. Ruzicka, J. Anal. Chem. 1983, 55, 1040A-1053A. Kariberg, B.; Thelander, S. Anal. Chim. Acta 1978, 9 8 , 1-7. Bergamin, H.; Medeiros, J. X.; Reis, 8 . F.; Zagatto, E. A. G. Anal. Chim. Acta 1978, 101, 9-16. Johansson, P. A,; Karlberg, B.; Thelander, S.Anal, Chim. Acta 1980, 114, 215-226. Deratani, A,; Sebille, B. Anal. Chem. 1981, 5 3 , 1742-1746. Nord, L.; Karlberg, B. Anal. Chim. Acta 1981, 125, 199-202. Ooms, P. C. A,; Leendertse. G. P.;Das, H. A,; Brinkman. U. A. T. J . Radioanal. Chem. 1981, 67, 5-14. Nord, L.; Karlberg, B. Anal. Chim. Acta 1983, 145, 151-158. Fossey, L.; Cantwell, F. Anal. Chem. 1985, 5 7 , 922-926. Sahiestrom, Y.; Karlberg, 8.Anal. Chim. Acta 1986, 185, 259-269. Castro, L. J . Autom. Chem. 1986, 8 , 56-62. Lucy, C. A.; Cantwell, F. F. Anal. Chem. 1986, 58, 2727-2731. Valcarcel, M.; Castro, L. J . Chromatogr. 1987, 393, 3-23. JaNinen, G. D.; Smith, 8. F.; Ritchey, J. M. Inorg. Chim. Acta 1987, 129, 139-148. Kotrly, S.; Sucha, L. Handbook of Chemical Equilibria in Analytical Chemistry; Ellis Horwood: New York, 1985; Chapters 2, 7. Draper, N.; Smith, H. Applied Regression Analysis, 2nd ed.; Wiiey: New York, 1981; Chapter 1.
RECEIVED for review September 14,1987. Accepted February 29, 1988.
Investigation of Poly(L-amino acids) by X-ray Photoelectron Spectroscopy Kenneth D. Bomben* T h e Perkin-Elmer Corporation, Physical Electronics Laboratory, 1161-C S a n Antonio Road, Mountain View, California 94043 Sukhendu B. Dev' Battelle Columbus Diuision, 505 King Auenue, Columbus, Ohio 43201 A systematlc Investigation of 11 homopolymeric amino acids by X-ray photoelectron spectroscopy wlll be dlscussed. The chemlcal shifts In the core levels and the relatlve Intensities of the atomlc environments, obtalned from least-squares curve fitting, can be shown to correspond to partlcuiar structural features. I n addltlon, the atomic concentratlons of the varlous elements have been obtalned and compared to those expected from the structures. Shake-up satellite intensltles from those compounds with aromatlc rlngs are also reported. Human serum albumin was examined, and the results are compared to the expected structure and to prevlously reported results. 1Present address: D e p a r t m e n t of A p p l i e d Biological Sciences, Massachusetts I n s t i t u t e of Technology, Cambridge, M A 02139.
Since the first reports of X-ray photoelectron spectroscopy (XPS, also known as electron spectroscopy for chemical analysis, ESCA) by Siegbahn et al. (1,2), the technique has been applied to numerous fields. The study of biological materials, which are chemically complex, presents special problems in the interpretation of the chemical shift data available from XPS. Nonetheless, over the last 2 decades there has been much work done on biological systems. For example, Siegbahn et al. reported the investigation of cystine hydrochloride (3) and insulin ( 4 ) ,and Millard (5-9) undertook a series of studies that have made use of XPS for the characterization of the surfaces of cells and microorganisms. Ratner et al. (10-12) have used the techniaue to investigate thin protein films' adsorbed on metal support surfaces.- Meisenheimer et al. (13) investigated blood, Klein and Kramer (14)
0003-2700/88/0360-1393$01.50/00 1988 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
Table I. Amino Acid Side Chain Structures Alanine
Arginine
-CH,
Methionine
7
-(CHJ:N-C-NH,
Hydrochloride
-(CH&S-CH3
Phenylalanine
“$I-
-CHjf@ C. H
Aspartic Acid (Sodium Salt)
Leuclne
301
0
‘ ‘0-Ne
Tyrosine
Hydrobromide
281
Flgure 1. C 1s spectrum superlmposed on the curve fit for poly(^tyrosine) Including the K-T* shake-up band.
FH3
-CHiC-H
6% Lysine
Binding Energy (eV)
-CH-C*
-(CH, ),-N$Br
Vallnr
-
have examined proteins in grains, and there is even a report by Hovland (15) of elemental mapping in biological specimens by using scanning XPS. Despite this body of work, only one previous study, that of Clark, Peeling, and Colling (16),has attempted to characterize biological compounds systematically. The results of that work were used as a standard in the evaluation of grain meals for the estimation of protein content (17). Unfortunately, Clark et al. report only the average binding energies for the atoms of interest in the amino acid structures. An XPS investigation of amino acid thin films on a silver substrate has been reported by Colton (18)for three monomeric amino acids. Further, Debies and Rabalais (19) and Klasinic (20) have examined amino acids by using ultraviolet photoelectron spectroscopy. We report here the results of a systematic XPS study of 11simple poly(amino acids). The purpose was to characterize the amino acids and to correlate the chemical shifts observed by XPS with the structures in the solid state. The results of this investigation were then used as a standard to help analyze human serum albumin. For all compounds, the investigation was conducted on the unsupported, insulating powder. Monomeric amino acids contain an amino group and a carboxyl group and have properties that are consistent with a dipolar ion structure. Dipeptides and polypeptides are formed by the interaction between the amino and carboxyl groups, generating an amide group with the loss of a water molecule. The characteristic organic side chains of the amino acids investigated in this study are shown in Table I. The central carbon in the backbone of each of these amino acids is optically active; all the compounds were levorotatory.
EXPERIMENTAL SECTION All spectra were obtained on a Perkin-Elmer PHI Model 5400 X-ray photoelectron spectrometer using Mg K a radiation (1253.6 eV) at 400 W. The samples were obtained from Sigma Chemical and were used without further purification. Thick coatings of the finely ground powders were pressed into double-sided tape and attached to the sample stub. The pressure in the analysis chamber was typically 4 x IO4 Pa. Curve fitting, to determine binding energies and relative intensities, was done with an iterative least-squares computer program using Gaussian line shapes. Prior to the curve fits, the spectra were corrected for the inelastic background shape by subtracting the contribution of the inelastic electrons (21). All spectra were then charge-corrected to bring the C Is hydrocarbon peak energy to 285.0 eV. The magnitude of the charge correction was between 0.2 and 3.0 eV. No evidence of differential charging was observed. Assignment of structural features to particular binding energies
---A
413
Binding Energy ( e V )
393
Figure 2. N 1s spectrum and curve fit for poly(L4ysine)hydrobromide.
4
I
n A t e n s * i
:: 545
Binding Energy (eV)
525
Figure 3. 0 1s spectrum and curve fit for poly(L-asparagine).
was based upon previous work (16)and comparison to published tables (22,23). Where ambiguities arose because of overlaps in the binding energies, the Auger parameter (24,25)was employed to reduce the number of possibilities. Atomic concentrations were calculated by determiningthe area under the peak and correcting for the relative atomic sensitivities.
RESULTS AND DISCUSSION Amino Acids. For each polyb-amino acid), a number of different atomic environments are expected. The amino acid backbone and its peptide linkage contain three different, easily detected elements. Furthermore, the two carbons of the backbone have distinguishable binding energies. The elements of the side chain give rise to additional peaks. Curve fitting was used to determine the binding energy of these peaks and to determine the relative abundance of each atomic environment. Figures 1-3 are the high-resolution spectra for the C ls, N 19, and 0 1s regions of some of the 11 poly(i-amino acids) examined in this work. For each figure, the raw data is superimposed on the least-squares curve-fit to that data. In general, the peaks are symmetric but broad (full width at half-maximum is greater than 2.0 eV). While the broadness is not unexpected for solids, it does pose some problems for
ANALYTICAL CHEMISTRY, VOL. 60,NO. 14, JULY 15, 1988
Table 11. C 1s Binding Energies with the Experimentally Determined Ratio of Carbons in Each Environment Compared to the Expected Ratio
compd alanine arginine hydrochloride asparagine aspartic acid 1eucin e lysine hydrobromide methionine phenylalanine tryptophan tyrosine valine av values: Clark et al.b this work
binding energies, eV C-H4 C-N C=O
ratios, % found expected
285.0 287.3 63:37 67:33 285.0 286.1 288.7 6916:15 67:17:17 285.0 287.8 64:36 5050 285.0 288.0 7624 5050 287.6 8317 285.0 83:17 285.0 286.4 288.1 66:24:11 67:17:17 287.6 83:17 285.0 8020 287.8 91:09 89:11 285.0 285.0 288.0 9O:lO 91:09 281.7 88:12 285.0 89:11 285.0 287.7 83:17 80:20 285.0 288.2 285.0 286.3 287.8
a The hydrocarbon was assigned to 285.0 eV as the internal reference. * Reference 16.
the curve fitting, particularly where multiple peaks might be expected. Attempts were made to fit the data to more peaks than the data might seem to warrant, but the results did not add any useful information. The elemental binding energies that result from the curve fitting are presented in Table 11. These binding energies have uncertainties of f 0 . 2 eV. Carbon. In every sample, more than one carbon peak could be identified, as expected. The cafbon at 285.0 eV is the hydrocarbon (C-Hand C-C), while the carbon a t about 287.8 eV is the amide carbon of the backbone. Additional carbon peaks were seen in arginine hydrochloride and lysine hydrobromide at about 286.3 eV and correspond to the carbon atom closest to the halide. These assignments are noted in Table 11,and the ratios of the areas from the c w e fits are compared with the theoretical ratios, on the basis of the structure. In general, it is found that the side chain carbons show up as hydrocarbons and as such add intensity to the 285.0 eV peak. For the most part, the agreement between the experimental and theoretical ratios is very good. In the three amino acids that have an aromatic ring in the R group, the r-r* shake-up transition is oberved and is about 2% of the total carbon intensity. The relative atomic concentrations, reported in Table 111, have this contribution added to the hydrocarbon line. Nitrogen and Oxygen. Binding energy shifts for either nitrogen or oxygen, shown in Table IV, are not easily discernible for these compounds. For nitrogen, only the presence of an ionic bromine atom (in lysine hydrobromide) shifts the nitrogen binding energy sufficiently to separate nitrogen components. The backbone nitrogen accounts for 55% of the nitrogen signal, while the side chain nitrogen makes up 45% of the total nitrogen signal. For oxygen, on the other hand, even the presence of the sodium ion is not sufficient to generate a shift in the binding energy. There are, however, two amino acids that show two oxygen peaks where only one is expected: arginine hydrochloride and lysine hydrobromide. Presumably, the "extran oxygen is adsorbed water in the ionic crystal matrix that arises with the presence of either hydrochloric or hydrobromic acid. In arginine hydrochloride this oxygen accounts for 22% of the oxygen signal, while in lysine hydrobromide it accounts for 12% of the total oxygen signal. Other Elements. The binding energies of the remaining elements are also presented in Table IV. The chlorine and bromine binding energies are consistent with halide salts. The sodium Auger parameter indicates that the sodium is present as an organic ion, and the sulfur binding energy is consistent
1395
Table 111. Atomic Concentrations compd alanine found expected arginine hydrochloride found expected asparagine found expected aspartic acid found expected 1eucin e found expected lysine hydrobromide found expected methionine found expected phenylalanine found expected tryptophan found expected tyrosine found expected valine found exDected
N
0
63 19 60 20
18 20
64 20 50 33
11
60 18 44 22
22 33
C
78 11 75 13
11
68 15 60 20
9.7 10
66 12 63 13
12 13
9.0 9.0
79 14 79 14 73 75
Br
C1
0.2 -
5.0 8.3 0.2 -
3.6 11
13 1.4
-
6.7
10
10
13
8.0 9.0 7.2 7.1
9.7 17 8.3 17
74 14 67 17
S
8.3
70 9.8 17 44 11 33
83 82
Na
0.1 -
13 17 ~
Table IV. Binding Energies (eV) for All the Remaining Elements, Referenced to Cls at 285.0 eV compd
N Is
0 Is
other
alanine arginine hydrochloride
399.5 399.8
166.3 S 2p 197.5 C1 2 ~ 3 1 2
asparagine aspartic acid
399.6 399.8
531.1 531.5 533.4 531.3 531.4
1eu cine lysine hydrobromide
399.8 399.9 401.5 399.5 400.1 400.4 400.1 399.7
531.5 531.7 533.5 531.4 531.7 532.1 532.4 531.2
400.6 399.8
532.6 531.6
methionine phenylalanine tryptophan tyrosine valine av values: Clark et al." this work
166.6 S 2p 1071.3 Na 1s 263.4 Na KLL 68.4 Br 3d 197.8 C1 2p3/2 163.4 S 2p 102.7 Si 2p
Reference 16. with a sulfide bridge. Of some surprise is the presence of trace amounts of unexpected elements in some of the compounds. While the presence of inorganic chloride in lysine hydrobromide may be explained by assuming the presence of lysine hydrochloride as a major contaminant, the presence of sulfur as a sulfide in alanine and asparagine is not so easily explained. The sulfur is assumed to be a residue from synthesis or purification that adheres to the surface. XPS is a surface-sensitive technique; hence the sulfur may not be representative of the bulk. Clark et al. (16)reported average binding energies for the polymeric amino acids in their study, and these are included in Tables I1 and IV. The set of compounds that they studied is not the same as the set used in the present study; however,
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 14, JULY 15, 1988
T a b l e V. S h a k e - u p S a t e l l i t e I n t e n s i t i e s as a F u n c t i o n o f Total Area compd
C
N
0
phenylalanine tryptophan tyrosine
2.0 2.5 1.5
4.5" 5.2 4.3
5.5
T a b l e VI. E l e m e n t a l Binding E n e r g i e s f o r H u m a n S e r u m
Albumin b i n d i n g energy, eV
this
element
6.1 4.3
N
285.0 286.4 288.2 400.0
0
531.7
S
164.0
C-H C-N
c-0
Estimated.
work
previous
work"
285.0 286.5 288.3
"Reference 11. T a b l e VII. A t o m i c C o n c e n t r a t i o n s f o r H u m a n S e r u m
t
Albumin
+
concentration, % element
this
Binding Energy ( e V )
as C-H as C-N as C-0
280
Figure 4. C Is spectrum and curve fit for human serum albumin.
N
15 0.7 a
Reference
26.
bReference
expectedu
65.3 56.4 21.4 22.2 14.2 18.1 2.4
15
0
S there is excellent agreement between the two sets for the average carbon binding energies and good agreement for the average nitrogen and oxygen binding energies. Atomic Concentrations. Table I11 shows the atomic concentrations determined for the amino acids in this study. There is general agreement with the concentrations expected on the basis of the polymeric structures. However, asparagine and aspartic acid show an excess of carbon and a depletion of oxygen on the surface. Both of these compounds have an esterlike carbon in the side chain. Furthermore, arginine hydrochloride shows an excess of carbon and a depletion of nitrogen, while lysine hydrobromide shows the same trend (increased carbon, decreased nitrogen) but to a lesser degree. Both of these compounds have an acid halide moiety. We assume that the atomic concentrations do not match the structure because of chemical interactions of adsorbates with these moieties. It should be further noted that XPS is not a bulk analysis technique; the correlations in chemistry and atomic concentration are, overall, excellent. Shake-up Satellites. Table V shows the shake-up intensities for C, N, and 0 as determined by the curve fit using an integrated background correction (21). Clark et al. (16) did not compensate for the inelastic background when they measured the shake-up intensities and, hence, they overestimate the intensity of these transitions. We find the T-T* shake-up intensity to be 2% for carbon, 4.7% for nitrogen, and 5.3% for oxygen. Human Albumin. An additional compound, human serum albumin (HSA), was examined in an effort to compare the amino acid characterization with that of a protein. The HSA chosen for this study was described by the manufacturer (Sigma Chemical) as "fatty acid free (less than 0.005%)". The C 1s spectrum and the curve fit are shown in Figure 4. The binding energy results are shown in Table VI. The atomic concentrations are shown in Table VI1 and are compared to the concentrations expected from the structure, as reported by Brown, Shockley, and Behrens (26). There is good agreement between the expected concentrations and the experimentally determined concentrations of this work and of previous work on HSA by Paynter and Ratner (12). The binding energies of this work also show good correlation with those of Paynter and Ratner. It should be noted that if lipids are present in the surface, they will play a role in the chemistry of the polypeptides' surface and that tl-e quantity of lipids
previous work"
69 56 25 19
C, a l l 300
work
63 65 10
26 16 20 0.95
11. ~~
~
in human serum albumin depends upon the blood samples collected. CONCLUSIONS
XPS has been used to systematically investigate biological compounds, despite the inherent complexity. In this study, poly(L-amino acid) powders were examined without differential charging or the need to provide a metal backing. The experimentally determined binding energies and elemental ratios are explained in terms of the chemistry of amino acids and correlate well with the values expected on the basis of structure. The XPS spectrum of human serum albumin was also found to correlate with its structure and with previous work. LITERATURE CITED (1) Siegbahn, K.; Nordling, C.; Fahlman, A,; Nordberg, R.; Hamrin, K.; Hedman, J.; Johansson, G.; Bergmark, T.; Karlsson. S. E.; Lindgren, J.; Lindberg, B. ESCA -Atomic, Molecular and Solid State Structure Studied by Means of Electron Spectroscopy; Almqvist and Wiksells: Upsalla, Sweden, 1967. (2) Siegbahn, K.; Nordling, C.; Johansson. G.; Hedman, J.; Heden, P. F.; Hamrin, K.; Gelius, U.; Bergmark, T.; Werme, L. 0.; Manne. R.; Baer, Y. ESCA Applied to Free Molecules; North Holland: Amsterdam, The Netherlands, 1969. (3) Fahiman, A.; Hamrin. K.; Hedman, J.; Nordberg, R.; Nordling, C.; Seigbahn, K. Nature (London) 1966,210, 4-8. (4) Seigbahn, K., Nordling, C.; Fahlman. A,; Nordberg, R.; Hamrin, K.; Hedman. J.; Johnsson, G.; Bergmark. T.; Karlsson, S. E.; Lindgren. I.; Lindberg, B. Nova Acta Regiae Soc. Sci. Ups. 1967, 20, 7-31. (5) Millard, M. M. "X-Ray Photoelectron Spectroscopic Studies of Biological Materials: Metal Ion Protein Binding and Other Analytical Applications" in Protein-Metal Interactions ; Friedman, M., Ed.; Plenum: New York, 1974;pp 589-619. (6) Millard, M. M.; Scherrer, R.; Thomas, R. S. Biochem. Biophys . Res. Commun. 1976,72, 1209-1217. (7) Millard, M. M.; Bartholomew, J. C. Anal. Chem. 1977, 49,
1290-1296. (8) Chu, D.;Tappel, A. L.;Millard, M. M. Arch. Biochem. Biophys. 1977, 784, 209-214. (9) Pickart, L.; Millard, M. M.; Beiderman, 8.; Thaler, M. M. Biochim. Biophys. Acta 1978, 544, 138-143. IO) Ratner, B. D.;Horbett, T. A,; Shuttleworth, D.; Thomas, H. R. J. Colloid Interface Sci. 1981,83, 630-642. I11 Paynter, R. W.; Ratner, B. D.; Horbett, T. A,: Thomas, H. R. J. Colloid Interface Sci. 1984, 101, 233-245. 12) Paynter, R. W.;Ratner, B. D. "The Study of Interfacial Proteins and Biomolecules by X-Ray Photoelectron Spectroscopy" in Surface and Interfacial Aspects of Biomedical Polymers ; Andrade, J. D., Ed.; Plenum: New York, 1985;Vol. 2, pp 189-216. 13) Meisenheimer, R. G.; Fischer. J. W.; Rehfeid, S. J. Biochem. Biophys. Res. Commun. 1976,68, 994-999.
Anal. Chem. 1988, 60, 1397-1400 (14) Klein, M. P.; Kramer, L. N. Symp.: Seed Proteins (Proc.) 1972, 19, 265-276. (15) Hovland, C. T. "SESCA: Scanning Electron Spectroscopy for Chemical Analysis" in Proc. 7th I n t . Vac. Congr. 1977, 3 , 2363-2366. (16) Clark, D. T.; Peeling, J.; Colling, L. 6iochim. Blophys. Acta 1976, 453, 533-545. (17) Peeling, J.; Clark, D. T.; Evans, I . M.; Boulter, D. J . Sci. Food Agric. 1976, 2 7 , 331-340. (18) Colton, R. J.; Murday, J. S.; Wyatt, J. R.; DeCorpo, J. J. Surf. Sci. 1979, 8 4 , 235-248. (19) Debies, T. P., Rabalais, J. W. J . Nectron Specrosc. Reiat. Phenom. 1974, 3 , 315-322. (20) Kiasinc, L. J . Nectron Spectrosc. Relat. Phenom. 1976, 8 , 161-164. (21) Shirley, D. A. Phys. Rev. 6: Solid State 1972, 5 , 4709-4715. (22) Wagner, C. D.: Rlggs, W. M.: Davis, L. E.; Moulder, J. F.: Muilenberg.
(23) (24) (25) (26)
1397
G. E. Handbook of X-Ray Photoelectron Spectroscopy; Perkin-Elmer: Eden Prairie, MN, 1978. Jolly, W. L.; Bomben, K. D.; Eyermann, C. J. At. Data. Nucl. Data Tables 1984, 3 1 , 433-493. Wagner, C. D. Faraday Discuss. Chem. SOC. 1975, 6 0 , 291-300. Wagner, C. D.; Gale, L. H.; Raymond, R. H. Anal. Chem. 1980, 52, 466-482. Brown, J. R.; Shockiey, P.; Behrens, P. Q. "Albumin: Sequence, Evolution and Structural Models" in The Chemistry and Physiology of the Human Plasma Proteins; Bing, D. H., Ed.; Pergamon: New York, 1979; pp 23-40.
RECEIVED for review October 20, 1987. Accepted March 8, 1988.
Liquid Chromatographic Separation of Phosphorus Oxo Acids and Other Anions with Postcolumn Indirect Fluorescence Detection by Aluminum-Morin S h o n E. Meek and D. J. Pietrzyk*
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242
Phosphorus oxo aclds lncludlng organodlphosphonates are Isolated and separated on a quaternary ammonlum anion exchange column (Hamllton PRP-X100). An alumlnum-morln solutlon was used as a postcolumn reagent for the lndlrect fluorometric detection of the analytes. The variables affecting detectlon were shown to be postcolumn reaction temperature, pH, and mlxlng. Others are postcolumn reagent solutlon organlc solvent, alumlnum:morln ratlo, buffer, and their concentratlons. When these varlbles were optlmlzed, (dlfluoromethylene)dlphosphonk acld was detected at 15 ng for a 2 1 slgnaknolse ratio; the h e a r range extended to about 800 ng. Detectlon is applicable to other dlphosphonates, condensed phosphates, sugar phosphates, nucleotides, other organophosphates, and non-phosphorus-contalnlng anlons such as fluorlde, sulfate, and polyprotlc organlc acids.
oxo acids include atomic absorption (10, 11) and flame emission (12). A conventional fluorometric method for the determination of phosphate was reported in 1965 which is based upon phosphate's ability to reduce fluorescence of the aluminummorin chelate due to an aluminum-phosphate reaction (13). The study described here was undertaken to determine (1) whether postcolumn indirect fluorometric detection (IFD) is a useful detection strategy in liquid chromatographic separations, (2) whether aluminum-morin as a postcolumn IFD reagent is applicable to the detection of trace levels of condensed phosphates, phosphonates, and related anions, and (3) if applicable, the optimum conditions for the post column detection of diphosphonates. While indirect fluorescence detection using fluorescent eluents has been shown to be practical in LC detection ( 1 4 , 1 5 ) ,little has been done with indirect fluorescence as a means of postcolumn detection (16,
17). Phosphorus oxo acids are usually determined by complexation reactions that involve the orthophosphate ion ( I ) . The condensed phosphates are converted to orthophosphate through acid hydrolysis while the lower oxo acids are oxidized. The most frequently used complexation methods are based upon the reaction of orthophosphate with molybdenum reagents to form a colored complex that can be detected spectrophotometrically (2). These methods have been adapted for use in HPLC by coupling the column effluent with flow injection analysis systems (3-5) or air-segmented automated analyzers (6, 7). A postcolumn LC detection system for the oxo acids of phosphorus has been reported that eliminates the need to convert the phosphorus compounds t o orthophosphate (8). This method involves a reaction between the oxo acid of phosphorus and Fe(III), which is monitored spectrophotometrically a t 340 nm. The detection limit reported is about 0.5 pg for the lower phosphates and phosphonates (8,9). Other online detection methods that have been used for phosphorous
* To whom correspondence should be addressed.
EXPERIMENTAL SECTION Reagents. Disodium (difluoromethy1ene)diphosphonate (F,MDP) was prepared by the method of Burton and co-workers (18). The disodium (dichloromethy1ene)diphosphonate (C1,MDP) and disodium 1-hydroxyethane-1,l-diphosphonate (EHDP) were obtained from the College of Pharmacy, University of Iowa, and used as received. The biological phosphate samples were obtained from Sigma Chemical Co. Morin hydrate was obtained from Aldrich Co. Acids, bases, A1(N0J3, and 95% ethanol were obtained as analytical grade when possible. HPLC grade water was prepared by passing distilled water through a Sybron-Barnstead Nanopure water purification system. Instrumentation. The chromatographic system consisted of a Du Pont Instruments 850 gradient controller and pump, a Rheodyne Model 7125 injector, a PRP-X100 4.1 mm X 150 mm, 10-pm anion-exchange column (Hamilton Co.), a postcolumn mixing unit, and a Kratos 9000-9501 fluorescence detector. The detector was operated with a Kratos mercury lamp (FSA 113) that was used in conjunction with a Kratos blue-band excitation filter (FSA 404)for excitation wavelengths between 400 and 470 nm. A Kratos high pass filter with 50% transmission at 480 nm was used as an emission filter. The detector output was recorded on a Hewlett-Packard integrator recorder, Model 3390A; peak areas
0003-2700/88/0360-1397$01.50/00 1988 American Chemical Society
