Anal. Chem. I N S , 58, 455-458
1f l
l a n e1.0
t/
L 0.01
0.02
0.03
0.04
CGallic acid3 mol/L
Flgure 5. Effect of gallic acid concentration on the initial rate: [Br03-] = 0.08 mol/L; [V(V)] = 25 pg/L; pH 3.4, Prideaux buffer.
Interference Study. Vanadium (15 pg/L) was determined in the presence of a large number of diverse ions: Mg(II), Ca(II), Sr(II), Ba(II), Fe(II), Fe(III), Pd(II), Cu(II), Ti(IV), Zn(II), Ni(II), Co(II), Cr(III), Mo(VI), AUIII), U(VI), Mn(II), Hg(II), Cd(II), Au(III), Bi(III), Pb(II), Ag(I), As(III), Ce(IV), Si(IV), Sb(III), Pt(IV), Sn(IV), W(VI), NO,, C1-, Sot-,tartrate, oxalate, and CO?-. In all instances, the determination of vanadium was not affected by any of the ions, when as much as A00 times excess was present. We wish to point out the serious Fe(III), Ti(IV), Mo(VI), and W(V1) interferences in the spectrophotometric method due to the formation of colored complexes between these ions and the gallic acid or the resulting compounds from the reaction. None of these reactions interfere in the thermometric method. Determination of Vanadium in Steel. The reliability of the proposed method was tested by determining vanadium
455
in steel (reference 64b from the Bureau of Analyzed Samples with the following composition: C, 0.90%; Cr, 4.55%; V, 1.99%; Mo, 4.95%; W, 7.05%). The results obtained agree well with the expected value of 1.98 f 0.03% V (result of two independent samples with four and five replicate determinations, respectively). Registry No. V, 7440-62-2; gallic acid, 149-91-7; steel, 12597-69-2.
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
Fishman, M. J.; Skougstad, M. W. Anal. Chem. 1984, 36, 1643. Costache, D. An. Unlv. Bucuresti. Chlm. 1972, 21, 145. Fuller, C. W.; Ottaway, J. M. Analyst (London) 1970, 95,41. Kreingol'd, S. U.; Panteleimonova, A. A.; Poponova, R. V. Zh. Anal. Khlm. 1973, 28, 2179. Il'icheva, I . A.; Degtereva, I . F.; Dolmanova, I. F.; Petrukhina, L. A. Metody Anal. Kontrolya Kach. Prod. Khim. Promsti. 1978, 4 , 65. Lisetskaya, G. S.; Bakal, G. F. Ukr. Khim. Zh. (Russ.Ed.) 1970, 36, 709. Motoharu, T.; Norio, A. Anal. Chim. Acta 1967, 39,485. Yamane, T.; Fukasawa, T. Bunseki Kagaku 1976, 25, 454. Jarabin, 2.; Szarvas, P. Acta Univ. Debrecen. Ludovico Kossuth Nominatae 1981, 7, 131. Weiguo, Q. Anal. Chem. 1983, 55, 2043. Grases, F.; Forteza, R.; March, J. G.; Cerdi, V. Anal. Chlm. Acta 1984, 158, 389. Grases, F.; March, J. G.; Forteza, R.; Cerdi, V. Thermochim. Acta 1884, 73, 181. Grases, F.; Forteza, R.; March, J. G.; Cerdl, V. Talanta 1985, 32, 123. Grases, F.; Fortera, R.; March, J. G. Analusis 1984, 12, 194. Grases, F.; March, J. G.; Forteza, R. J . Thermal. Anal. 1984, 29, 1397. Lumbiarres, J.; Mongay, C.; Cerdi, V. J . Thermal. Anal. 1981, 22, 275. Mongay, C.; Cerdi, V. Talanta 1977, 24, 747.
RECEIVED for review April 22,1985. Accepted October 1,1985.
Determination of Stereoisomers by Circular Dichroism Soon M. Han and Neil Purdie* Chemistry Department, Oklahoma State University, Stillwater, Oklahoma 74078
Clrcular dictdsm spectra in aqueous buffer clearly distingulsh between the following pairs of compounds: quinlne-qulnldine, clnchonlne-clnchonldlne, atropine-hyoscyamine, and piiocarpine-lsopllocarplne. Blnary mixtures prepared from these pairs can be determlned wlthout performlng any separatlon. The method has the potential for the recognltlon of an enantiomeric excess in a simple experlment and for quality control In the pharmaceutical Industry.
It is commonly understood that stereoisomers are indistinguishable in all respects but one, their interaction with circularly polarized light. The resultant optical activity can be observed either as the rotation of the plane of linearly polarized light or as the difference in absorption between left and right circularly polarized light, called circular dichroism (CD). In spite of this, polarimetry is not often encountered among the instrumental procedures used for their identification and determination. The reason for this is easily appreciated since total separations from all other optically active compounds would be required and would have to be com-
pletely ensured for positive identification and quantitation. For that reason chromatographic methods are obviously preferred. Compounds such as these about to be discussed here are routinely identified by paper or thin-layer chromatography and by gas or liquid chromatography (I)and confirmed by IR (1). Since 1969, distinction between enantiomers has been made possible by NMR spectroscopy (2). Even in these instances, confirmation can sometimes still be difficult if the samples are available as mixtures of the stereoisomers. This is frequently the case among pharmaceutical products and preparations and in organic syntheses. It is known that therapeutic effects and toxicity levels can be radically different even for enantiomeric pairs, so positive identifications are of crucial importance (3). A rapid reliable method for isomeric confirmation and determination of enantiomeric excess is needed. Provided the stereoisomers contain a chromophore, circular dichroism might fulfill the need. In this work a CD study of the diastereoisomers of the quinine and cinchonine alkaloids, of pilocarpine and isopilocarpine, and of mixtures of atropine (DL-hyoscamine) with L-hyoscyamine in aqueous buffer has been done. No chromatographic separations were performed. In some instances
0003-2700/86/0358-0455$01.50/00 1986 Amerlcan Chemical Society
458
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
Table I. Simultaneous Determinations for Mixtures of Atropine and L-Hyoscyamine
a
b
103 x [atropine],
%
M
error
1.56 1.54 1.70 1.83 1.47 0.53
2.3 -0.5
1O4[~-hyoscyamine], % M error
1.0
1.2 3.6 2.1
3.74 3.70 2.97 2.54 5.08 6.69
%
excessn A%b
5.5 4.5 -4.0 -4.2 -4.2 -5.5
19.3 19.3 14.9 12.2 25.7 56.9
0.5 0.7 -0.7 -0.6 -1.5 -0.9
Calculated as 100[~-hyoscyamine]/([~-hyoscyamine] + [atropine]). Difference between calculated and known percent excess. Table 11. Simultaneous Determinations for Mixtures of Quinine and Quinidine 1
C6H5
d
C
Flgure 1. Molecular structures for (a) quinine-quinidine, (b) cinchonine-cinchonidine, (c)L-hyoscyamine,and (d) pilocarpine-isopilocrpine.
UV absorption and CD were combined in order to fully determine the system; in others, only CD data were required.
EXPERIMENTAL SECTION Quinine and quinidine as hydrochlorides, cinchonine, cinchonidine, atropine, L-hyoscyamine, and pilocarpine nitrate were obtained from Sigma Chemical Co. Isopilocarpine nitrate was obtained from Aldrich Chemical Co. All of the standard chemicals were used without further purification. UV absorbance and CD data were collected for the analytes dissolved in aqueous solutions buffered to pH 8 (pHydrion). Mixtures were prepared by weight from the standard materials. UV absorption measurements were made on a Hitachi Model 100-80 spectrophotometer. CD spectra were obtained on a JASCO-500A automatic recording spectropolarimeter fitted with a DP-500N data processor. The instrument was calibrated daily with a standard solution of androsterone in dioxan as recommended. Scan rates, sensitivities, and repeat functions were selected to give optimum signal to noise ratios. Calibration curves were prepared for each of the analytes using five standard solutions. RESULTS AND DISCUSSION Molecular structures for the analytes are shown in Figure 1. At pH 8, the amine nitrogen in these alkaloids is deprotonated so the UV spectra, &s shown in Figure 2, compare quite favorably with literature data for the analytes in basic media ( 4 ) . Only for the quinine-quinidine pair are the molar absorbance values different throughout the spectra, for example, 6300 and 5600, respectively, a t the 331-nm maximum. Distinction between the quinine and cinchonine alkaloids is readily done from the UV spectral differences, although the only structural difference is the methoxy substituent a t position 6. CD spectra are shown in Figure 3 for all but atropine, which, being a racemate, is achiral. Opposite polarities are observed for the diastereoisomeric pairs. Distinction among all four of the cinchona1 alkaloids is now possible. The CD spectrum of L-hyoscyamine in aqueous base was reported before (5). Little difference is observed with pH change. The typical aromatic triplet observed in the UV spectrum is so low in CD intensity to be of little assistance to quantitative analysis. Instead the more intense band with maximum at 222 nm is used in the calculation of enantiomeric excesses. For the pilocarpines, the CD data a t short wavelengths are of such poor quality that their only value is in the qualitative distinction between the isomers. The low signal to noise ratio is a consequence of the combination of low
mixture ratio" 20180 20/80b 40160 40/60b 50150 50/50* 60140 60/40b 80120 80/20b
104 x [quinine], M
%
error
0.64
1.9
1.26
0.8
1.47
1.4
1.70 0.49 2.37 1.78
-2.3 -7 2 -4.8 -29
104 x [quinidine], M
error
2.44 1.76 1.85 0.54 1.50 0.90 1.14
-2.0 -29 -1.1 -71 3.4 -38 -1.5
0.64
2.0
%
Ratio expressed as [quinine]/[quinidine]. bData treated as due only to the analyte in excess. rotatory power with high absorbance by the molecules. At best CD data alone would reveal which isomer is present in excess but could not be used for absolute purity determinations. During the extraction of L-hyoscamine from the natural source, atropia belladonna, racemization to atropine occurs (6). This system was chosen as a model for the application of UV data combined with CD data in the determination of enantiomeric excess. The total analyte concentration, atropine plus hyoscyamine, was determined from the measured absorbance at 257 nm, and the hyoscyamine alone was determined from the CD data at 222 nm for the same solution. Overall the agreements between the calculated and prepared resulta are better than 2.0%for atropine, and better than 4.5% for L-hyoscyamine, which, for the most part, is present in much lower concentrations (Table I). When the excess of Lhyoscyamine is defined as a mole fraction percent, the agreement between the prepared and calculated values is better than 1%. For mixtures of quinine and quinidine or cinchonine and cinchonidine, where both analytes would contribute toward the overall CD signal, either the purity or the percent composition of the sample can be established from CD data alone. This is demonstrated for a number of in-house mixtures of quinine and quinidine, Table 11. The simultaneous solution was carried out as before (7) using a least-squares fit of the spectra for the mixtures to a weighted algebraic sum of the spectra for the standards, which minimized the coefficients in the solution of a set of linear equations. Ellipticity data were taken at every nanometer over the absorption range. Once again agreements between the prepared and calculated quantities are within 2.4% for both analytes. Molar ellipticities for the quinine alkaloids are opposite in sign but not equal in magnitude, which is consistent with the observation already made with regard to the molar absorbances. The reason for this is unknown.
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
e
cc 1
457
C
+ a
190
230 ,I'
\\
x
270
\
\
-
\\ S'
$ \ '\
220
260
300
a
I"\
stereoisomers (A) quinine or quinidine, (B) clnchonine or cinchonidine, (C) atropine or L-hyoscyamine, and (D) pliocarplne or isopilocarpine. Molar absorbance data are avaliable in ref 4. The procedures discussed for these in-house mixtures are very effective and one can conclude that for a mixture of enantiomers the excess can be determined in a single CD experiment if the molar ellipticity value for either one is known. For a mixture of diastereoisomers the calculation is only certain if the molar values for both are known. A more cautious approach should be taken for the determination of an unknown that is suspected to consist of a mixture of enantiomers or diastereoisomers. Since the CD spectra of the standards are mirror images of each other, the spectrum for the mixture, which is the algebraic sum of the two, might be mistaken for that of a standard at a lower concentration. The
I,I
1 I
../ I
I
e
(87)
n 240
l
,.-
D
l I
1
Flgure 2. UV spectra in aqueous buffer pH 8, representatlve of the
I
(-268)
1
CD)
/
I
Flgure 3. CD spectra in aqueous buffer, pH 8, for (A) quinine (---) and quinidine (-), (B) cinchonlne (- -) and cinchonldine (-), (C) L-hyoscyamine, and (D) pilocarpine (- -) and isopilocarpine (-). Figures in
-
parentheses are the molar ellipticity values, 8, at the maxima. data must be analyzed to cover both possibilities. This has been done for the quinine-quinidine in-house mixtures in order to compare the relative standard deviations for a single-component vs. a two-component determination, Table 11. As expected, very distinct differences in correspondence are observed, and the sensitivity of the CD method is such that the distinction between the two options is clearly unambiguous.
ACKNOWLEDGMENT We wish to acknowledge the assistance of Robert Kroutil, US Army, Aberdeen Proving Grounds, MD, for developng the
458
Anal. Chem. 1986, 58,458-462
BASIC program used in the analyses. Registry No. Quinine, 130-95-0;quinidine, 56-54-2; cinchonine, 118-10-5;cinchonidine, 485-71-2; atropine, 51-55-8; hysocyamine, 101-31-5; pilocarpine, 92-13-7; isopilocarpine, 531-35-1.
LITERATURE CITED (1) Clarke, E. G. C. “Isolation and Identification of Drugs”; The Pharmaceutlcal Press: London, 1978.
(2) Williams, T.; Pitcher, R. G.; Bommer, P.; Gutzwiiler, J.; Uskokovlc, M. J . Am. Chem. SOC.1060, 91, 1871. (3) Goto, J.; Goto, N.; Nambara, T. J. J . Chromatogr. 1982, 239, 559. (4) Slek, T. J.; Osiewicz, R. J.; Bath, R. J. J . Forensic Scl. 1076, 21, 525. (5) Han, S. M.; Purdle, N. Anal. Chem. 1084, 56, 2827. (6) “Merck Index”, 9th ed.; Merck & Co. Inc.: Rahway, NJ, 1976. (7) Han, S. M.; Purdie, N. Anal. Chem. 1988, 5 8 , 113.
RECEIVED for review July 15, 1985. Accepted September 23, 1985.
Determination of Oxygen/Uranium Ratio in Irradiated Uranium Dioxide Based on Dissolution with Strong Phosphoric Acid Hideyo Takeishi, Hiroshi Muto, Hisao Aoyagi, Takeo Adachi, Kimie Izawa, Zenko Yoshida,* and Hiroshi Kawamura‘ Analytical Chemistry Laboratory, Japan Atomic Energy Research Institute, Tokai, Ibaraki 319-11, Japan
Sorin Kihara The Institute for Chemical Research, Kyoto University, Uji-shi, Kyoto 611, Japan
Appllcablllty of a chemical method to the determlnatlon of O N ratlo In lrradlated uranlum dloxlde, UO,+, , was lnvestlgated. A sample piece was taken from the Irradiated U02+, pellet wlth a dlamond cutter and dlssolved In strong phosphoric acid (SPA). The O/U ratio was calculated from the amounts of U( IV) and U(VI) In the solution. Oxidation states of uranium after sampllng and dlssolutlon were conflrmed to be the same as those In the pellet. The stablllty of U( IV) and U(V1) In the SPA solutlon with radloactlvlty was lnvestlgated. For the determination of U(IV) and U(VI), two methods based on dlfferent prlnclples, flow coulometry and spectrophotometry, were employed to avold unexpected error. The Interference from plutonlum and fission products In the determlnatlon was reduced by preelectrolysls. The O/U ratlo of >2.001 in U02+, with < 1 X lo4 MW days/ton burnup was deterrnlned wlth flow coulometrlc detectlon. Reliablllty of the results was verified by spectrophotometry.
The oxygen/uranium ratio, O/U ratio, in nonstoichiometric uranium dioxide, UOz+,, as a nuclear fuel is one of the most important factors that influences the compatibility between the cladding and the fuel, the fuel safety, and the fuel economy ( I , 2). The O/U ratio and also the distribution of the ratio in the pellet may change during neutron irradiation due to the growth of fission product elements (FPs) and/or the thermal gradient inside the pellet. Therefore, it is desired to know the O/U ratio in UO,+, not only before but also after the irradiation. Methods for the determination of the O/U ratio in the nonirradiated UO2+, can be classified into physical and chemical methods (3, 4). Such physical methods as X-ray diffraction analysis (5),X-ray photoemission spectrometry (6, 7), and the measurement of electromotive force (3, 8, 9) substantially need reference matmials having identical comPresent address: Department of JMTR Project, Japan Atomic Energy Research Institute, Oarai, Ibaraki 311-13,Japan.
position with that of the sample in order to calibrate the methods. One of the chemical methods, which does not require reference material, is gravimetry (3, l o ) ,and the other is grounded on a principle that UOz+l can be dissolved by some kinds of solvents such as strong phosphoric acid, SPA (3, 11-13), and the molten salts mixture of aluminum chloride and potassium chloride (14) producing uranium ions whose oxidation states are the same as those in the solid UOZ+,. The O/U ratio caJi be calculated from the amounts of U(1V) and U(V1) in the solution determined by spectrophotometry (15, 161, polarography (11-13), coulometry (17,18), or titrimetry (3, 19). For the determination of the O/U ratio in the irradiated UOz+,, physical methods are hardly used because (i) it is difficult to prepare reference materials containing many kinds of elements such as plutonium and FPs and (ii) the radiation may affect the measurement. Gravimetry is not applicable, because the amounts and chemical states of coexisting elements are not known. In the present work, we investigated the applicability of the chemical method based on the dissolution with SPA, which seems to be the most promising, to the determination of the O/U ratio in irradiated UO,,,.
EXPERIMENTAL SECTION Chemicals. Uranium Dioxide Pellet. Enriched uranium (1.5%
,%U)dioxide pellet of 10.6 mm diameter and 10.0 mm length with of specific gravity, a product of Nuclear Fuel In10.41 g dustries Co. (Japan),was used. The O/U ratio of the pellet before irradiation was determined by two-step flow-coulometry reported previously (18) to be 2.002-2.020 in the surface layer, less than 0.05 pm, and 2.000 in the bulk, more than 0.1 pm from the surface of the pellet. Hence, the average O/U ratio in the nonirradiated UOz+xwas 2.000. The UOz+zpellet, which had been packed in a Zircaloy cladding under helium atmosphere, was irradiated for 40 days with an average neutron flux of 1.1 X 1014n cm-2 s-l in the Japan Materials Testing Reactor (JMTR), and cooled for about 3 years. The burnup of the irradiated UO2+, was around 2 X lo3 MW days/ton. Strong Phosphoric Acid (SPA). Reagent grade phosphoric acid was heated in a Teflon flask under reduced pressure with the aid 0 1986 American Chemical Society
