may be precisely proportioned and mixed within a very wide volume range, thus enabling the use of relatively small ionexchange columns even at the end of long coupled series. Further advantages are simple construction and freedom from the risk of corrosion, which mean reliable operation for long periods. The main disadvantage of the machine might be the need for careful refilling of solutions between each run owing to the lack of a suction-side. A modified type of this kind of apparatus, suitable for the present chemical separations as well as for selective sorption experiments in common, will possibly be commercially available from a firm in the near future.
ACKNOWLEDGMENT We are greatly indebted to Erik Haeffner, Head of the Chemistry Department who, through his active and stimulating interest, made this work possible. Skillful technical assistance was performed by Sigrid Hackbart, Christine Hellmer, Agneta Hesselgren, Gun Jacobson, Ulla Lundgren, and Ingegerd Sundquist. RECEIVED for review June 6, 1967. Accepted August 8, 1967. Financial support was given by the Swedish Technical Research Council and Malmfonden, the Swedish Foundation for Scientific Research and Industrial Development.
Quantitative Determination of Rare Earths in Yttrium Oxide by Spectrophotoluminescence Lyuji Ozawa and Takao Toryu Research Department, Dai Nippon Toryo Co., Ltd., Chigasaki, Kanaguwa, Japan A technique for the quantitative determination of small amounts of rare earth impurities in YzOs(powder form) is described. Rare earths (RE) detected by spectrophotoluminescence in the visible and near ultraviolet spectral region were Pr, Sm, Eu, Gd, Tb, Dy, Ho, Er, and Tm; other rare earths in Y203had no luminescence. The emission and excitation spectra of rare earth ions in Y203 indicated a sharp difference in the spectra with regard to the energy distribution. The emission intensities increased linearly with rare earth concentration up to about 1 x mole (RE)z03 per mole Yzo3, except for Gd (up to about 2 X mole). The lower limit for emission detection was in the region between 10-9 and mole, depending on the rare earth element; the lower limit for Gd and Sm was mole because of poor emission. No correction of the calibration curves had to be made when the coexistence of total impurities of other rare earths in Y203 was below mole. The data indicated that spectrophotoluminescence is a suitable technique for the quantitative analysis of rare earth impurities in highly pure Yzo3.
To DETERMINE small amounts of rare earth impurities in pure yttrium oxide, an analytical technique is necessary which has high sensitivity, sharp selectivity, and ease of operation. There are several techniques for the determination of rare earths: polarography, spectrophotometry, spectrography, x-ray absorption and emission spectrometry, radiochemical techniques, and atomic absorption spectrometry. However, levels of detectability and procedure of operation for these techniques are not comparable. The number of papers dealing with spectrophotofluorometric determination of small amounts of rare earth elements in organic or inorganic solution has increased recently (I-.?), but little has been presented regarding rare earth impurities in rare earth compounds. Recently, the high luminesence sensitivity of dysprosium, terbium, europium, and gadolinium (1) E. C . Stanley, B. I. Kinnerberg, and L. P. Varga, ANAL.CHEM., 38, 1362 (1966). (2) G. Alberti and M. A. Massucci, Zbid.,38, 214 (1966). (3) T. Taketatsu, M. A. Carey, and C . V. Banks, Tulunta, 13, 1081 (1966).
in yttrium oxide crystal under x-ray irradiation has been reported by Linares et al. (4). The limits of detectability by this technique are 0.02 to 1 ppm. During a study on the concentration quenching mechanism of the luminescence of rare earth-activated yttrium oxide phosphors under the excitation of light, it was noted that the photoluminescence of Pr, Sm, Eu, Gd, Tb, Dy, Ho, Er, and Tm in yttrium oxide was sensitive and sharply selective. The possibility of adapting the technique for the quantitative determination of small amounts of rare earths in yttrium oxide has been studied in detail. The results of this work are presented here, as are details of the excitation and emission properties. EXPERIMENTAL Yttrium oxide used was 99.999 % pure (Shin-Etsu Chemical Industry Co., Ltd., Japan); other rare earths introduced as impurities were 99.9% pure. From the analytical results of the procedure described in this paper, rare earth impurities in the yttrium oxide were 1.7 X lo-' mole Tb203and 1.5 x 10-6 mole Dy203; other rare earth impurities were not detected. A desired amount of yttrium oxide and rare earth oxide impurity was dissolved in hot nitric acid to make a mixture of ions in solution. A 10% oxalic acid solution at 60" C was used as the precipitant. The precipitate was dried at 100" C and heated in air at 1200" C for 1 hour to form the oxides, using open, pure silica crucibles. Average particle size of the samples obtained was about 5 p . The emission and excitation spectra of rare earths in yttrium oxide were measured with a Hitachi Fluorescence Spectrometer MPF-2 in combination with an R-136 photomultiplier, Hamamatsu T. V. Co., Ltd., Japan. A 45" viewing mode was provided with the instrument for the measurement of the powder sample. For measuring the emission spectra, an ultraviolet band pass filter, Corning No. 7-54, which absorbed the stray light from the first monochromator, was placed before the sample holder. For measuring the excitation spectra, a suitable c ,t filter which absorbed the reflected ultraviolet radiation on the sample surface and passed the (4) R. C . Linares, J. B. Schroeder, and L. A. Hurlbut, Spectrochim. Acta, 21, 1915 (1965). VOL 40, NO. 1, JANUARY 1968
187
Table I. Main Emission and Excitation Bands of Rare Earths in YzOaand Detection Range of Impurities Transition Element Emission, mp From To Excitation, mp Detection range, mole/mole Pr 630 4 x 10-4 - 5 x 10-4 Sm 565 1 x 1 0 - 5 - 1 x 10-3 Eu 611 1 x 10-7 - 1 x 10-3 Gd 315 2 x 10-6 - 1 x 10-2 Tb 543 2 x 10-8 - 1 x 10-3 572.5 DY 1 x 10-1 - 3 x 10-3 Ho 550 4 x 10-8 - 2 x 10-3 Er 563 1 x 10-7 - 2 x 10-3 Tm 453 1 x 10-7 - 2 x 10-3
fluorescence to a second monochromator was placed behind the sample holder. A small amount (0.2 gram) of the sample to be used for the spectral measurement was taken from the center portion of the fired sample in the crucible. All of the spectra were obtained with the monochromator set to provide a resolution of 1.5 mp. Each emission spectrum was determined under light excitation that gave maximum emission intensity. The excitation spectrum was determined for the main emission band, and the measurements were made at room temperature. RESULTS AND DISCUSSION
Rare earths in yttrium oxide matrix which produced photoluminescence in the visible and near ultraviolet spectral region , Er+3, and were Pr+3, Sm+3, E d 3 , Gd+3, Tbf3, D Y + ~Ho+3, Tm+3; other rare earths had no luminescence. Inspection of the energy diagram ( 5 ) of trivalent rare earths reveals that a spectrometer sensitive to an emission in the near infrared spectral region would be required to observe intense photoluminescence of Nd+3 and Yb+3. The spectra of emissive rare earths in Y203 were generally in agreement with those reported by Ropp (6). The luminescent properties observed in this investigation are as follows: Pr+3. An intense red fluorescence of Pr+a in Y203 was observed under 280-mp excitation. A weak broad fluorescence of Pr+3in Y203 was observed at 77" K (4,but this was not detected at room temperature (6). The emission spectrum consisted of a number of narrow bands in the vicinity of 630 mp. No change of the emission spectrum was observed under other excitation wavelengths. The excitation spectrum for the red fluorescence consisted of a broad band with a maximum at 283 mp and weak bands in the spectral region from 452 mp to 499 mp. Srn+3. The emission spectrum of Y203: Smia consisted of three groups of narrow emission bands in the regions near 568, 608, and 656 mp. The groups should be assigned as the transitions from a lower radiative level 4G5/2to 6Hsi2, 6H712,and 6H9/2levels of Sm+3,respectively. Relative emission spectrum were dependent upon the wavelengths of the exciting light. The relative emission intensities in the group, which were assigned to the transition between 4G6,2and 6H5/2, were most noticeably changed. When the sample was excited by wavelengths other than that utilized in Table I, the emission band at 565 mp distinctly decreased and the band at 608 mp remained as the strongest band. The emission band at 608 mp under the excitation at 350, 375, or 410 mp was hidden in the stray light of the spectrometer at samar( 5 ) G. H. Dieke and H. M. Crosswhite, Appl. Opt., 2,675 (1963). (6) R. C. Ropp, J. Electrochem. Soc., 111, 311 (1964).
188
ANALYTICAL CHEMISTRY
ium concentrations below 1 X mole, while the 565 mM emission band was independent of stray light. Eu+~.The emission and excitation spectra of Eu+3 in Y203agreed with those reported by Chang (7,8)and by Ropp (6) who have studied in detail the spectra of the Y203:Eu phosphor. Gd+3. The main emission band of Gd+a in YzO3 is at 315 mp (9). This emission band split into two bands when the measurement was made with a spectrometer which had a resolving power greater than 1.5 mp, Tb+a. The emission spectrum of Tb+3 in Y203 consisted of a series of groups of narrow bands in the regions near 483,543, 583,623, and 666 mp. The intensities of the groups at 623 and 666 mp were weak-below one-hundredth of the intensity of main band at 543.5 mp. The relative emission intensities of the bands in groups and of the groups were changed in some degree with terbium concentrations, The excitation spectrum for the green emission at 543 mp consisted of two broad bands with maximum at 280 and 304 mp. The 304 mp band was less effective in the excitation of the 483 mp emission, so that the relative emission intensities of the groups were also changed by the exciting light. Dy+*. The main emission band of D Y +in~ Y z O at ~ 572.5 mp was narrow and the wavelength position differed somewhat from that previously reported (6). The excitation spectrum for 572.5 mp emission consisted of a broad band with a maximum at 231 mp and many narrow bands which could be identified as transitions within D Y + ~ . H O + ~ .The emission spectrum of H O +in~ Y 2 0 3consisted of a series of narrow bands in the vicinity of 550 mp. The excitation spectrum for the 550-mp emission band consisted of a large number of narrow bands ranging from ultraviolet to blue. Er+3. The emission spectrum of Er+3in Y203 in the visible spectral region consisted of a large number of narrow bands near 563 mp. The strong excitation band for the most intense emission band at 563 mp occurred at 380 mp. Tm+8. A sample activated with Tm+a had a blue fluorescence when the sample was excited by ultraviolet light from a high pressure mercury discharge lamp. The emission spectrum of Y203:Tm+3consisted of several narrow bands near 453 mp. The excitation spectrum for the 453 mp emission also consisted of several narrow bands. In Table I are given the main emission and excitation bands of the rare earths in yttrium oxide crystal. The wavelengths of the main emission and excitation bands of rare earths de(7) N. C. Chang, J. Appl. Phys., 1963, p. 3500. (8) N. C. Chang and J. B. Gruber, J. Chem. Phys., 41, 3227(1964). (9) K. A. Wikersheim and R. A. Lefever, J. Electrochem. SOC.,
111, 47 (1964).
Table 11. Accuracy of Spectrophotoluminescence Determination of D Y +in~ YzOa to Which Was Added DyzOz at a Concentration Mole per Mole of YZO3 of 1 X Sample A1 A2 A3 Bi B2 BJ c1 cz CS Av. Dy detected, mole X 0.92 1.12 0.95 1.02 1.00 1.07 1.08 1.15 1.02 1.04
tected by spectrophotoluminescence were specific and characteristic for each rare earth. The main excitation bands of Pr, Sm, Eu, and Tb were broad bands. To ascertain the origin of the broad excitation bands, Y203 : Eu phosphors, which each had a broad band and a large number of narrow bands, were prepared at various europium concentrations. The position of the main excitation band of Y203:Euphosphor shifted linearly to longer wavelengths with increasing europium concentrations, while the positions of the narrow excitation bands (which could be assigned to the transitions within 4f6 configuration) were independent of europium concentrations. It is inferred from the above results that the origin of the broad excitation band of in Y 2 0 3corresponds to transitions from f electron to d electron levels (7), because only the d electron levels are easily perturbed by surrounding ions. Similarly, the origin of the broad excitation bands of other rare earths should be assigned tof-delectron transitions. The broad excitation bands of Gd+3 and H o + ~at about 220 mp are attributable to host lattice excitation bands because the absorption edge of pure yttrium oxide crystal is at 220 mp (IO). The broad excitation band for Dy+3 in Y 2 0 3is composed of two bands which can be observed with samples containing small amounts of dysprosium. As an example, the excitation spectrum of Y203:Dy (1 X mole) is shown in Figure 1. The 220-mp band in the figure could be identified as host lattice excitation and the 231-mp band (of which the intensity was increased with dysprosium concentration) was attributable to transitions within trivalent dysprosium. All of the main excitation bands of rare earths in yttrium oxide are ascribed to direct excitation of the lanthanide ion; other excitation mechanism, such as energy transfer or energy transport ( I ] ) , are not involved in the luminescence because energy transfer between rare earth ions (12-14) is negligible at concentrations below 10-5 mole. This is of particular importance for the estimation of the concentration of rare earth ions in Y 2 0 3 . Calibration Curves. To investigate the possibility of adapting the spectrophotoluminescence of rare earths to analytical determinations, a sample of Y 2 0 3with various concentrations of rare earths was prepared. The main emission intensities were determined as a function of concentration under light excitation (Table I). The emission intensities were detected using a spectrometer with a resolution power of 3 mp. The emission intensities increased linearly with rare earth concentrations up to about 1 X mole (RE)*03 per mole Y203,except to about 2 x mole for Gdf3; at higher concentrations saturation occurred. This saturation referred to as concentration quenching, may result from a decrease in the concentration of isolated single activator ions ~
~~
(10) N. C. Chang, J . Chem. Phys., 44, 4044 (1966). Th. P. J. Botden, Philips Res. Rept., 6 , 425 (1951). (12) L. F. Johnson, L. G. Van Uitert, J. J. Rubin, and R. A. Thomas, Phys. Reu., 133, A494 (1964). (13) L. G. Van Uitert and S. Iida, J. Chem. Phys., 37,986 (1962). (14) L. G. Van Uitert and L. F. Johnson, Ibid.,44, 3514 (1966). (11)
0N O PJN
M
.-
E,
200
U
m
300 Wavelength
400 in
my
Figure 1. Excitation spectrum for 572.5-mp emission of YzO3 :Dy 1X
mole/YnOa
(15). However, account must also be taken of the presence
of a filter effect due to the absorption of exciting light by the luminescent centers. At a concentration below about 1 X mole per mole YzO3, all data fit well on a straight line of approximately unity slope. From these results, it should be predicted that the exciting light is absorbed by a constant volume of the powder sample. The linear region of the curves can serve as calibration curves in analytical determinations. Lower limits for emission detection are between 10-9 and 10-8 mole, depending on the rare earth element; the higher limits of detection for gadolinium and samarium are mole because of poor emission. The emission intensities are conventional values indicated by a recorder, and are not absolute values. The values, therefore, change with the measuring conditions such as slit width of spectrometer, intensity of exciting light source, and photomultiplier voltage. The values are also sensitive to preparation conditions of the samples, such as firing temperature, firing times, and precipitating conditions. Therefore, the detectable limits of the emission may be extended to lower concentrations than those mentioned, but the extending investigation is not included in this paper. Correction of the calibration curve was not necessary when yttrium oxide, which contained total rare earth impurities at about 10-5 mole, was used for the host crystal. The presence of small amounts of rare earths in yttrium oxide would have little effect on the spectra if the samples were excited by wavelengths of the main excitation bands. The emission intensities were somewhat decreased when the yttrium oxide contained total impurities above mole. Details of these interferences are being studied. If samples contain various amounts of Tb+3, Dyi3, H o + ~and , Erf3, which are simultaneously excited by the (15) L. G. Van Uitert, “Luminescence of Inorganic Solids,” Chap. 9, Academic Press, 1966. VOL. 40, NO. 1 , JANUARY 1968
189
Elements Impurity, ppm
Table 111. Rare Earth Impurities in High Purity YzO3 Determined by Spectrophotoluminescence Pr Sm Eu Gd Tb DY Ho Er 25
...
0.56
...
same exciting wavelength and which have overlapping emission bands, good analytical results are obtained by determination of the respective excitation spectra which differ markedly. Error. To estimate the error caused ‘Jy sample preparation, nine samples were prepared by three persons (A, B, C ) . Residual rare earth impurities in the Y 2 0 3used in this test were 4 X mole Dy203and 9 X 10-6 mole Tbz03; the added impurity was dysprosium at 1 X mole. The dysprosium concentrations were used to estimate the error. Data obtained are given in Table 11. Accuracy of determination by spectrophotoluminescence was good with an error +lox. The time for one sample preparation was 3 to 4 hours ; operation time for the spectrophotoluminescence determination was 10 to 20 minutes. If several samples are prepared simultaneously, time requirements may be reduced.
0.11
1.1
0.38
0.74
Trn
...
In Table I11 is given the result of a quantitative determination of rare earth impurities in 99,999x pure Y203 (Spex Industries, Inc.). Determinations of Dy, Er, Pr, Tb, Eu, Sm, and Tm were made by the emission spectra. Sm, Gd, and Tm were below sensitivity limits. ACKNOWLEDGMENT
The authors thank Satoru Nishikawd for his assistance in sample preparation, and measurements and also Kiyoshi Morita and Hiroshi Sunahara, Government Industrial Research Institute, Nagoya, for their helpful discussions. RECEIVED for review March 1, 1967. Accepted September 22, 1967.
Homovanillic Acid as a Fluorometric Substrate for Oxidative Enzymes Analytical Applications of the Peroxidase, Glucose Oxidase, and Xanthine Oxidase Systems George G. Guilbault, Paul Brignac, Jr., and M a r y Zimmer
Department of Chemistry, Louisiana State Unicersity in New OrIeans, Lakefront Campus, New Orleans, La, 70122 Fluorescence methods are described for the assay of the oxidative enzymes-peroxidase, glucose oxidase, and xanthine oxidase-based on the conversion of the nonfluorescent homovanillic acid to the highly fluorescent 2,2’-dihydroxy-3,3‘-dimethoxybiphenyl-5,5’-diacetic acid. The initial rate of formation of this fluorescent compound is measured and related to the activity of the enzymes. The substrates of these enzyme systems-peroxide, glucose, hypoxanthine and xanthine-can be determined in microgram concentrations in biological samples as blood and urine with a precision and accuracy of less than 3%. Finally, analytical methods are described for various inorganic and organic substances which inhibit these enzyme systems: cyanide, sulfide, dichromate, sulfite, Cu(ll), Fe(ll or Ill), Mn(ll), Pb(lI), Co(ll), Cd(lI), hydroxylamine, and cystine. The reagent homovanillic acid is very stable and can be used for several months.
SEVERAL ARTICLES have described the development of analytical procedures for the determination of various inorganic and organic substances using enzymes as analytical reagents ( I ) . Advantage is taken of the extreme sensitivity (frequently in the nanogram to picogram region) and specificity of the enzyme, thus giving the two factors most desirable in any analytical technique. In general the acceptance of enzymes as useful analytical reagents has been slow, mainly because of the lack of good sensitive analytical methods for their determination. In this (1) G.
1%
Guilbault, ANAL.CHEM., 38, 527R (1966). 0
ANALYTICAL CHEMISTRY
vein Guilbault and Kramer have been particularly active in developing fluorescence methods for enzyme systems based on a measurement of initial reaction rates (2-6). Such analyses resulted in a two- to threefold increase in sensitivity over conventional colorimetric, manometric, or pH techniques, and in a considerable saving in analysis time (inasmuch as a typical reaction rate analysis can be performed in 1 to 3 minutes). Several enzymic methods have been used for the determination of peroxide ( 7 - l l ) , glucose (12), and xanthine (13) based on conventional spectrophotometric procedures. (2) G. Guilbault and D. N. Kramer, ANAL.CHEM., 36, 409 (1964). (3) Ibid., 37, 120 (1965). i4j Ibid.;p. i m . (5) D. N. Kramer and G. Guilbault, Ibid.,35, 588 (1963). (6) Ibid., 36, 1662 (1964). (7) B. Chance and A. C. Maehly, “Methods in Enzymology,” S. Collowick and N. Kaylan, Eds., Vol. 2, Academic Press, New York, 1955, p. 764. (8) B. Chance and A. C. Maehly, “Biochemists Handbook,” C. Long, Ed,, Van Nostrand, Princeton, N. J., 1961, p. 384. (9) N. B. Chapman and B. C. Saunders, J . Chem. SOC.,1941, p. 496. (10) G. G. Guilbault and D. N. Kramer, ANAL.CHEM., 36, 2494 (1964). (11) R. Wilstatter and A. Stoll, Ann., 416, 21 (1927). (12) H. Bergmeyer, “Methods of Enzymic Analysis,” Academic Press, New York, 1963, p. 123. (13) Ibid.,p. 495.
