only a small fraction of the reactant is protonated, resulting in a small heat rise, and errors in correction terms cause loss in precision and accuracy. In this case, the heat rise (and thus precision and accuracy) can be increased by increasing the titrant and solution concentrations. When the overall reaction constant lies between about 10-1 and lo4, conditions can be adjusted so that there is both sufficient curvature in the thermometric titration curve and a large enough fraction of the reactant protonated to allow the calculation of precise and accurate log K values. When the overall reaction constant is larger than lo4,there is not sufficient curvature in the thermometric titration curve to allow calculation of precise and accurate log K values for the reaction. Also, small systematic errors in the solution and titrant concentrations cause large errors in the calculated log K values for the reaction. However, precise and accurate A H o values can still be obtained from the thermometric titration data, Small systematic errors may cause large errors in AH" when log K is less than approximately - 1 because of insufficient heat released by the reaction. The use of thermometric titration data to determine log K values for proton ionization reactions is advantageous because there is no liquid junction potential involved in the measurements and the log K value can be determined in non-
aqueous solvents (31) without concern for the effect of the solvent on the sensing elements. It has the further advantage that A H and AS values are determined simultaneously with log K for the reaction. The only prerequisites for application of the entropy titration method are that the reaction constant be in the range from about 10-l to l o 4 for the reaction, reactant titrant = product, and that the heat of this reaction is not equal to zero.
+
ACKNOWLEDGMENT
The authors express their appreciation to Lee. D. Hansen, Department of Chemistry, University of New Mexico, for many helpful suggestions in the preparation of this manuscript. RECEIVED for review August 28, 1967. Accepted October 23, 1967. Work supported by National Institutes of Health Grant RG-9430-06, and (in part) by Public Health Service Research Career Development Awards to James J. Christensen (Award No. 1-K3-GM-24, 361-01) and Reed M. Izatt (Award No. 1-K3-GM-35, 250-01). (31) B. Nelander, G. Olofsson, and S. Sunner, Abstracts, 21st Annual Calorimetry Conference, Boulder, Colo., p. 23 (1966).
An Automatic Group Separation System for the Simultaneous Determination of a Great Number of Elements in Biological Material Recovery and Reproducibility Studies Knut Samsah1,l Per Olov Wester, and Ove Landstrom AB Atonienergi, Stockholin, Sweden An automatic group separation system for the determination of about 40 elements has been tested on biological material. The method is based on ionexc ha ng e se pa ratio n a nd extractio n c hro matog r a p hy combined with gamma spectrometry. Sixteen groups of elements suitable for gamma spectrometry are obtained. The reproducibility and recovery of added activities to biological samples of the following 32 elements have been studied: Ag, As, Ba, Br, Ca, Cd, Ce, Co, Cr, Cs, Cu, Fe, Ga, Hf, Hg, In, K, La, Mn, Mo, Na, P, Rb, Sb, Sc, Se, Sm, Sr, Th, U, W, and Zn. Recovery values 590% were obtained for all elements except In, Se, Th, and U. A standard error of the mean value 5 3% was obtained for all elements except Ba and Th. The reproducibility of the method was also tested by repeated analysis of a biological standard. The precision was better than 10% for all elements except Sb and Th, and for most of the elements better than 5%.
THEADVANTAGE and usefulness of neutron activation analysis in the determination of trace elements in various materials have been thoroughly documented. The most simple way of performing the activation analysis is by instrumental analysis-viz., by measurement of the radioactivity directly Present address, Gesellschaft fur Strahlenforschung m.b.H., 8042 Neuherberg, Munich, West Germany.
after the activation of the sample, without any chemica treatment. This technique is limited mainly to the determination of a few elements depending on the sample composition. The activation analysis of most trace elements in biological material, or in other materials of complicated composition, generally requires some kind of chemical separation, Several chemical methods have therefore been worked out for the determination of one or a few trace elements at a time, commonly involving some kind of precipitation technique. In recent years different group separation systems have been described which permit the simultaneous determination of a great number of trace elements (1-5). In this laboratory we have developed and applied Samsahl's group separation system for trace element studies of biological (1) G. Aubouin, J. Diebolt, E. Junod, and J. Laverlochere, Proc. Intern. ConJ Modern Trends in Actication Analysis, College Station, Texas, 1965, in press. (2) F. Girardi, M. Merlini, J. Pauly, and R. Pietra, Proc. Symp. Radiochemical Methods of Analysis, Vol. 2, p. 3, Salzburg, 1964. (3) V. V. Moiseev, R. A. Kusnetsov, and A. J. Kalinin, Proc. Intern. Cot$. Modern Trends in Actication Analysis, College Station, Texas, 1965, in press. (4) W. J. Ross, ANAL.CHEM., 36, 1114 (1964). ( 5 ) K. Samsahl, Aktiebolaget Atomenergi, Stockholm, Repts. AE-54 (1961), AE-56 (1961), and AE-82 (1962). VOL. 40, NO. 1, JANUARY 1968
181
(6-13) and geological materials (14). A great disadvantage, however, has been that, like most other group separation methods, it is very time-consuming. To reduce this disadvantage an automatic separation system has been developed (15-17).
The purpose of the present study was to apply the system to trace element determinations in biological material and to
investigate the recovery and reproducibility of the method. EXPERIMENTAL
Group Separation System. APPARATUS.The dissolution and distillation were performed with a 15-ml pear-shaped borosilicate flask, surrounded by a glass tube to ensure sufficient isolation during the heating cycle. The flask, heated by a Bunsen burner, was connected to a 30-ml receiver, and the latter to a 150-mm-long reflux condenser and a 5 4 NaOH-absorption trap. The apparatus used for ion-exchange and extraction chromatography is pictured in Figure 1 and consists of one unit (500 mm long, 300 mm high, and 300 mm broad). The central parts of the apparatus are a system of glass barrels with pistons and three series of ion-exchange or extraction chromatographic columns. The pistons, driven by compressed air, will simultaneously deliver the sample solution and the washings at the beginning of a series, as well as appropriate solutions between the columns. The mixture of the effluent from a column and the injected amount of solution are flowed through a mixing coil before entering the next column in the series, thus ensuring homogeneity of the influent. The apparatus, which performs the separations almost fully automatically, is made of Perspex with a few selected parts of Teflon. A more comprehensive description of the working principle, as well as the constructional details of this kind of apparatus, have been given elsewhere (15). REAGENTS.Buffer solution; 600 ml of 4N CH,COONa is mixed with 120 ml of 10N NaOH and 80 ml of 5N NaBr. CHELATING RESINS. Bio-Rad Chelex 100, 100-200 mesh, preparation with acid solution (13, Figure 2). About 100 ml of the resin is thoroughly freed from colloidal particles. The rest is treated with a composite solution, corresponding to the influent in practical runs, and consists of 1 part by volume of 8N NaOH, 2 parts of 8N HCI and 4 parts of the buffer solution described above. After the resin has ceased to shrink it is kept in stock in the same solution, The suitability of storage periods exceeding 2 to 3 days has not been tested. Bic-Rad Chelex 100, 100-200 mesh, preparation with alkaline solution (14, Figure 2). The resin i s conditioned and stored in the same way as described above except that twice the amount of 8N NaOH is used for the preparation of the washing and storage solution. ANIONEXCHANGE RESINS. Dowex 2 X 10, chloride form, 2 W O O mesh (3-9, 12, Figure 2). About 200 ml of resin is (6) K. Samsahl and R. Soremark, Proc. Intern. Cmf. Modern Trends in Acfiwlion Analysis, College Station, Texas, 1961, p. 149. (7) K. Samsahl, D. Brune, and P. 0. Wester, Intern. J. Appl. Radiation Isotopes, 16, 273 (1965). (8) P. 0. Wester, Smnd. 3. Lab. C h . Inoesr., 357, 17 (1965). (9) P. 0. Wester, Biochim. Biophys. Acta, 109, 283 (1965). (10) P. 0. Wester, Acta Med. Smnd., 178,765 (1965). (11) Ibid., p. 789. (12) Ibid., Suppl. 439. (13) D. Brune, K. Samsahl, and P. 0. Wester, Clin. Chim. Acta, 13, 285 (1966). (14) 0. Landstrom and C. G. Wenner, Akfiebolagef Afomenergi, Stockholm, Repls. AE-204 (1966) and AE296 (1967). (15) K. Samsahl, Nukleonik, 8, 252 (1966). (16) K. Samsahl, Akriebolanef Afomenergi, Stockholm, Rept.
AE247 (1966). (17) K. Samsahl, ANAL.CHEM., 39,1480(1967).
182
ANALYTICAL CHEMISTRY
Figure 1. Apparatus used for automated chemical separations
suspended in water and colloidal particles are removed by several decantations. The rest is treated in a column with 1 liter of 1N HCI at a rate of 4 ml/minute and washed with 3 to 4 column-volumes of water. The prepared resin is stored under distilled water in a stoppered polyethylene bottle. All the column fillings described below were stored in the same way. Dowex 2 X 10, sulfate form, MO-400 mesh (2, Figure 2). Fifty milliliters of resin is prepared with 0.5 liter of 1 : l O H S 0 4 and washed with the same amount of distilled water. INORGANIC ION-EXCHANGER. Bio-Rad ZP-1, 100-200 mesh (15, Figure 2). About 200 ml of the material, after removal of colloidal particles, is transferred to a column and slowly washed with 4N sodium acetate until the pH of the effluent falls to between 4 and 5. The exchanger is finally rinsed with distilled water and may be stored for at least one week. PARTITION CHROMATWRAPHIC COLUMN MATERIAL(10, 11, Figure 2). Celite 545 (Johns-Manville) is mixed with water several times to remove colloidal particles. After drying, the kiselguhr is siliconized with dimethyldichlorosilane vapors and again dried. Fifteen grams of the material is then stirred in a solution of 1.5 grams of di-(2-ethylhexyl) orthophosphoric acid (HDEHP) in 50 ml of isopropyl ether. The hulk of the solvent is volatilized by standing at room temperature and the last traces are removed at reduced pressure. CHEMICAL SEPARATION. The biological sample was added to the distillation flask together with carriers corresponding to 50 pg each of As, Hg, Sb, and Se and 1 pg of the rest of the elements studied. Exceptions were N p and Pa, which were studied in the carrier-free state. The organic material was charred with 2 ml of 30 to 33% fuming H2S04,and the hulk was destroyed with 1 ml of 30% H202. After heating to incipient fumes of SOa,the solution was made completely clear by adding H,O, in small drops. As, Hg, Sb, and Se were then distilled from the solution as bromides with 48% HBr added in three 0.5-ml portions. Accompanying 82Br activity was volatilized from the receiver flask after HIOz addition and finally absorbed in 6N NaOH. Subsequently the strongly acid distillate was adjusted to about 3.5N in HSO, and 0.1N in HCI. From this solution Hg was s e p arated on the first column in a series of three small coupled anion-exchange columns. Sb is retained on the second column from stronger HCI, whereas As and Se are separated from a strong mixture of HCI and HBr (Figure 2). The adjustment of the influent solutions is in these cases, as well as in the following ones, automatically effected by means of the machine with piston drive described above. Further Group Separation. The trace elements remaining in the HISOl solution in the distillation flask are split into
1. NaOH
distillation
____HpOp
+ KBr
H O
absorption B r , (1) ,(Os
-52-
A
I
-
5 . Dovex 2 ( I r ) , b, Pa,
w
-
10. DEW-extr.
chromatogr. Hf, S c , ( Z r )
J
I
---'-t 0.025
2 HBr
1 pH : 3.5
I
r
-
KBr
6. m v e x
2
Cd
_.
- 0.3%
XBr
-- 4.5
t
LE-
2.3 Ii H C 1 HBr
-
4 . Dovex
12. Dovex 2
2
A s , Se
Ag >
NaOH 4
PH = 5-5.5
8. Dovex 2 Fe, Ca, I n
I-
IiaOH
pH :14
9. Dovex 2 Co, Cu, Np
pH = 4-4.5
,
-W O 3 ,
;!ai!* PO4
Elements in brackets have n o t been i n v e s t i g a t e d i n t h e p r e s e n t study.
11
Figure 2. Scheme of the chemical separation system
VOL. 40, NO. 1, JANUARY 1968
183
Element Ag As Ba Br Ca cd Ce co Cr cs
As76
Ba131 Bra2
Fe Ga Hf Hg In
Ca47 Cd113 Ceir1 COG0 Cr51 Cs'34 cue4 FeS9 Ga7 Hf181 Hg 2o In114m
K
K42
La Mn Mo Na P Rb Sb sc Se Sm Sr Th
Lalro Mn56 Tcggm Na24 P32 Rbs6 Sb1Z4 Sc46 Se15 Sm153
cu
U W
Zn a
Table I. Recovery and Reproducibility Test with Added Activities to Biological Samples Standard Standard Energy Mean value deviation of a error of Nuclide used measured MeV of yield single value mean value Agllo 0.66 90 7 2
Srs3
Pa233 NpZ39 W 187
Zn63
0.55 0.22 0.17 1 31 0.52 0.14 1.33 0.32 0.60 0.51 1.29 0.83 0.48 0.28 0.19 1.53 1.60 0.84 0.14 1.38, 2.76
Bremsstrahlung 1.08 0.60 1.12 0.26 0.10 0.51 0.31 0.106 0.68 1.11
90 90 92 98 93 97" 99 97 96 98 91 90 92 96 70 93 91" 92 97 96 92 97 91 95 85 92" 95 79 84 90 100
6 10 7 5 5 7 9 2 2 1 6 3 4
1 6 6 7 2 5 5 5 4 2 5 5 5 4 14 5 5 3
2 4 2 2 2 2 3 1 1 2 2 1 2 3 2 2 3 1 2 2 2 1 1 2 2 2 2 5 2 2 1
Number of determinations 8 6 7 10 8 10 7 11 7 6 10 7
1 6 8 9 10 6 6 6 7 7 6 7 5 9 5 6 7 10 6 11
The values are added from ion-exchange columns 11, 12, and 13, Figure 2.
two main groups by means of selective sorptions on three coupled anion-exchange columns from 8N HCI solution (5, 6, 7, Figure 2). The effluent from the last column is continuously passed through a second stack of columns, which automatically subdivides the radioactive constituents into seven groups. According to Figure 2 , Hf and Sc are first separated from 8N HC1 solution by extraction chromatography, using a small column of kiselguhr impregnated with HDEHP (10, Figure 2). Lanthanum and rare earths are then adsorbed in a similar way from the neutralized effluent (11, Figure 2). In the third step anion-exchange resin selectively retains silver as bromide complex (12, Figure 2). Further Chelex 100 chelating resin separates chromium and manganese from faintly acid solution (13, Figure 2 ) and the alkaline earths from strong alkali (14, Figure 2 ) . Finally the sample solution is taken back to the buffering interval of the acetate system and flowed through a column of zirconium phosphate. The exchanger retains traces of K, Rb, and Cs (15, Figure 2), whereas radioactive Na and P in the effluent form the seventh group of elements separated (16, Figure 2). The trace elements initially adsorbed on anion-exchange resin from 8N HCl(5, 6, 7, Figure 2) are split into five groups by combining an H2SOaelution step with selective sorptions on small anion-exchange columns from successively increasing concentrations of halogen acids. Thus 25 ml of 0.5N H8.04 containing 0.3 H202 will, a t a rate of 0.5 ml/minute, elute all the trace elements absorbed on the first column in the series except Mo, Pa, and W ( 5 , Figure 2). The machine with piston drive described above will simultaneously adjust the influent solutions in the series, thus effecting the separation of Cd from 0.025N HBr (6, Figure 2), of Zn from 0.7N HCl 184
ANALYTICAL CHEMISTRY
(7, Figure 2), of Fe, Ga, and In from 3.6N HCl(8, Figure 2) and of Co, Cu, and Np, finally, from 8.1N HCI (9, Figure 2 ) . The time needed for performing a complete separation by a single person is 2 hours, provided that 45 minutes are used for the dissolution and distillation step. However, for studies of only one or a few trace elements the procedure may be shortened in various ways, depending on the problem. The chemical separation method has been or will be described in greater detail elsewhere (15, 16, 17). MEASUREMENT TECHNIQUE. The gamma spectrometric measurements were performed with a 512-channel pulse height analyzer attached to a 3- x 3-inch NaI well-type crystal. After the separations the different resins were well mixed and transferred to plastic cans, which were then measured either in the well or close to the crystal. For quantitative evaluation of the activity the areas of characteristic photopeaks were integrated, the energies of which are given in column 3, Table I. In the case of complex spectra, a spectrum stripping technique was applied or duplicate measurements were made. Recovery and Reproducibility Tests with Added Activities. Elements or compounds of analytical reagent grade were irradiated for periods of 2 to 5 days with a thermal neutron flux of about 2 x 1013 n/cm2 second. The activated samples were in most cases individually dissolved in mixtures of H2S04and Hz02. After heating to incipient fumes of SO3 appropriate dilutions were done with concentrated H2S04. In a few cases HC1 or weak NaOH was used as an aid in the dissolution process. One hundred microliters of the different standard solutions was used for a single run. The weight amounts of the standards were in each case chosen as small
Table 11. Reproducibility Test by Repeated Analysis of a Biological Standard Values expressed in counts per 100 mg standard.
. 103
5.21 5.75 5.35 2.73. Br 2.82 2.91 3.15 . Ca 2.99 2.65 2.36. Cd 2.16 2.10 4.40. Ce 4.53 4.07 1.00. c o 1.02 1.09 3.48. Cr 3.38 3.59 3.59. Cs 4.08 3.76 3.23. c u 3.34 3.34 2.32 Fe 2.22 2.57 5.88. Ga 5.38 5.40 4.31 Hg 4.82 4.46 8.60. K 9.21 9.54 7.30 La 7.86 7.28
As
5.44
. 103 i 0.28 . 103
2.82
. 105 i 0.07 . 105
2.93
. 104 =t0.26 . 104
2.21
. 103
4.33
. 103
1.04
. 104 i 0.05
3.48
. 103 i 0 . 1 0 .
3.81
. 103 + 0.25
105
104
103
103
104
103
103
0 . 1 4 . 103
i:0 . 2 4 . 103
. 104
. 103
3 . 3 0 . 1 0 3 h 0 . 0 6 . 103
2.37
. 103 i 0.18 . 103
5.55
. 103 * 0.22 . 103
4.53
-
9.12
. 103 i 0.48
7.48
. 103 =t0.33 . 103
103
. 103 103
. 103
lo3
i 0.26
e
. 103
.
.
5.79 103 i 0.48 103 5.33 3 . 4 2 . 103 Mo 3.68 3.58 . 103 i= 0 . 1 4 . 103 3.64 7.32 . 104 Na 7.45 7.42 104 A 0.09 . 104 7.50 1.42 . 106 P 1.32 1.36 . 106 i 0.05 105 1.35 7 . 5 5 . 103 Rb 7.50 7.58 103 i 0.10 . 103 7.69 5 . 5 8 . 103 Sb 4.36 5.37 103 0.93 . 103 6.18 2.82 103 Sc 3.04 2.93 . 103 i= 0.11 . 103 2.92 7.90 . 102 Se 9.34 8.73 . 102 i 0.75 * 102 8.96 3 . 2 6 . 103 Sm 3.04 3 . 1 9 . 103 =k 0.13 103 3.27 6 . 0 0 . 104 Sr 5.90 5.72 104 i= 0.41 104 5.25 1.62 103 Th 1.61 1 . 4 4 . 103 i 0 . 3 0 . 103 1.10 2.73 . 104 W 2.73 2 . 7 4 . 104 i 0.01 . 104 2.75 1.41 104 Zn 1.42 1.41 . 104 i 0.01 . 104 1.41
.
+
.
.
.
103
103
. 103
6.30
Mn 5.73
lo3
. 103
as possible in order to obtain test solutions with trace element concentrations of the same order of magnitude as in practical runs with irradiated biological material. Human heart and liver tissue were chosen as examples of biological material. This material was added to the distillation flask together with appropriate amounts of activities from different standard solutions. The chemical separation was then performed as described. The same amounts from the same standard solutions were used as reference activities. To be comparable with the different resins obtained during the experimental runs, the reference samples were carefully prepared to ensure equal geometry. Reproducibility Tests by Repeated Analysis of Activated Biological Material. For this test a biological standard sample (scale provided by H. J. M. Bowen) was used, About 300 mg was irradiated in the R2 reactor at Studsvik with a thermal neutron flux of 2 x 1013 n/cm2second for 48 hours. The sample was then thoroughly mixed by shaking and three parts were taken out and weighed. The three samples were then analyzed as described above.
+
.
.
.
RESULTS
The results of the recovery and reproducibility tests with added activities are presented in Table I. The 32 elements investigated are listed alphabetically in column 1 and the nuclides used are in column 2. The energies used .in the activity measurements are given in column 3. Column 4 contains the mean values of the yield expressed in per cent of the added activities. The standard deviation and the standard error, presented in columns 5 and 6, were calculated by means of the formulas d/s(x - 5)z/(n - 1) and d s ( x - 5)"n(n - l), respectively. The last column contains the number of determinations. As seen from column 4, the recovery values obtained were, with a few exceptions, equal to or better than 90%. The values of some elements, however-viz. In, Se, Th, and U-were found to be lower than 90% and in the case of In as low as 70%. The standard error of the mean value was, for nearly all of the elements VOL 40, NO. 1, JANUARY 1968
185
studied, equal to or less than 3 % and for most of the elements 2 or less. A somewhat higher standard error was, however, obtained for Ba and Th. The results of the reproducibility tests by repeated analysis of the biological standard are given in Table 11. Twentyseven elements were determined in three samples. The values presented express the total number of integrated counts, recalculated to be valid for a 100-mg irradiated sample. The total number of counts in every measurement was, if possible, of an order of magnitude such as to reduce the statistical uncertainty in the measurement to less than 1%. Different times of measurement varying from 1 to 200 minutes were accordingly used. The mean and the standard deviation are presented beside the values of the elements in the table. A reproducibility better than 3 ~ 1 0 %was obtained for all but a few elements, and for more than half of the elements the reproducibility was equal to or better than 5 %. Some elements, howevere.g., Sb and Th-showed a high deviation. Accuracy Test. All the elements studied in the reproducibility test of the biological standard have also been determined quantitatively. The possibility of determining simultaneously a large number of trace elements in different materials of high complexity-e.g., biological-has been very useful (6-14), but the methods have been very time-consuming. In recent years much effort has been devoted to the development of more rapid techniques. Measurement techniques, evaluation techniques, and chemical separation techniques have been subjected to efficiency studies. Sophisticated methods have been developed in the field of measurement techniques-e.g., multidimensional gamma-ray spectrometry (18). Using this technique Thomas succeeded in simultaneously determining 8 trace elements and 3 bulk elements in cancerous and noncancerous tissues (19). Other authors-e.g., Aubouin and Laverlochere ( I ) , Girardi, et al. (2) and Kalinin et al. (3)-have developed automatic or semiautomatic group separation systems for different kinds of materials. With the present automatic group separation system for biological material about 40 elements may be detected (Figure 1). This study has, however, been restricted to the 32 elements which we have earlier determined in biological material. The number of quantitatively determinable elements, however, depends on the composition of the material. Thus, in the reproducibility studies of the biological standard, 27 elements were determined. The high activity of Ca4’ and Srs5in this material disturbed the measurement of Ba131. Hflsl was poorly detected in view of the Sc content. Further, the activities of Co60 and Cu64 made the measurement of the low activity of Np*39 uncertain. The activities of the elements Ag and In were too low to allow a satisfactory reproducibility test. With the nonautomatic group separation system which we have used up to now, the separation time has been 1.5 to 3 days. The corresponding time for the automatic system is about 2 hours. However, the total time for performing a single analysis has, of course, not diminished on the same scale. The measurements and the calculations have not yet been subjected to efficiency studies. The short separation time in the automatic method, however, enables measure-
ments to be made several days earlier than is possible with the nonautomatic method. This brings both a gain of time in the measurements and permits the determination of shorterlived nuclides. Further, the possibility of simultaneous use of several automatic apparatus may also be mentioned. The reproducibility and recovery of the nonautomatic method have been tested in an earlier study and were found to be satisfactory (20). The results of a corresponding study with added activities by the automatic method show recovery values and standard errors of the mean of the same order of magnitude-viz. recovery values of 90% or better and standard errors of 3 or less for most-of the elements. The recovery value of Se was in the previous study 80% and in the present study somewhat better, With the automatic system some more elements have been studied than by the previous method-viz. Ga, Hf, In, Mn, Sr, Th, and U. Among these new elements, Ga, Hf, Mn, and Sr showed recovery values of 90% or better. In, Th, and U, however, showed recovery values of less than 90% and in the case of In as low as 70 %. These three elements were distributed in the chemical separation system (Figure 2). In was found mainly on ion-exchange column 8 but also to some extent in the eluate after column 9, Th(Pa) mainly on column 5 but also on columns 8 and 9, and U(Np) mainly on column 9 but also on columns 5 and 6. In most irradiated biological material, at least in human tissues, the activities of In114m, Np239,and Pa233are extremely small and hardly detectable. Thus the distribution of these elements cannot cause any major masking-effect problems. Some other elementsviz. Ce, La, and Sm-were also found to be distributed in the automatic separation system. They were mainly adsorbed on ion-exchangecolumn 11 but also to some extent on columns 12 and 13. The distribution was irregular, and it was necessary to add together the activities on the different ion-exchange columns. Thus the recovery values of Ce, La, and Sm presented in Table I are added values. In practical runs this distribution will hardly cause masking effects. In the reproducibility test of the biological standard (Table 11) the values of all elements except Sb and Th were found to differ less than *lOz and in most cases less than +5%. In the case of Th a large deviation was also found in the reproducibility test with added activities. We have no explanation of the large deviation of Sb found in the reproducibility test of the biological standard, and it is not in agreement with the reproducibility found for Sb in the test with added activities. Further studies of the reproducibility of Sb are therefore planned. In determining the amounts of the different elements in the biological standard various sources of error were considered-e.g., flux-gradients, flux-depression, self-shielding and interfering reactions. Because of the low uranium content of the biological standard the neutron-induced fission products could be neglected. About three years of practical experience has shown that the apparatus used for the chemical separations has several important advantages as compared to peristaltic pumps, which may also in principle do the same work (16). First, the present machine with piston drive will, in general, work in a much more exact way and almost completely independent of the strength of acids and alkalis used, as well as of differences in flow-resistance of the ion-exchange columns. Second, depending on the diameters of the barrels, liquids
(18) R. W. Perkins, Nucl. Instr. and Methods, 33, 71 (1965). (19) C . W. Thomas, Battelle Northwest, Richland, Wash., Rept. BNWL-235-2, p. 164 (1966).
(20) P. 0. Wester, D. Brune, and K. Samsahl, Intern. J . Appl. Radiation Isotopes, 15, 59 (1964).
DISCUSSION
186
0
ANALYTICAL CHEMISTRY
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
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