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An improved method for the ion-exchange chromatographic separation of 32P-labelled oxo-anions of phosphorus produced in neutron-irradiated orthophosphates
J. inorg, nucl. Chem., 1972, Vol. 34. pp. 1409-1415.
AN
Pergamon Press.
Printed in Great Britain
IMPROVED METHOD FOR THE ION-EXCHANGE CHROMATOGRAPHIC SEPARATION O F 32p_ LABELLED OXO-ANIONS OF PHOSPHORUS PRODUCED IN NEUTRON-IRRADIATED ORTHOPHOSPHATES
M A M O R U T O M I N A G A , * T E R U M A S A N A K A M U R A and S H I G E R U O H A S H I Department of Chemistry, Faculty of Science, Kyushu University, Hakozaki, Fukuoka, Japan (Received 18 May 1971)
A ~ t r a c t - T h e separation of 3sP-labelled oxo-anions of phosphorus produced in neutron-irradiated orthophosphates by means of ion-exchange chromatography was investigated. The fifteen kinds of oxo-anions of phosphorus were separated by using Dowex 1 - × 8 resin and sodium chloride solution as eluent in the order of 'P-, ~P,-, 3p., 4p_4p_, 2p_4p_ .~p_o_3p_, 3p_o_~p_" 5p_, ~p_3p,4p_, 3p_o_4p_4p_ ' 5P-O-4p-4p-, 5P3-, sP4-, 5P5- and 5P3m-anions. This method was applied to hot-atom chemistry of phosphorus and a complex mixture of 3zp recoil species in neutron-irradiated potassium dihydrogen orthophosphate was successfully separated. INTRODUCTION
IT IS WELL known that various kinds of 32P-labelled oxo-anions of phosphorus are produced by recoil reactions in neutron-irradiated solid inorganic phosphorus compounds [ 1]. In our previous paper [2], it was indicated that some kinds of oxoanions of phosphorus must be added as carriers to a sample solution in order to separate 3~p recoil species in neutron-irradiated inorganic phosphates by means of anion-exchange chromatography. A rough estimate obtained by this method for the distribution of 32p recoil species in neutron-irradiated alkali and ammonium orthophosphates was also reported/3/. The anion-exchange chromatography employed in the previous work [3] gives a good separation of the following species; ,p, ~p,_, 3p_, 4p_4p_, 2p_4p_, P-O-P (as overlapped peaks), 5P3-, '5P4-, 5P5- and 5P 6a n i o n s (as for these abbreviated notations, see the experimental section). It fails, however, to seperate the three kinds of P-O-P-anions, i.e. 3P-O-3p-, 3P-O-sP- and '~P-O-~P- (5P2-) anions, and to give evidence for the existence of other expected a2p recoil species such a s 3 p - o - 4 p - 4 p - , 5P-O-4p-4p-anions and some analogues to triand/or tetraphosphate, e.g. 3P-O-~P-O-sP- and 3P-O-sP-O-aP-anions, in which one or two 5P-atoms of triphosphate are replaced with 3P-atoms. Fenger[4] has suggested that the peaks located between 3P-O-~P- and sP,-anions in the paper electrophoretic analysis of neutron-irradiated ammonium dihydrogen orthophosphate may be assigned to the latter two species. It is important to determine that detailed distributions of 32p recoil species in *Present address: National Research Institute for Pollution and Resources, 188, Kotobukicho, Kawaguchi, Saitama, Japan. I. M. Halmann, Chem. Rev. 64, 689 (1964). 2. K. Ujimoto, T. Nakamura, H. Asada, N. Yoza, Y. Takashima and S. Ohashi, J. inorg, nucl. Chem. 32, 3177 (1970). 3. T. Nakamura, K. Ujimoto, N. Yoza and S. Ohashi, J. inorg, nucl. Chem. 32, 3191 (1970). 4. J. Fenger, Radiochim. Acta 12, 186 (1969). 1409
1410
M. T O M I N A G A , T. N A K A M U R A and S. O H A S H I
order to discuss hot-atom reactions of solid inorganic phosphorus compounds. The purpose of the present work is the development of an improved ion-exchange chromatographic technique for the separation of all kinds of a2p recoil species mentioned above. Pollard et al. [5] have separated several kinds of lower oxo-anions of phosphorus by gradient-elution chromatography using Dowex 1- × 8 resin and potassium chloride solution as eluent. The separated species are 1p, 5p1_, 3p_, 4p.4p_, 2p 4p., 3p.o_3p_ ' 3p_o_sp_ ' 5P2_' and 4p-3p-4p-anions. On the other hand, Ohashi et al. [6] have obtained a good separation of linear phosphates from ortho- to octadecaphosphate, using Dowex 1 - × 4 resin and potassium chloride solution as eluent. Anselmo[7] has applied Pollard's method to hot-atom chemistry of several kinds of oxo-acids of phosphorus, replacing potassium chloride as eluent with sodium chloride in order to avoid the disturbance of 4°K activity in the measurement of 32p activity in the effluent, but his result is not sufficient for the present purpose. These results suggest that the successful separation of a2p recoil species will be obtained by precisely controlling chloride concentration of eluent. After the preliminary tests for the elution conditions, the present authors succeeded in separating the fifteen kinds of oxo-anions of phosphorus by anionexchange chromatography, using Dowex 1- × 8 resin and sodium chloride solution adjusted to pH 6.8 as eluent. EXPERIMENTAL
Reference substancesfor oxo-anions of phosphorus The following materials were used to determine the elution positions of individual oxo-anions of phosphorus in anion-exchange chromatography; NaPH202"H20 (lp), Na2PHO2"5H20 (3p), NaH2PO4"2HzO (5P1), Na3PzHOs"12H20 (zp.4p), Na2H2P2Oe'6HzO (4p_4p), Na,2P2HzO5 (aP-O-3P), Na3PzHOo'4H~O (3p-o-sP), Na4PzOT' 10HzO (sP-O-sP or 5P2), NasP308' 14H20 (4p_3p.4p), Na4P3HOs' H20 (3P-O-4p-4p), (NH4)sP309"xH20 (5p-o-4p-4p), glassy sodium polyphosphates, Na~+2PnO.~+l with ~ of 4 and 4.5, Na3P309"6H20 (5p3m)and Na4P40~z'4H20 (SP4m). The abbreviated notations in the parentheses are employed for the respective oxo-anions of phosphorus in the present paper. Tri-, tetra-, penta- and hexaphosphate are represented by 5P3, 5P4, 5P5 and 5P6.
A nion-exchange chromatography Anion-exchange chromatography was carried out with a column of Dowex 1- × 8, and gradientelution technique was employed. Eluent concentration was increased exponentially during the course of elution. The gradient of the concentration of potassium or sodium chloride as eluent was changed successively at suitable effluent volumes by replacing one reservoir with another which contained the eluent of higher chloride concentration. Six kinds of elution systems summarized in Table 1 were examined for the separation of oxo-anions of phosphorus. The effluent was collected into 5 ml fractions with an automatic fraction collector of the weight type, and an aliquot of each fraction was used for the determination and identification, if necessary, of each oxo-anion of phosphorus. As described in the previous paper [2], the colorimetric determination of each oxo-anion of phosphorus was carried out with the formation of orthophosphoric and/or hypophosphoric heteropoly blue. In the present work, 1 ml of a molybdenum(V)-molybdenum(VI) reagent and 1 ml of 1 M sodium hydrogen sulfite, if needed, were added to each fraction in a test tube, and it was heated at 100°C for 1 hr in a water bath. After cooling and adjusting the volume of the solution to 20ml with water, the absorbance was measured by a Hitachi spectrophotometer-101 at 830 m/z for orthophosphoric heteropoly blue and at 630 m/x for hypophosphoric heteropoly blue. 5. F. H. Pollard, G. Nickless, D. E. Rogers and M. T. Rothwell, J. Chromatog. 17, 157 (1965). 6. S. Ohashi, N. Tsuji, Y. Ueno, M. Takeshita and M. Muto, J. Chromatog. 50, 349 (1970). 7. V. C. Anselmo, U. S. Atomic Energy Comm., COO-1618-I (1967).
Ion-exchange separation of 32P recoil oxo-anions
141 I
Table 1. Elution conditions for anion-exchange chromatography. Resin; Dowex 1 - × 8 (100-200 mesh) in chloride form. Column dimension; ~b 1.3 × 63 cm. Flow rate; 1 ml/min, pH of eluent: 6.8 (buffered with 25 ml of 2 M ammonium acetate/l, eluent). Temperature; Room temperature Reservoirs
Elution system Eluent A B C D E F
KCI KCI NaCI NaCI NaCI NaCI
Mixing bottle M Cz
CR,
V/, R~
CR~
0"05M 0.10 0-10 0.10 0.075 0.07
0"20"~r 0.20 0.20 0.20 0.20 0.20
1400m' 1265 1389 1436 1350
0-40 '~l 0.40 0.38 0.38 0.38
R,
R~ Vn~ R~,
VR~ .~
R3
S
Cn:,
Cs
3013 mj 2622 2350 ml
0.70 '~f 0.70 2-O~
Elution diagram Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Figs 6-8
The volume of eluent in the mixing bottle was kept to 750 ml in all elution systems. CR~and Vn~ R , mean the concentration of eluent in the reservoir R~ and the volume of effluent when the reservoir R~ was replaced with the reservoir R , , respectively. S means the simple elution with 0-70 M sodium chloride and Vn, s means the effluent volume when the simple elution was started.
Neutron irradiation Commercially available reagent grade potassium dihydrogen orthorphosphate, KH2PO4, was used as a target material without further purification. Each sample, about 20 mg, was sealed in a polyethylene tube and cooled with dry ice. Then, one or two samples were packed together with powdered dry ice in a polyethylene rabbit and irradiated in pneumatic tube No. 3 of KUR (the Kyoto University Reactor) for 30 min at 1 MW operation. The nominal values of thermal neutron flux and y-dose rate in this pneumatic tube were 5 × 10 lz n/cm z. sec and 2.7 × 10rR/hr, respectively. The neutron-irradiated samples were stored in dry ice before dissolution.
A nalysis of neutron-irradiated samples The neutron-irradiated samples were dissolved in 5 ml of the respective carrier solutions (Table 2) and stored below 5°C in a refrigerator before analysis. The carrier solutions were prepared by dissolving appropriate amounts of sodium salts of oxo acids of phosphorus listed in Table 2 so as to contain Table 2. Composition of the sample solutions Number of chromatographic run
Elution diagram
1 2 3 4 5
Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5
6
Fig. 6
7 8
Fig. 7 Fig. 8
Oxo-anions of phosphorus 5p1' 3p, 4p_4p, 2p_4p, 3p.o_3p, 3p.o_sp, 5p2 5p1' 3p, 4p.4p, 2p.4p, ap_o.ap, 3p.o_sp, 5p2' G.P. (~ = 4) * 1p, 5p1' ap, 4p4p, zp4p, 3p_o 3p, 3p.o_sp, 5p2' G .P. (~ = 4)* N . - I . KHzPO4t, ap, 3p_o.3p, 5p2' G.P. (~ = 4)* N. --I. KH2PO4t, 1p, 3p, 4p_4p, 2p_4p, 3p.o_3p, ~p_o_sp, 5P2, 3P-O-4P-4P, 5P-O-4p-4p, G.P. (,~ = 4) ,5P3m 1p, ~Pl, 3p, 4p_4p, 3p.o_3p, 3p.o_sp, 5p2' 4p 3p_4p, 3P-O-4p-4p, ~P-O-4P-4P, G.P. (h = 4'5)*, ~P:~,, ~P,, 5P2, 5P3, ~P4m,~P.~ N. --I. KH2PO4~', '~P-O-'~P,5p~, G.P. (,} = 4.5 ) *
*The glassy sodium polyphosphate with the average chain length described in the parentheses. tN eutron-irradiated potassium dihydrogen orthophosphate.
1412
M. TOMINAGA, T. NAKAMURA and S. OHASHI
0"1-0.2 mg of phosphorus/ml for each species. The anion-exchange chromatographic separation of 3~precoil species was practiced by the procedures mentioned above. For the measurement of a2p activity, 1 ml of each fraction was transferred to a stainless steel planchet and dried under an infrared lamp. Then in order to settle sodium chloride crystals in the planchet, a small amount of acetone solution of synthetic adhesive "vinyl cemedine" was added and dried again. A 27r low-background gas-flow counter, Aloka LBC-22B, was used for activity measurements. RESULTS AND DISCUSSION T h e separation of m o n o - and diphosphorus oxo-anions was first carried out by m e a n s of Pollard's m e t h o d (elution s y s t e m A in T a b l e 1). T h e resulting elution c u r v e shown in Fig. 1 indicates that 5P1-, 3p_, 4p.4p_, 2p_4p_, 3p_o.3p_ ' 3p.o_sp. and 5P2-anions can be almost completely separated, but a large volume o f eluent s e e m s to be needed for the separation of a m o r e c o m p l e x mixture o f o x o - a n i o n s of p h o s p h o r u s if this m e t h o d is applied. In order to separate a c o m p l e x mixture o f lower oxo-anions o f p h o s p h o r u s and linear p h o s p h a t e s efficiently by one operation, a c o m b i n e d gradient elution was a d o p t e d as shown by s y s t e m B in T a b l e 1 and Fig. 2. By this elution system, 5p1. ' zp_, 4p_4p_, 2p_4p_, 3p_o_3p_ ' 3p_o_sp_ ' 5p2_' 5P3_' 5p4. ' 5p5_ and 5pc-anions can be separated. T h e p r e s e n t authors then e x a m i n e d the use o f sodium chloride as eluent instead o f potassium chloride. T h e alteration o f eluent f r o m p o t a s s i u m chloride to sodium chloride gave no effect on the elution pattern. T h e elution curve thus obtained by this elution s y s t e m C is shown in Fig. 3. T h e r e f o r e , in the following experiments sodium chloride was used as eluent, because one can avoid the disturbance of 4°K in the m e a s u r e m e n t of ~2p activity. 5
P2
3
i~'
4
P-O-P
P
s
[1"0
/~
5
_*
i I000 Effluent
2000 volume,
mE
Fig. 1. Chromatographic run No. 1. ( ; the elution curves obtained by colorimetry, - - - - - ; concentration of eluents, - . . . . ; the elution curves obtained by radioactivity measurement, in this figure and in the following ones.)
Lo4
-I.0
//~g-o4 2 4
-t
5
IOO0 Effluent
~
2000 volume,
mt
Fig. 2. Chromatographic run No. 2.
5
-0.5
L) X:
Ion-exchange separation ofazP recoil oxo-anions
1413
For the improvement of the separation of linear phosphates, the elution system D was introduced. As indicated in Fig. 4, the separation of linear phosphates obtained by this system is much better than that obtained by elution system C, while 1p_ and 5Pl-anions cannot be separated from one another. The elution system E was applied to separate these two monophosphorus anions and a complex mixture of oxo-anions of phosphorus including 3p-o-4P-4P-, ~P-O-4P-4P - and ~P3m-anions. Fig. 5 is the elution pattern obtained by the elution system E. Two kinds of lower oxo-anions containing three phosphorus atoms in a molecule, 3P-O-4P-4P- and ~P-O-4P-4P-anions are eluted between 5P2- and 5p3_ anions, and the ring-formed ~P3m-anions give their own peak different from that of the higher polyphosphates. 3 3 P-O-P
/A
8
--i0
ko-#
c t3
o 44
24
7 -05
2 / 1 ~ . & ....
O z
. . . .
I000
2000
Effluent
volume,
mt
Fig. 3. Chromatographic run No. 3. 5
P' PI Activity,
counts/min
55
P4
300
-i0
5
~P2
3 P-O-P/~ 3
,
3
P-0-P P
I
IIlP, ~>~
I^l
P 2_P_P
F
L ~
. . . . . . . .
.. . . . .
~j
--
i
200 05
~>
"7
5m
i00
{D o Z
.~-~ 2000
I000
Effluent volume,
~3 0 0 0
mt
Fig. 4. Chromatographic run No. 4. Activity, counts/rain
5
5004
5
4
4 Iq" 15
2ooc
g 2~ <
P-O-P 3
5
5
HIQh
P5
poly
IO
I
Ji'
l/i!
IO0-
.-.
I000
2000
Effluent volume,
mt
Fig. 5. Chromatographic run No. 5.
L_
05
_2 L) o Z
1414
M. T O M I N A G A , T. N A K A M U R A
and S. O H A S H I
For more complete separation of linear and cyclic phosphates, the elution system F was carried out. Figures 6-8 are the elution curves obtained by this elution system. Figure 6 shows the almost complete separation of 1p_, 5P1_' 3p_, 4p_4p_, 3p_o_3p_ ' 3p_o.sp_ ' 5P2_' 4p_3p_,p_, 3p.o_4p_4p_ ' 5p.o.4p_4p_ ' 5p3., 5p4_, 5P5-, 5P6- and 5P3m-anions in the elution order. Figure 7 suggests that 5P4m-anions will appear as an overlapped peak with 5P6-anions and higher polyphosphates. An example for the application of this gradient elution system to the analysis of neutron-irradiated potassium dihydrogen orthophosphate is shown in Fig. 8. The elution curves shown in Figs 4, 5 and 8 indicate that 1p_, 5p1_' 3p_, 2p_4p_, 3p-o-5p-, 5P2-, 5P3-, 5P4-, 5P5- and 5P3m-anions are present as 32p recoil species in the neutron-irradiated potassium dihydrogen orthophosphate. The 32p recoil species which were eluted between 5P 2- and 5P3- anions in the elution curves of 3 3 P-O-P
--15
/
P,5
-I.O
~3
I~~~
5
P-O'P4
3 4
~-O-~-~ /~ ~ ~4
High /;/'/'/" po~,_ ~m
d --0'5
44
roo 0
2000
Effluent volume,
mt
Fig. 6. Chromatographic run No. 6.
5
5
P4m
5
./
g
t" /
.Q <
1000 Effluent
-15
P3rn
2000 volume,
mt
./
-I.0
¢.)
I
-0 '5
30OO
Fig. 7. Chromatographic run No. 7.
IO00
~4
-1.5
5
E
ZK
/
PI ii ill
rl
!i
5OO
v l
,,I
!I ,~ IP
It
5
2
P2 ~ ~[',,, P-O-
/ .
i
/"
-I.O
',~ / ~2__// ~
-05
<
IO00 Effluent volume,
2000 m(
Fig. 8. Chromatographic run No. 8.
3000
Z
Ion-exchange separation of 32p recoil oxo-anions
14 15
Figs 4, 5 and 8 have not yet been decidedly identified. These recoil species were first considered to be 3P-O-4p-4p- and 5P-O-4p-4P-anions from their elution positions. The preliminary experiments, however, demonstrated that the hydrolytic products of these recoil species under the mild conditions were composed of only radioactive 3p_ and sPl-anions in both cases, contrary to the presumption that radioactive 3p_ and 4P-4p-anions or 5p1. and 4p-4p-anions are produced, if the recoil species are 3P-O-4p-4p- or ~P-O-4p-4p-anions, respectively. These results indicate the possibility of the presence of analogues to triphosphate such as ~P-O-~P-O-~P - and 3P-O-~P-O-3P-anions, in which one or two ~P-atoms of triphosphate are replated with 3P-atoms. The replacement of 5p with 3p is identical with that of a P-O- bond with a P-H bond in a molecule, and it means that anionic charge of these analogues are smaller than that of triphosphate. Since authentic samples for :~P-O-sP-O-~P - and 3P-O-sP-O-3P-anions have not been synthesized, one cannot identify them directly. However, the elution positions of the .~2p recoil species in question suggest the presence of the analogues mentioned above. The distributions of 32p recoil species in the irradiated orthophosphates indicate that the yield of P-O-P- bonds is much higher than that of P-P-bonds. This fact also supports the presumption that the formation of the analogues to triphosphate is more conceivable than the formation of 3P-O-4p-4p- and 5P-O-4p-4P-anions. Acknowledgement-The authors wish to express their thanks to the staffs of the Research Reactor Institute, Kyoto University for their assistance in the irradiations.
AN
Pergamon Press.
Printed in Great Britain
IMPROVED METHOD FOR THE ION-EXCHANGE CHROMATOGRAPHIC SEPARATION O F 32p_ LABELLED OXO-ANIONS OF PHOSPHORUS PRODUCED IN NEUTRON-IRRADIATED ORTHOPHOSPHATES
M A M O R U T O M I N A G A , * T E R U M A S A N A K A M U R A and S H I G E R U O H A S H I Department of Chemistry, Faculty of Science, Kyushu University, Hakozaki, Fukuoka, Japan (Received 18 May 1971)
A ~ t r a c t - T h e separation of 3sP-labelled oxo-anions of phosphorus produced in neutron-irradiated orthophosphates by means of ion-exchange chromatography was investigated. The fifteen kinds of oxo-anions of phosphorus were separated by using Dowex 1 - × 8 resin and sodium chloride solution as eluent in the order of 'P-, ~P,-, 3p., 4p_4p_, 2p_4p_ .~p_o_3p_, 3p_o_~p_" 5p_, ~p_3p,4p_, 3p_o_4p_4p_ ' 5P-O-4p-4p-, 5P3-, sP4-, 5P5- and 5P3m-anions. This method was applied to hot-atom chemistry of phosphorus and a complex mixture of 3zp recoil species in neutron-irradiated potassium dihydrogen orthophosphate was successfully separated. INTRODUCTION
IT IS WELL known that various kinds of 32P-labelled oxo-anions of phosphorus are produced by recoil reactions in neutron-irradiated solid inorganic phosphorus compounds [ 1]. In our previous paper [2], it was indicated that some kinds of oxoanions of phosphorus must be added as carriers to a sample solution in order to separate 3~p recoil species in neutron-irradiated inorganic phosphates by means of anion-exchange chromatography. A rough estimate obtained by this method for the distribution of 32p recoil species in neutron-irradiated alkali and ammonium orthophosphates was also reported/3/. The anion-exchange chromatography employed in the previous work [3] gives a good separation of the following species; ,p, ~p,_, 3p_, 4p_4p_, 2p_4p_, P-O-P (as overlapped peaks), 5P3-, '5P4-, 5P5- and 5P 6a n i o n s (as for these abbreviated notations, see the experimental section). It fails, however, to seperate the three kinds of P-O-P-anions, i.e. 3P-O-3p-, 3P-O-sP- and '~P-O-~P- (5P2-) anions, and to give evidence for the existence of other expected a2p recoil species such a s 3 p - o - 4 p - 4 p - , 5P-O-4p-4p-anions and some analogues to triand/or tetraphosphate, e.g. 3P-O-~P-O-sP- and 3P-O-sP-O-aP-anions, in which one or two 5P-atoms of triphosphate are replaced with 3P-atoms. Fenger[4] has suggested that the peaks located between 3P-O-~P- and sP,-anions in the paper electrophoretic analysis of neutron-irradiated ammonium dihydrogen orthophosphate may be assigned to the latter two species. It is important to determine that detailed distributions of 32p recoil species in *Present address: National Research Institute for Pollution and Resources, 188, Kotobukicho, Kawaguchi, Saitama, Japan. I. M. Halmann, Chem. Rev. 64, 689 (1964). 2. K. Ujimoto, T. Nakamura, H. Asada, N. Yoza, Y. Takashima and S. Ohashi, J. inorg, nucl. Chem. 32, 3177 (1970). 3. T. Nakamura, K. Ujimoto, N. Yoza and S. Ohashi, J. inorg, nucl. Chem. 32, 3191 (1970). 4. J. Fenger, Radiochim. Acta 12, 186 (1969). 1409
1410
M. T O M I N A G A , T. N A K A M U R A and S. O H A S H I
order to discuss hot-atom reactions of solid inorganic phosphorus compounds. The purpose of the present work is the development of an improved ion-exchange chromatographic technique for the separation of all kinds of a2p recoil species mentioned above. Pollard et al. [5] have separated several kinds of lower oxo-anions of phosphorus by gradient-elution chromatography using Dowex 1- × 8 resin and potassium chloride solution as eluent. The separated species are 1p, 5p1_, 3p_, 4p.4p_, 2p 4p., 3p.o_3p_ ' 3p_o_sp_ ' 5P2_' and 4p-3p-4p-anions. On the other hand, Ohashi et al. [6] have obtained a good separation of linear phosphates from ortho- to octadecaphosphate, using Dowex 1 - × 4 resin and potassium chloride solution as eluent. Anselmo[7] has applied Pollard's method to hot-atom chemistry of several kinds of oxo-acids of phosphorus, replacing potassium chloride as eluent with sodium chloride in order to avoid the disturbance of 4°K activity in the measurement of 32p activity in the effluent, but his result is not sufficient for the present purpose. These results suggest that the successful separation of a2p recoil species will be obtained by precisely controlling chloride concentration of eluent. After the preliminary tests for the elution conditions, the present authors succeeded in separating the fifteen kinds of oxo-anions of phosphorus by anionexchange chromatography, using Dowex 1- × 8 resin and sodium chloride solution adjusted to pH 6.8 as eluent. EXPERIMENTAL
Reference substancesfor oxo-anions of phosphorus The following materials were used to determine the elution positions of individual oxo-anions of phosphorus in anion-exchange chromatography; NaPH202"H20 (lp), Na2PHO2"5H20 (3p), NaH2PO4"2HzO (5P1), Na3PzHOs"12H20 (zp.4p), Na2H2P2Oe'6HzO (4p_4p), Na,2P2HzO5 (aP-O-3P), Na3PzHOo'4H~O (3p-o-sP), Na4PzOT' 10HzO (sP-O-sP or 5P2), NasP308' 14H20 (4p_3p.4p), Na4P3HOs' H20 (3P-O-4p-4p), (NH4)sP309"xH20 (5p-o-4p-4p), glassy sodium polyphosphates, Na~+2PnO.~+l with ~ of 4 and 4.5, Na3P309"6H20 (5p3m)and Na4P40~z'4H20 (SP4m). The abbreviated notations in the parentheses are employed for the respective oxo-anions of phosphorus in the present paper. Tri-, tetra-, penta- and hexaphosphate are represented by 5P3, 5P4, 5P5 and 5P6.
A nion-exchange chromatography Anion-exchange chromatography was carried out with a column of Dowex 1- × 8, and gradientelution technique was employed. Eluent concentration was increased exponentially during the course of elution. The gradient of the concentration of potassium or sodium chloride as eluent was changed successively at suitable effluent volumes by replacing one reservoir with another which contained the eluent of higher chloride concentration. Six kinds of elution systems summarized in Table 1 were examined for the separation of oxo-anions of phosphorus. The effluent was collected into 5 ml fractions with an automatic fraction collector of the weight type, and an aliquot of each fraction was used for the determination and identification, if necessary, of each oxo-anion of phosphorus. As described in the previous paper [2], the colorimetric determination of each oxo-anion of phosphorus was carried out with the formation of orthophosphoric and/or hypophosphoric heteropoly blue. In the present work, 1 ml of a molybdenum(V)-molybdenum(VI) reagent and 1 ml of 1 M sodium hydrogen sulfite, if needed, were added to each fraction in a test tube, and it was heated at 100°C for 1 hr in a water bath. After cooling and adjusting the volume of the solution to 20ml with water, the absorbance was measured by a Hitachi spectrophotometer-101 at 830 m/z for orthophosphoric heteropoly blue and at 630 m/x for hypophosphoric heteropoly blue. 5. F. H. Pollard, G. Nickless, D. E. Rogers and M. T. Rothwell, J. Chromatog. 17, 157 (1965). 6. S. Ohashi, N. Tsuji, Y. Ueno, M. Takeshita and M. Muto, J. Chromatog. 50, 349 (1970). 7. V. C. Anselmo, U. S. Atomic Energy Comm., COO-1618-I (1967).
Ion-exchange separation of 32P recoil oxo-anions
141 I
Table 1. Elution conditions for anion-exchange chromatography. Resin; Dowex 1 - × 8 (100-200 mesh) in chloride form. Column dimension; ~b 1.3 × 63 cm. Flow rate; 1 ml/min, pH of eluent: 6.8 (buffered with 25 ml of 2 M ammonium acetate/l, eluent). Temperature; Room temperature Reservoirs
Elution system Eluent A B C D E F
KCI KCI NaCI NaCI NaCI NaCI
Mixing bottle M Cz
CR,
V/, R~
CR~
0"05M 0.10 0-10 0.10 0.075 0.07
0"20"~r 0.20 0.20 0.20 0.20 0.20
1400m' 1265 1389 1436 1350
0-40 '~l 0.40 0.38 0.38 0.38
R,
R~ Vn~ R~,
VR~ .~
R3
S
Cn:,
Cs
3013 mj 2622 2350 ml
0.70 '~f 0.70 2-O~
Elution diagram Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Figs 6-8
The volume of eluent in the mixing bottle was kept to 750 ml in all elution systems. CR~and Vn~ R , mean the concentration of eluent in the reservoir R~ and the volume of effluent when the reservoir R~ was replaced with the reservoir R , , respectively. S means the simple elution with 0-70 M sodium chloride and Vn, s means the effluent volume when the simple elution was started.
Neutron irradiation Commercially available reagent grade potassium dihydrogen orthorphosphate, KH2PO4, was used as a target material without further purification. Each sample, about 20 mg, was sealed in a polyethylene tube and cooled with dry ice. Then, one or two samples were packed together with powdered dry ice in a polyethylene rabbit and irradiated in pneumatic tube No. 3 of KUR (the Kyoto University Reactor) for 30 min at 1 MW operation. The nominal values of thermal neutron flux and y-dose rate in this pneumatic tube were 5 × 10 lz n/cm z. sec and 2.7 × 10rR/hr, respectively. The neutron-irradiated samples were stored in dry ice before dissolution.
A nalysis of neutron-irradiated samples The neutron-irradiated samples were dissolved in 5 ml of the respective carrier solutions (Table 2) and stored below 5°C in a refrigerator before analysis. The carrier solutions were prepared by dissolving appropriate amounts of sodium salts of oxo acids of phosphorus listed in Table 2 so as to contain Table 2. Composition of the sample solutions Number of chromatographic run
Elution diagram
1 2 3 4 5
Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5
6
Fig. 6
7 8
Fig. 7 Fig. 8
Oxo-anions of phosphorus 5p1' 3p, 4p_4p, 2p_4p, 3p.o_3p, 3p.o_sp, 5p2 5p1' 3p, 4p.4p, 2p.4p, ap_o.ap, 3p.o_sp, 5p2' G.P. (~ = 4) * 1p, 5p1' ap, 4p4p, zp4p, 3p_o 3p, 3p.o_sp, 5p2' G .P. (~ = 4)* N . - I . KHzPO4t, ap, 3p_o.3p, 5p2' G.P. (~ = 4)* N. --I. KH2PO4t, 1p, 3p, 4p_4p, 2p_4p, 3p.o_3p, ~p_o_sp, 5P2, 3P-O-4P-4P, 5P-O-4p-4p, G.P. (,~ = 4) ,5P3m 1p, ~Pl, 3p, 4p_4p, 3p.o_3p, 3p.o_sp, 5p2' 4p 3p_4p, 3P-O-4p-4p, ~P-O-4P-4P, G.P. (h = 4'5)*, ~P:~,, ~P,, 5P2, 5P3, ~P4m,~P.~ N. --I. KH2PO4~', '~P-O-'~P,5p~, G.P. (,} = 4.5 ) *
*The glassy sodium polyphosphate with the average chain length described in the parentheses. tN eutron-irradiated potassium dihydrogen orthophosphate.
1412
M. TOMINAGA, T. NAKAMURA and S. OHASHI
0"1-0.2 mg of phosphorus/ml for each species. The anion-exchange chromatographic separation of 3~precoil species was practiced by the procedures mentioned above. For the measurement of a2p activity, 1 ml of each fraction was transferred to a stainless steel planchet and dried under an infrared lamp. Then in order to settle sodium chloride crystals in the planchet, a small amount of acetone solution of synthetic adhesive "vinyl cemedine" was added and dried again. A 27r low-background gas-flow counter, Aloka LBC-22B, was used for activity measurements. RESULTS AND DISCUSSION T h e separation of m o n o - and diphosphorus oxo-anions was first carried out by m e a n s of Pollard's m e t h o d (elution s y s t e m A in T a b l e 1). T h e resulting elution c u r v e shown in Fig. 1 indicates that 5P1-, 3p_, 4p.4p_, 2p_4p_, 3p_o.3p_ ' 3p.o_sp. and 5P2-anions can be almost completely separated, but a large volume o f eluent s e e m s to be needed for the separation of a m o r e c o m p l e x mixture o f o x o - a n i o n s of p h o s p h o r u s if this m e t h o d is applied. In order to separate a c o m p l e x mixture o f lower oxo-anions o f p h o s p h o r u s and linear p h o s p h a t e s efficiently by one operation, a c o m b i n e d gradient elution was a d o p t e d as shown by s y s t e m B in T a b l e 1 and Fig. 2. By this elution system, 5p1. ' zp_, 4p_4p_, 2p_4p_, 3p_o_3p_ ' 3p_o_sp_ ' 5p2_' 5P3_' 5p4. ' 5p5_ and 5pc-anions can be separated. T h e p r e s e n t authors then e x a m i n e d the use o f sodium chloride as eluent instead o f potassium chloride. T h e alteration o f eluent f r o m p o t a s s i u m chloride to sodium chloride gave no effect on the elution pattern. T h e elution curve thus obtained by this elution s y s t e m C is shown in Fig. 3. T h e r e f o r e , in the following experiments sodium chloride was used as eluent, because one can avoid the disturbance of 4°K in the m e a s u r e m e n t of ~2p activity. 5
P2
3
i~'
4
P-O-P
P
s
[1"0
/~
5
_*
i I000 Effluent
2000 volume,
mE
Fig. 1. Chromatographic run No. 1. ( ; the elution curves obtained by colorimetry, - - - - - ; concentration of eluents, - . . . . ; the elution curves obtained by radioactivity measurement, in this figure and in the following ones.)
Lo4
-I.0
//~g-o4 2 4
-t
5
IOO0 Effluent
~
2000 volume,
mt
Fig. 2. Chromatographic run No. 2.
5
-0.5
L) X:
Ion-exchange separation ofazP recoil oxo-anions
1413
For the improvement of the separation of linear phosphates, the elution system D was introduced. As indicated in Fig. 4, the separation of linear phosphates obtained by this system is much better than that obtained by elution system C, while 1p_ and 5Pl-anions cannot be separated from one another. The elution system E was applied to separate these two monophosphorus anions and a complex mixture of oxo-anions of phosphorus including 3p-o-4P-4P-, ~P-O-4P-4P - and ~P3m-anions. Fig. 5 is the elution pattern obtained by the elution system E. Two kinds of lower oxo-anions containing three phosphorus atoms in a molecule, 3P-O-4P-4P- and ~P-O-4P-4P-anions are eluted between 5P2- and 5p3_ anions, and the ring-formed ~P3m-anions give their own peak different from that of the higher polyphosphates. 3 3 P-O-P
/A
8
--i0
ko-#
c t3
o 44
24
7 -05
2 / 1 ~ . & ....
O z
. . . .
I000
2000
Effluent
volume,
mt
Fig. 3. Chromatographic run No. 3. 5
P' PI Activity,
counts/min
55
P4
300
-i0
5
~P2
3 P-O-P/~ 3
,
3
P-0-P P
I
IIlP, ~>~
I^l
P 2_P_P
F
L ~
. . . . . . . .
.. . . . .
~j
--
i
200 05
~>
"7
5m
i00
{D o Z
.~-~ 2000
I000
Effluent volume,
~3 0 0 0
mt
Fig. 4. Chromatographic run No. 4. Activity, counts/rain
5
5004
5
4
4 Iq" 15
2ooc
g 2~ <
P-O-P 3
5
5
HIQh
P5
poly
IO
I
Ji'
l/i!
IO0-
.-.
I000
2000
Effluent volume,
mt
Fig. 5. Chromatographic run No. 5.
L_
05
_2 L) o Z
1414
M. T O M I N A G A , T. N A K A M U R A
and S. O H A S H I
For more complete separation of linear and cyclic phosphates, the elution system F was carried out. Figures 6-8 are the elution curves obtained by this elution system. Figure 6 shows the almost complete separation of 1p_, 5P1_' 3p_, 4p_4p_, 3p_o_3p_ ' 3p_o.sp_ ' 5P2_' 4p_3p_,p_, 3p.o_4p_4p_ ' 5p.o.4p_4p_ ' 5p3., 5p4_, 5P5-, 5P6- and 5P3m-anions in the elution order. Figure 7 suggests that 5P4m-anions will appear as an overlapped peak with 5P6-anions and higher polyphosphates. An example for the application of this gradient elution system to the analysis of neutron-irradiated potassium dihydrogen orthophosphate is shown in Fig. 8. The elution curves shown in Figs 4, 5 and 8 indicate that 1p_, 5p1_' 3p_, 2p_4p_, 3p-o-5p-, 5P2-, 5P3-, 5P4-, 5P5- and 5P3m-anions are present as 32p recoil species in the neutron-irradiated potassium dihydrogen orthophosphate. The 32p recoil species which were eluted between 5P 2- and 5P3- anions in the elution curves of 3 3 P-O-P
--15
/
P,5
-I.O
~3
I~~~
5
P-O'P4
3 4
~-O-~-~ /~ ~ ~4
High /;/'/'/" po~,_ ~m
d --0'5
44
roo 0
2000
Effluent volume,
mt
Fig. 6. Chromatographic run No. 6.
5
5
P4m
5
./
g
t" /
.Q <
1000 Effluent
-15
P3rn
2000 volume,
mt
./
-I.0
¢.)
I
-0 '5
30OO
Fig. 7. Chromatographic run No. 7.
IO00
~4
-1.5
5
E
ZK
/
PI ii ill
rl
!i
5OO
v l
,,I
!I ,~ IP
It
5
2
P2 ~ ~[',,, P-O-
/ .
i
/"
-I.O
',~ / ~2__// ~
-05
<
IO00 Effluent volume,
2000 m(
Fig. 8. Chromatographic run No. 8.
3000
Z
Ion-exchange separation of 32p recoil oxo-anions
14 15
Figs 4, 5 and 8 have not yet been decidedly identified. These recoil species were first considered to be 3P-O-4p-4p- and 5P-O-4p-4P-anions from their elution positions. The preliminary experiments, however, demonstrated that the hydrolytic products of these recoil species under the mild conditions were composed of only radioactive 3p_ and sPl-anions in both cases, contrary to the presumption that radioactive 3p_ and 4P-4p-anions or 5p1. and 4p-4p-anions are produced, if the recoil species are 3P-O-4p-4p- or ~P-O-4p-4p-anions, respectively. These results indicate the possibility of the presence of analogues to triphosphate such as ~P-O-~P-O-~P - and 3P-O-~P-O-3P-anions, in which one or two ~P-atoms of triphosphate are replated with 3P-atoms. The replacement of 5p with 3p is identical with that of a P-O- bond with a P-H bond in a molecule, and it means that anionic charge of these analogues are smaller than that of triphosphate. Since authentic samples for :~P-O-sP-O-~P - and 3P-O-sP-O-3P-anions have not been synthesized, one cannot identify them directly. However, the elution positions of the .~2p recoil species in question suggest the presence of the analogues mentioned above. The distributions of 32p recoil species in the irradiated orthophosphates indicate that the yield of P-O-P- bonds is much higher than that of P-P-bonds. This fact also supports the presumption that the formation of the analogues to triphosphate is more conceivable than the formation of 3P-O-4p-4p- and 5P-O-4p-4P-anions. Acknowledgement-The authors wish to express their thanks to the staffs of the Research Reactor Institute, Kyoto University for their assistance in the irradiations.