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Labeling of 131I-hippuran by ultraviolet excitation
MICROCHEMICAL
14,481-485
JOURNAL
Labeling
(1969)
of “‘I-Hippuran Excitation E. HALLABAAND
Nuclear
by Ultraviolet M.
RAIEH
Chemistry Department, Isotope Division, Atomic Energy Establishment, U.A.R. Received
April
18, 1969
The successful application of ultraviolet irradiation of rose bengal (I) with radioiodine encouraged us to investigate the possibility of labeling hippuran. The spectrophotometric study of hippuran solution before and after ultraviolet irradiation for 6 hours at 25°C revealed that the two spectra are identical and show maximum absorption at 273 mp. EXPERIMENTAL
METHODS
The labeling of hippuran was studied using two media, one aqueous and the other ethereal. (a) Labeling in aqueous medium. A 2-ml portion of ~0.14 M hippuran solution in double distilled water with trace H,O, (30% w/v) (2 lambdas) and 0.1 mCi of Na1311was irradiated by ultraviolet radiation in a quartz transparent tube (0.8 cm diam and 4 cm length). The tube was closed by a ground stopper and placed at 2 cm distance from a Hanovia high pressure quartz mercury lamp. A fan was used to lower the temperature to 24°C. The yield of exchange was determined by paper chromatography using n-butanol:acetic acid:water (4:l:l) as solvent. The effect of time of irradiation on the yield of exchange and the change in pH of the aqueous solution during irradiation were investigated. (h) Labeling in organic solvent. It was found that ether can extract both iodine and ortho-iodohippuric acid. A hippuran solution adjusted to pH 3 with 1 N HCl can be extracted by ether (80% ) due to the conversion of the sodium hippurate to ortho-iodohippuric acid. The labeling procedure consists in adding to the aqueous solution (1 ml of 0.1 mCi of Na*311,1 mg of KI, and 0.5 of KIO,) 2 ml of ether and acidifying with few drops 1 N HCl, whereas all activity is extracted in the ether layer. The 2 ml of ether containing Ia11is mixed with 8 ml of ether containing 40 mg of ortho-iodohippuric acid in a quartz tube which is closed with a cooling condenser, then we proceed as previously. (c) The light source. The UV lamp used for irradiation is the Han-
482
HALLABA
AND RAIEH
ovia utility quartz lamp SH 616A.l It is a 100 W high pressure Hg vapor lamp operating on 220 V, 50 cycle AC through a reactive transformer. The arc lamp generates a high UV over a broad spectral range. In fact the complete spectrum is transmitted : 1894-28OOA (far), 2800-32OOA (middle) and 3200-4OOOA (near). Some of the principal lines with their quantum energy are given in Table 1. The short wavelength lines are absorbed by the air gap by the quartz. (d) Purification of the final product. The ether layer is evaporated and the residue is dissolved in dilute alkali whereas the irradiated aqueous solution is adjusted to pH 8. Both solutions could be purified from unreacted iodide and iodine atoms by passing either on a column of silver chloride adosrbed on silica gel (0.5 I#I x 10 cm) or on a small TABLE SOME PRINCIPAL RESONANCE ENERGY OF THE
Line (A): Quantum (kcal) Energy (eV)
a.950
1
LINE-S WITH THEIR
QUANTUM
SH.616A Hg LAMP
3660
3130
2967
2752
2571
2432
78.11 3.48
91.34 3.97
96.35 4.19
103.52 4.5
111.2 4.83
115.19 5
t
0.900
-
0.050
-
0.800
-
I 210
FIG.
I
I 210
I
1
!
150
1. Ultraviolet
, 270
1
1 290
/
I 310
1
L 330
I
! 350~-
i.uvu
390
Ia0
WNE
LENGTH
my
spectrum of inactive and labeled hippuran.
1 Hanovia Inc. Lamp Division Newark 5, New Jersey Special Energy Distribution of quartz Hg-vapor Arc tube 616A-13 type SH.
uv
LABELING
0~
483
1311-~IP~U~~N
column (0.2 + x 2 cm) of Dowex l-X8 in the chloride form. Elution of adsorbed labeled hippuran could be achieved by eluting with 0.09% saline. The radiochemical purity of the final product exceeds 99%, it was checked by spectrophotometry and paper chromatography. A maximum absorption spectra is attained at 273 rnp which agrees with that of the inactive hippuran of same concentration (Figs. 1 and 2). RESULTS
AND
DISCUSSION
By ultraviolet irradiation of the two hippuran solutions, aqueous and ethereal, at 24’C the yield of exchange at different time intervals of irradiation is illustrated in Table 2. A blank solution of 0.14 M hippuran,
FIG.
2. Paper
chromatogram
of irradiated orrho-oiodohippuric
acid with radioiodine
in ether. TABLE EFFECX
OF IRRADIATION
-0.14
M
TIME
ON THE YIELD
OF EXCHANGE
Aqueous solution
Yield (%) of labeled hippuran
Time (nin)
PH
15 30
6.3 6
22 28
60
5.7 5.4 5.3 5.25
44 57 66 70
120 180 240
BY UV
2
Observation
Slight yellow coloration
-0.01 Time (min)
AND
pH
OF SOLUTION
M Ethereal solution
Yield (%) of labeled hippuran
30 60
32 38
210 270
50 74
Observation
Faint yellow coloration
484
HALLABA
AND
RAIEH
containing all chemicals and radioactivity, but not irradiated, contained 5% of labeled hippuran after 300 minutes. The final labeled hippuran has a similar spectrum as the inactive one which means that no sideproducts were detected by either spectrophotometry or chromatography. The ethereal solution of hippuran which is 10 times less concentrated gives the same yield of exchange as the aqueous one, with very faint coloration meaning less photolysis and higher purity of the final product. A weaker solution of hippuran than 0.14 M gave a lower yield of exchange. Hippuran is a very stable organic compound with a high m.p. 172°C. The iodine content is about 35% existing in a stable organically bound state. The faint yellow coloration obtained by UV irradiation of the exchange mixture may be mainly due to slight photolysis on its lowest energy bond, viz, the C-I bond (2) forming a dissociated radical and an excited iodine atom as: RI + hv G-‘
R’ + I’.
This has been checked by irradiating a blank hippuran solution of same concentration without additives, whereas the same yellow coloration as well as decrease of pH are noticed. The quantum yield of the photolysis step is only about 10e2 (4 = 0.02). This low yield in a liquid is due in that radicals formed in a dissociation reaction are surrounded by a cage of closely packed molecules. They recombine within a time not much greater than lo-l3 of a second, a typical vibration period, even if they may diffuse away from each other the chance is high that the same pair soon meet again and recombine (geminative recombination). The presence of a trace of an oxidizing agent catalyzed the labeling reaction. In a previous study (I) of labeling rose bengal with 1311 by UV, absence of H,O, lowered the yield from 70 to 17% during 4-hours irradiation time. The process resulting from the absorption of UV radiation by iodide ions may be described, similarly to Hayon (3), as follows: (I-H,O)
--fr-,
(I-H20)‘,
(I-H,O)’
------+ (I-H,O)
(I-HZO)’
------, I + HzO-,
(3)
(I-HZO)’
------, I + H + OH-.
(4)
(1) + heat,
(2)
This means that atomic radioiodine is formed as well as H+ and HI, as found by McDonald (S), on irradiating iodobenzene. Trace iodide solution under UV irradiation does not show any coloration nor decrease in
uv
LABELINGOF
f311-~~~p~R~N
485
pH due to the very low concentration of the iodide ion. The decrease in pH may be mainly due to the photolysis of hippuran where the liberated iodine atom can undergo the radiolytic steps described by Hayon. Therefore, we can summarize the overall exchange mechanism as: hv
l?* ----,
21*,
RI + I* ------,
RI*
(1) + I,
1 + 1* ------, I + I *, I*
+
I*
-_----
l
(4
(3)
Iz**
(4) Noyes (5) proposed 2 alternative mechanisms for such exchange reactions thermodynamically similar: one involves a direct substitution by an iodine atom and the other involves the formation of a carbonium radical. REFERENCES 1. Hallaba, E. and Raieh, M., Photoinduced labeling of rose bengal with 1131 by UV radiation. Intern. 1. Appl. Radiation Isotopes. 18, 533-535 (1967). 2. Moore, W. .I., “Physical Chemistry,” Prentice-Hall, Englewood Cliffs, New Jersey, 1965. 3. Hayon, E., The photochemistry of iodide ion in aqueous solution. J. Phys. Chem. 66, 1937 (1961). 4. Blair, J. McD., and Smith, D. B., Liquid phase photolysis (iodobenzene). 1. CAem. Sot. 20, 1788 (1960). 5. Noyes, R. M., Mechanism of exchange reaction between elementary iodine and organic iodides. J. Am. Chem. Sot. 75, 767 (1953).
14,481-485
JOURNAL
Labeling
(1969)
of “‘I-Hippuran Excitation E. HALLABAAND
Nuclear
by Ultraviolet M.
RAIEH
Chemistry Department, Isotope Division, Atomic Energy Establishment, U.A.R. Received
April
18, 1969
The successful application of ultraviolet irradiation of rose bengal (I) with radioiodine encouraged us to investigate the possibility of labeling hippuran. The spectrophotometric study of hippuran solution before and after ultraviolet irradiation for 6 hours at 25°C revealed that the two spectra are identical and show maximum absorption at 273 mp. EXPERIMENTAL
METHODS
The labeling of hippuran was studied using two media, one aqueous and the other ethereal. (a) Labeling in aqueous medium. A 2-ml portion of ~0.14 M hippuran solution in double distilled water with trace H,O, (30% w/v) (2 lambdas) and 0.1 mCi of Na1311was irradiated by ultraviolet radiation in a quartz transparent tube (0.8 cm diam and 4 cm length). The tube was closed by a ground stopper and placed at 2 cm distance from a Hanovia high pressure quartz mercury lamp. A fan was used to lower the temperature to 24°C. The yield of exchange was determined by paper chromatography using n-butanol:acetic acid:water (4:l:l) as solvent. The effect of time of irradiation on the yield of exchange and the change in pH of the aqueous solution during irradiation were investigated. (h) Labeling in organic solvent. It was found that ether can extract both iodine and ortho-iodohippuric acid. A hippuran solution adjusted to pH 3 with 1 N HCl can be extracted by ether (80% ) due to the conversion of the sodium hippurate to ortho-iodohippuric acid. The labeling procedure consists in adding to the aqueous solution (1 ml of 0.1 mCi of Na*311,1 mg of KI, and 0.5 of KIO,) 2 ml of ether and acidifying with few drops 1 N HCl, whereas all activity is extracted in the ether layer. The 2 ml of ether containing Ia11is mixed with 8 ml of ether containing 40 mg of ortho-iodohippuric acid in a quartz tube which is closed with a cooling condenser, then we proceed as previously. (c) The light source. The UV lamp used for irradiation is the Han-
482
HALLABA
AND RAIEH
ovia utility quartz lamp SH 616A.l It is a 100 W high pressure Hg vapor lamp operating on 220 V, 50 cycle AC through a reactive transformer. The arc lamp generates a high UV over a broad spectral range. In fact the complete spectrum is transmitted : 1894-28OOA (far), 2800-32OOA (middle) and 3200-4OOOA (near). Some of the principal lines with their quantum energy are given in Table 1. The short wavelength lines are absorbed by the air gap by the quartz. (d) Purification of the final product. The ether layer is evaporated and the residue is dissolved in dilute alkali whereas the irradiated aqueous solution is adjusted to pH 8. Both solutions could be purified from unreacted iodide and iodine atoms by passing either on a column of silver chloride adosrbed on silica gel (0.5 I#I x 10 cm) or on a small TABLE SOME PRINCIPAL RESONANCE ENERGY OF THE
Line (A): Quantum (kcal) Energy (eV)
a.950
1
LINE-S WITH THEIR
QUANTUM
SH.616A Hg LAMP
3660
3130
2967
2752
2571
2432
78.11 3.48
91.34 3.97
96.35 4.19
103.52 4.5
111.2 4.83
115.19 5
t
0.900
-
0.050
-
0.800
-
I 210
FIG.
I
I 210
I
1
!
150
1. Ultraviolet
, 270
1
1 290
/
I 310
1
L 330
I
! 350~-
i.uvu
390
Ia0
WNE
LENGTH
my
spectrum of inactive and labeled hippuran.
1 Hanovia Inc. Lamp Division Newark 5, New Jersey Special Energy Distribution of quartz Hg-vapor Arc tube 616A-13 type SH.
uv
LABELING
0~
483
1311-~IP~U~~N
column (0.2 + x 2 cm) of Dowex l-X8 in the chloride form. Elution of adsorbed labeled hippuran could be achieved by eluting with 0.09% saline. The radiochemical purity of the final product exceeds 99%, it was checked by spectrophotometry and paper chromatography. A maximum absorption spectra is attained at 273 rnp which agrees with that of the inactive hippuran of same concentration (Figs. 1 and 2). RESULTS
AND
DISCUSSION
By ultraviolet irradiation of the two hippuran solutions, aqueous and ethereal, at 24’C the yield of exchange at different time intervals of irradiation is illustrated in Table 2. A blank solution of 0.14 M hippuran,
FIG.
2. Paper
chromatogram
of irradiated orrho-oiodohippuric
acid with radioiodine
in ether. TABLE EFFECX
OF IRRADIATION
-0.14
M
TIME
ON THE YIELD
OF EXCHANGE
Aqueous solution
Yield (%) of labeled hippuran
Time (nin)
PH
15 30
6.3 6
22 28
60
5.7 5.4 5.3 5.25
44 57 66 70
120 180 240
BY UV
2
Observation
Slight yellow coloration
-0.01 Time (min)
AND
pH
OF SOLUTION
M Ethereal solution
Yield (%) of labeled hippuran
30 60
32 38
210 270
50 74
Observation
Faint yellow coloration
484
HALLABA
AND
RAIEH
containing all chemicals and radioactivity, but not irradiated, contained 5% of labeled hippuran after 300 minutes. The final labeled hippuran has a similar spectrum as the inactive one which means that no sideproducts were detected by either spectrophotometry or chromatography. The ethereal solution of hippuran which is 10 times less concentrated gives the same yield of exchange as the aqueous one, with very faint coloration meaning less photolysis and higher purity of the final product. A weaker solution of hippuran than 0.14 M gave a lower yield of exchange. Hippuran is a very stable organic compound with a high m.p. 172°C. The iodine content is about 35% existing in a stable organically bound state. The faint yellow coloration obtained by UV irradiation of the exchange mixture may be mainly due to slight photolysis on its lowest energy bond, viz, the C-I bond (2) forming a dissociated radical and an excited iodine atom as: RI + hv G-‘
R’ + I’.
This has been checked by irradiating a blank hippuran solution of same concentration without additives, whereas the same yellow coloration as well as decrease of pH are noticed. The quantum yield of the photolysis step is only about 10e2 (4 = 0.02). This low yield in a liquid is due in that radicals formed in a dissociation reaction are surrounded by a cage of closely packed molecules. They recombine within a time not much greater than lo-l3 of a second, a typical vibration period, even if they may diffuse away from each other the chance is high that the same pair soon meet again and recombine (geminative recombination). The presence of a trace of an oxidizing agent catalyzed the labeling reaction. In a previous study (I) of labeling rose bengal with 1311 by UV, absence of H,O, lowered the yield from 70 to 17% during 4-hours irradiation time. The process resulting from the absorption of UV radiation by iodide ions may be described, similarly to Hayon (3), as follows: (I-H,O)
--fr-,
(I-H20)‘,
(I-H,O)’
------+ (I-H,O)
(I-HZO)’
------, I + HzO-,
(3)
(I-HZO)’
------, I + H + OH-.
(4)
(1) + heat,
(2)
This means that atomic radioiodine is formed as well as H+ and HI, as found by McDonald (S), on irradiating iodobenzene. Trace iodide solution under UV irradiation does not show any coloration nor decrease in
uv
LABELINGOF
f311-~~~p~R~N
485
pH due to the very low concentration of the iodide ion. The decrease in pH may be mainly due to the photolysis of hippuran where the liberated iodine atom can undergo the radiolytic steps described by Hayon. Therefore, we can summarize the overall exchange mechanism as: hv
l?* ----,
21*,
RI + I* ------,
RI*
(1) + I,
1 + 1* ------, I + I *, I*
+
I*
-_----
l
(4
(3)
Iz**
(4) Noyes (5) proposed 2 alternative mechanisms for such exchange reactions thermodynamically similar: one involves a direct substitution by an iodine atom and the other involves the formation of a carbonium radical. REFERENCES 1. Hallaba, E. and Raieh, M., Photoinduced labeling of rose bengal with 1131 by UV radiation. Intern. 1. Appl. Radiation Isotopes. 18, 533-535 (1967). 2. Moore, W. .I., “Physical Chemistry,” Prentice-Hall, Englewood Cliffs, New Jersey, 1965. 3. Hayon, E., The photochemistry of iodide ion in aqueous solution. J. Phys. Chem. 66, 1937 (1961). 4. Blair, J. McD., and Smith, D. B., Liquid phase photolysis (iodobenzene). 1. CAem. Sot. 20, 1788 (1960). 5. Noyes, R. M., Mechanism of exchange reaction between elementary iodine and organic iodides. J. Am. Chem. Sot. 75, 767 (1953).