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Compensation of fluorescence quenching by extrapolation of fluorescence intensity at time zero
125
Analyt&za Chrmzca Acta, 264 (1992) 125-129
Elsevier Saence Pubhshers B V , Amsterdam
Compensation of fluorescence quenching by extrapolation of fluorescence intensity at time zero Sang-Mock Lee, Jeong-Moog Km, Jang-Soo Shm and Cheol-Jung fim Korea Atomc
Energy Research Instrtute, P 0 Box 7, Daeduk-danIt,
(Received 20th December 1991, revised manumpt
DaeJeon 305404
(Korea)
received 27th February 1992)
Abstract
A method to compensate for fluorescence quenchmg caused by quenchmg elements or temperature fluctuations was studled Thus method calculates the fluorescence intensity at time zero, which 1s unaffected by the changes m fluorescence hfetlme due to various quenchmg effects The method uses the integrated signal at two gates with an appropriately selected delay interval on an exponentially decaymg fluorescence curve Induced by a mtrogen pulsed laser Apphcatlon to the measurement of uranmm concentration showed the exact compensatton for quenching caused by other ions or temperature fluctuations within f 5% error range Keywords
Fluonmetry, Fluorescence quenching, Uranium
Compared with other photometnc methods, fluornnetry provides some of the lowest detection lumts However, fluorunetnc measurements can suffer mterferences from quenchers [l-31 or sample temperature fluctuations Although a number of mechamsms for such quenching reactions exist, they produce the same result, 1 e , a reduced quantum efficiency [4,.5] owmg to a consequent loss in fluorescence mtenslty Correction for quenching effects 1s very dlfflcult because the concentration of the quenching agent is usually unknown There are several methods avadable for the ebmlmatlon of these large mterfermg effects, mcludmg dilution of the sample, the use of an internal standard or the separation of a fluorophore from the quencher However, these methods are very time consuming The development of lasers with high spectral brightness, dlrectlonahty and short pulse length Correspondence to Dr Sang-Mock Lee, Korea Atomic Energy Research Institute, P 0 Box 7, Daeduk-danjl, DaeJeOn, 305606 (Korea)
provides a time-resolved fluorlmetnc techmque to solve the problems of quenching effects In this study, the time-resolved fluorlmetrlc technique was also used, but it was improved to derive the fluorescence mtenslty at trme zero adequately This method derives a signal at time zero using the integrated values at two gates with appropriately selected delay intervals and two precise analog integrators As a result, it was ascertained that the effects of quenching or temperature fluctuation could be efficiently ehmlnated
THEORETICAL
BACKGROUND
Various types of theoretxal bases are possible for derlvmg the mtenslty gf fluorescence at time zero A theoretically snnple method is to use a signal rntenslty at two pomts m the signal decay curve for calculating the mtenslty at time zero However, this method 1s difficult to use because of serious electrical noise interference
0003-2670/92/$0_5 00 0 1992 - Elsevler Science Publishers B V All rights reserved
S-M Lee et al /Anal
Chun Rcta 244 (1992) 1.25-129
IS the fluorescence mtenslty at time zero and T IS the fluorescence hfetlme With d as the delay interval and g as the gate time, the first area, S,, 1s given by
S, = adagio e-‘/’ m;
dt
s-~:-;~~;(;-‘:,’ 9
S,=~2f+~2*&
eat/'
- 1)
(2)
27
dt
*d*hS*d*P'
= -_1# e -(2d+g)/~(e-&+ - 1) Derwatlon of fluorescence mtenslty at time zero by mtegratlon of two mtervals
Equation 2 dlvlded by Eqn 3 becomes
As regards this point, a method desgned to Integrate two mtervals m a slgnal decay curve usmg two precise analog Integrators and a twochannel A/D converter for each interval was used Thus method can solve the noise problems and directly use the received mformatlon meluded m the decay cmve Figure 1 shows the general shape of an exponentially decaymg fluorescence signal A general exponential function 1s expressed as follows 1~1, e-t/7 (1)
’ = ln( S,/S,)
d+s
where I IS the fluorescence intensity at tune t, Z,
97-l
(3)
(4)
On the other hand, Eqn 2 can be formulated as follows, I, = S,/[ 7 ePd17( 1 - e-8/T)] = &S1
In(S,/S,)(S,/S,)dffd+gf
x [ 1 - (S,/S,)
--g’(d+g)]-’
(5)
In Eqn 5, the intensity of fluorescence at time zero, lo, IS unaffected by changes m fluorescence
r-----l merowmputer
pie
Dwcardad Fig 2 SchematIc dlagtam of a fluorescence analyser PMT = photomultrpher tube (Hamamatsu type lP28A), Cell = quartz cell (10 mm x 10 mm x 40 mm), L = quartz blconvex lens (L,, f= 75 mm, L,, f= 45 mm), N, laser = Model VSL-337,120 ,uJ per pulse, pulse vndth (FWHM) < 3 ns, Interface board = DIO and 4-channel A/D converter, ~~111~~ = LeCroy 9400 ~~d~dth 12.5MHz)
S 44 Lee et al /Anal
127
Chrm Acta 264 (1992) 125-129
lifetime caused by quenching effects To obtam the optimum delay and gate times, partial drfferentlatlon of Eqn 5 agamst S, and Sz is performed and the equation for AZ,/Z, IS as follows, AZ,,/Z, = A&/S,((2d -g/[(d
+ 2g + T)/(d
+g)(l
+g)
-e-g’?]}
=
Fig 3 Clrcultry of fluorescence signal Integrator with two CMOS analog mtches and an MF capacitor
+AS,/S,{(d+g+~)/(d+g) -g/[(d
=
+g)(l - eWg/‘)])
If g/T .=K1, then AZ,/Z, = AWS,(2 + =
- 1/[2(1+
AWS,(l -
d/g)]}
1/[2(1+
d/g)]}
K1(AS,/&) + K,( A%/%)
(6)
In Eqn 6, the relative change m fluorescence mtenslty at tnne zero, AZO/Z,, is mmnnlzed as long as the delay mterval d becomes shorter and the gate time g becomes longer However, the delay Interval has to be adjusted so that the noise at the begmnmg of the laser pulse 1s excluded from integration Also, consldermg other factors such as ripple frequency and AS,/& ratio, it seems appropriate to select a gate time of about r/4 N r/2 In comparison with a slmllar method [61,where the delay mterval d should be larger than gate time g, this approach can give better results It can shorten the delay Interval without reducing the gate time It 1s therefore possible to select a long gate time and ehmmate the effects of npple noise easily Also, because it can shorten the delay Interval d, a higher AS,/& ratio can be obtamed
APPARATUS
This theory was applied to a fluorescence agnal analyser developed m this laboratory Figure 2 shows a scheme of the apparatus The fluorescence slgnal emitted from a sample cell IS detected by a photomultlpher tube (PMT) (lP28A, Hamamatsu) and mtenslfled by an amplrfier The ygnal-processmg circuitry consists of two mtegrators, a DIO (digital mput/output), and A/D
converter (resolution 8 bits, 2 channels) and a computer The signal profile 1s observed with a LeCroy 9400 dIgIta storage oscilloscope (band width 125 MHz) A detailed descrlptlon of the apparatus was given in a previous paper [7] The Integrator uses a low-leakage MF (metallzed film) capacrtor and two CMOS analog mtches wrth a FET element m an mput port The input port of the integrator usmg a CMOS analog switch has an input impedance of more than 1 GR when It turns off Hence the mtegrated values of the signal stored m an mtegratmg capacitor can be mamtamed over several nulhseconds The parts of the delay and gate trme crrcmtry consist of two g-bit digital counters and two magnitude comparators, which can be controlled to 1 ps Figure 3 shows the integrator used m this expernnent
RESULTS AND DISCUSSION
Effect of quenchers The method for derwmg fluorescence mtenslty at time zero usmg integrated values at two mtervals was apphed to a pulsed mtrogen laser-mduced uranium fluorescence measurement m aqueous solution To observe the effect on uramum fluorescence quenchers [l-31, appropriate amounts of 0 02 M Mn2+ or 0 04 M Ca’+ were added to an aqueous solution of uranium (1 mg UO;+ l-l, pH 6) and 0 4 ml of Fluran (Scmtrex), Figure 4 shows the fluorescence signal from the solution The fluorescence intensities at time zero of each of three fluorescence signals were the
128
S -M Lee et al /Anal Chm Acta 264 (1992) 125-129
Fluorescence
Decay Tune (10&&v )
Shot
Shot
Fig 4 Fluorescence signals m uramum aqueous solution with and wlthout hfetlme quenchers Top, neat UO$+ solution, middle, Ca2+ added, bottom, MI? added
same but the fluorescence hfetlme was shortened by the addltlon of quencher solutions (Mn2+ or Ca2+) Because the hfetune of uranium fluorescence was about 38 ps, a 9+s delay and 11-ps gate time were chosen for integration Two mtegrated values were processed to obtam the fluorescence intensity at time zero using a 16-bit IBM-compatible personal computer Table 1 shows the relative devlatlon of the fluorescence Intensity at time zero The smgle-mterval integrated value, which relatively reflects the degree of quenchmg, 1s also shown for comparison The values were added over 20 times and averaged As shown m Table 1, the value obtained with the fluorescence intensity at time zero gave a much smaller devlatlon than the
Fig 5 Fluorescence signal with respect to temperature vanabon Top, 25”C, middle, 33’C, bottom, 43°C
uncorrected value obtained by smgle-interval mtegration Eff;ect of temperature jluctuatwn
Figure 5 shows the uramum fluorescence agnals from an aqueous solutlon (1 mg UO;+ l-l, pH 6) measured at 25, 33 and 43°C The fluorescence hfetlme appeared to be shortened conslderably as the temperature increased Thrs 1s considered to be due to the serious quenching caused by frequent colhslons at higher temperature It can be seen that the fluorescence mtenslty at
TABLE 1 Relative compamon of the fluorescence. Intensity at time zero with the smgle Interval mtegrated values with addltlon of quenchers Appropriate quantltles of 0 02 M Mn2+ or 0 04 M CaZ* were added Quencher
Smgle Interval mtegrated value (S,)
Fluorescence mtenslty at time zero (1,)
Uranium standard solutlon (ref ) Mn2+ added Ca2+ added
1
1
0 836 0 928
0 999 0 994
b
Fig 6 Dependence of relative devlatlon on temperature variatlons (0) for I,, (0) for S,
S -M Lee et al /Anal
Chum Acta 264 (1992) 125-129
time zero of each of the three fluorescence agnals 1s the same lrrespectlve of the temperature fluctuations In this experunent, owmg to the rapld lifetime shortening, the delay and gate tune were set at 8 and 10 ps, respectively The value obtained for the fluorescence mtenslty at time zero was also compared with the single-mterval Integrated value, S,, using a uramum standard solution (1 mg UOi+ l-‘, pH 6) The temperature of a 1 pg ml-’ uranium sample solution was heated gradually from 25 to 53°C The relative devlatlon with respect to the value at room temperature was plotted m Frg 6 The devlatlon obtamed using the fluorescence mtenslty at time zero IS much smaller than that obtamed using a single-interval integrated value, and has an error range wlthm f5% Conclwlons
A method for analysis of fluorescence mduced by a pulsed laser was developed It allows a more precise measurement of the fluorescence signal affected by various types of quenching than the usual method [6] It makes it possible to select a shorter delay interval and a longer gate time and to reduce the effect of ripple noise Also, a shorter
129
delay interval can be chosen and a higher signalto-noise ratlo obtamed for the integration A uranium fluorescence analyser that employed this method appeared to be apphcable to samples m a complicated matrm or with temperature vanatlons within a f5% error range The authors are grateful to the Mlmstry of Science and Technology of Korea for financial support
REFERENCES 1 M Monyasu, Y Yokoyama and S Ikeda, J Inorg Nucl Chem ,39 (1977) 2240 2 M Morlyasu and Y Yokoyama, J Inorg Nucl Chem, 53 (1977) 2211 3 Y Yokoyama, M Morlyasu and S Ikeda, J Inorg Nucl Chem , 38 (1976) 1329 4 J C Veselsky, B Kwlecmska, E Wehrstem and 0 Suschny, Analyst, 113 (1988) 451 5 G H Hleftje and G R Haugen, Anal Chum Acta, 123 (1981) 255 6 J C Robbms, prehmmary draft for dehvely to 30th Annual Conference on Bioassay, AnalytIcal and Envuonmental ChemlstIy, Cmcmnatl, OH, October 10-12, 1984 7 KW Jung, J M IOm, C J &m and J M Lee, J Kor Nucl Sot , 19 (1987) 242
Analyt&za Chrmzca Acta, 264 (1992) 125-129
Elsevier Saence Pubhshers B V , Amsterdam
Compensation of fluorescence quenching by extrapolation of fluorescence intensity at time zero Sang-Mock Lee, Jeong-Moog Km, Jang-Soo Shm and Cheol-Jung fim Korea Atomc
Energy Research Instrtute, P 0 Box 7, Daeduk-danIt,
(Received 20th December 1991, revised manumpt
DaeJeon 305404
(Korea)
received 27th February 1992)
Abstract
A method to compensate for fluorescence quenchmg caused by quenchmg elements or temperature fluctuations was studled Thus method calculates the fluorescence intensity at time zero, which 1s unaffected by the changes m fluorescence hfetlme due to various quenchmg effects The method uses the integrated signal at two gates with an appropriately selected delay interval on an exponentially decaymg fluorescence curve Induced by a mtrogen pulsed laser Apphcatlon to the measurement of uranmm concentration showed the exact compensatton for quenching caused by other ions or temperature fluctuations within f 5% error range Keywords
Fluonmetry, Fluorescence quenching, Uranium
Compared with other photometnc methods, fluornnetry provides some of the lowest detection lumts However, fluorunetnc measurements can suffer mterferences from quenchers [l-31 or sample temperature fluctuations Although a number of mechamsms for such quenching reactions exist, they produce the same result, 1 e , a reduced quantum efficiency [4,.5] owmg to a consequent loss in fluorescence mtenslty Correction for quenching effects 1s very dlfflcult because the concentration of the quenching agent is usually unknown There are several methods avadable for the ebmlmatlon of these large mterfermg effects, mcludmg dilution of the sample, the use of an internal standard or the separation of a fluorophore from the quencher However, these methods are very time consuming The development of lasers with high spectral brightness, dlrectlonahty and short pulse length Correspondence to Dr Sang-Mock Lee, Korea Atomic Energy Research Institute, P 0 Box 7, Daeduk-danjl, DaeJeOn, 305606 (Korea)
provides a time-resolved fluorlmetnc techmque to solve the problems of quenching effects In this study, the time-resolved fluorlmetrlc technique was also used, but it was improved to derive the fluorescence mtenslty at trme zero adequately This method derives a signal at time zero using the integrated values at two gates with appropriately selected delay intervals and two precise analog integrators As a result, it was ascertained that the effects of quenching or temperature fluctuation could be efficiently ehmlnated
THEORETICAL
BACKGROUND
Various types of theoretxal bases are possible for derlvmg the mtenslty gf fluorescence at time zero A theoretically snnple method is to use a signal rntenslty at two pomts m the signal decay curve for calculating the mtenslty at time zero However, this method 1s difficult to use because of serious electrical noise interference
0003-2670/92/$0_5 00 0 1992 - Elsevler Science Publishers B V All rights reserved
S-M Lee et al /Anal
Chun Rcta 244 (1992) 1.25-129
IS the fluorescence mtenslty at time zero and T IS the fluorescence hfetlme With d as the delay interval and g as the gate time, the first area, S,, 1s given by
S, = adagio e-‘/’ m;
dt
s-~:-;~~;(;-‘:,’ 9
S,=~2f+~2*&
eat/'
- 1)
(2)
27
dt
*d*hS*d*P'
= -_1# e -(2d+g)/~(e-&+ - 1) Derwatlon of fluorescence mtenslty at time zero by mtegratlon of two mtervals
Equation 2 dlvlded by Eqn 3 becomes
As regards this point, a method desgned to Integrate two mtervals m a slgnal decay curve usmg two precise analog Integrators and a twochannel A/D converter for each interval was used Thus method can solve the noise problems and directly use the received mformatlon meluded m the decay cmve Figure 1 shows the general shape of an exponentially decaymg fluorescence signal A general exponential function 1s expressed as follows 1~1, e-t/7 (1)
’ = ln( S,/S,)
d+s
where I IS the fluorescence intensity at tune t, Z,
97-l
(3)
(4)
On the other hand, Eqn 2 can be formulated as follows, I, = S,/[ 7 ePd17( 1 - e-8/T)] = &S1
In(S,/S,)(S,/S,)dffd+gf
x [ 1 - (S,/S,)
--g’(d+g)]-’
(5)
In Eqn 5, the intensity of fluorescence at time zero, lo, IS unaffected by changes m fluorescence
r-----l merowmputer
pie
Dwcardad Fig 2 SchematIc dlagtam of a fluorescence analyser PMT = photomultrpher tube (Hamamatsu type lP28A), Cell = quartz cell (10 mm x 10 mm x 40 mm), L = quartz blconvex lens (L,, f= 75 mm, L,, f= 45 mm), N, laser = Model VSL-337,120 ,uJ per pulse, pulse vndth (FWHM) < 3 ns, Interface board = DIO and 4-channel A/D converter, ~~111~~ = LeCroy 9400 ~~d~dth 12.5MHz)
S 44 Lee et al /Anal
127
Chrm Acta 264 (1992) 125-129
lifetime caused by quenching effects To obtam the optimum delay and gate times, partial drfferentlatlon of Eqn 5 agamst S, and Sz is performed and the equation for AZ,/Z, IS as follows, AZ,,/Z, = A&/S,((2d -g/[(d
+ 2g + T)/(d
+g)(l
+g)
-e-g’?]}
=
Fig 3 Clrcultry of fluorescence signal Integrator with two CMOS analog mtches and an MF capacitor
+AS,/S,{(d+g+~)/(d+g) -g/[(d
=
+g)(l - eWg/‘)])
If g/T .=K1, then AZ,/Z, = AWS,(2 + =
- 1/[2(1+
AWS,(l -
d/g)]}
1/[2(1+
d/g)]}
K1(AS,/&) + K,( A%/%)
(6)
In Eqn 6, the relative change m fluorescence mtenslty at tnne zero, AZO/Z,, is mmnnlzed as long as the delay mterval d becomes shorter and the gate time g becomes longer However, the delay Interval has to be adjusted so that the noise at the begmnmg of the laser pulse 1s excluded from integration Also, consldermg other factors such as ripple frequency and AS,/& ratio, it seems appropriate to select a gate time of about r/4 N r/2 In comparison with a slmllar method [61,where the delay mterval d should be larger than gate time g, this approach can give better results It can shorten the delay Interval without reducing the gate time It 1s therefore possible to select a long gate time and ehmmate the effects of npple noise easily Also, because it can shorten the delay Interval d, a higher AS,/& ratio can be obtamed
APPARATUS
This theory was applied to a fluorescence agnal analyser developed m this laboratory Figure 2 shows a scheme of the apparatus The fluorescence slgnal emitted from a sample cell IS detected by a photomultlpher tube (PMT) (lP28A, Hamamatsu) and mtenslfled by an amplrfier The ygnal-processmg circuitry consists of two mtegrators, a DIO (digital mput/output), and A/D
converter (resolution 8 bits, 2 channels) and a computer The signal profile 1s observed with a LeCroy 9400 dIgIta storage oscilloscope (band width 125 MHz) A detailed descrlptlon of the apparatus was given in a previous paper [7] The Integrator uses a low-leakage MF (metallzed film) capacrtor and two CMOS analog mtches wrth a FET element m an mput port The input port of the integrator usmg a CMOS analog switch has an input impedance of more than 1 GR when It turns off Hence the mtegrated values of the signal stored m an mtegratmg capacitor can be mamtamed over several nulhseconds The parts of the delay and gate trme crrcmtry consist of two g-bit digital counters and two magnitude comparators, which can be controlled to 1 ps Figure 3 shows the integrator used m this expernnent
RESULTS AND DISCUSSION
Effect of quenchers The method for derwmg fluorescence mtenslty at time zero usmg integrated values at two mtervals was apphed to a pulsed mtrogen laser-mduced uranium fluorescence measurement m aqueous solution To observe the effect on uramum fluorescence quenchers [l-31, appropriate amounts of 0 02 M Mn2+ or 0 04 M Ca’+ were added to an aqueous solution of uranium (1 mg UO;+ l-l, pH 6) and 0 4 ml of Fluran (Scmtrex), Figure 4 shows the fluorescence signal from the solution The fluorescence intensities at time zero of each of three fluorescence signals were the
128
S -M Lee et al /Anal Chm Acta 264 (1992) 125-129
Fluorescence
Decay Tune (10&&v )
Shot
Shot
Fig 4 Fluorescence signals m uramum aqueous solution with and wlthout hfetlme quenchers Top, neat UO$+ solution, middle, Ca2+ added, bottom, MI? added
same but the fluorescence hfetlme was shortened by the addltlon of quencher solutions (Mn2+ or Ca2+) Because the hfetune of uranium fluorescence was about 38 ps, a 9+s delay and 11-ps gate time were chosen for integration Two mtegrated values were processed to obtam the fluorescence intensity at time zero using a 16-bit IBM-compatible personal computer Table 1 shows the relative devlatlon of the fluorescence Intensity at time zero The smgle-mterval integrated value, which relatively reflects the degree of quenchmg, 1s also shown for comparison The values were added over 20 times and averaged As shown m Table 1, the value obtained with the fluorescence intensity at time zero gave a much smaller devlatlon than the
Fig 5 Fluorescence signal with respect to temperature vanabon Top, 25”C, middle, 33’C, bottom, 43°C
uncorrected value obtained by smgle-interval mtegration Eff;ect of temperature jluctuatwn
Figure 5 shows the uramum fluorescence agnals from an aqueous solutlon (1 mg UO;+ l-l, pH 6) measured at 25, 33 and 43°C The fluorescence hfetlme appeared to be shortened conslderably as the temperature increased Thrs 1s considered to be due to the serious quenching caused by frequent colhslons at higher temperature It can be seen that the fluorescence mtenslty at
TABLE 1 Relative compamon of the fluorescence. Intensity at time zero with the smgle Interval mtegrated values with addltlon of quenchers Appropriate quantltles of 0 02 M Mn2+ or 0 04 M CaZ* were added Quencher
Smgle Interval mtegrated value (S,)
Fluorescence mtenslty at time zero (1,)
Uranium standard solutlon (ref ) Mn2+ added Ca2+ added
1
1
0 836 0 928
0 999 0 994
b
Fig 6 Dependence of relative devlatlon on temperature variatlons (0) for I,, (0) for S,
S -M Lee et al /Anal
Chum Acta 264 (1992) 125-129
time zero of each of the three fluorescence agnals 1s the same lrrespectlve of the temperature fluctuations In this experunent, owmg to the rapld lifetime shortening, the delay and gate tune were set at 8 and 10 ps, respectively The value obtained for the fluorescence mtenslty at time zero was also compared with the single-mterval Integrated value, S,, using a uramum standard solution (1 mg UOi+ l-‘, pH 6) The temperature of a 1 pg ml-’ uranium sample solution was heated gradually from 25 to 53°C The relative devlatlon with respect to the value at room temperature was plotted m Frg 6 The devlatlon obtamed using the fluorescence mtenslty at time zero IS much smaller than that obtamed using a single-interval integrated value, and has an error range wlthm f5% Conclwlons
A method for analysis of fluorescence mduced by a pulsed laser was developed It allows a more precise measurement of the fluorescence signal affected by various types of quenching than the usual method [6] It makes it possible to select a shorter delay interval and a longer gate time and to reduce the effect of ripple noise Also, a shorter
129
delay interval can be chosen and a higher signalto-noise ratlo obtamed for the integration A uranium fluorescence analyser that employed this method appeared to be apphcable to samples m a complicated matrm or with temperature vanatlons within a f5% error range The authors are grateful to the Mlmstry of Science and Technology of Korea for financial support
REFERENCES 1 M Monyasu, Y Yokoyama and S Ikeda, J Inorg Nucl Chem ,39 (1977) 2240 2 M Morlyasu and Y Yokoyama, J Inorg Nucl Chem, 53 (1977) 2211 3 Y Yokoyama, M Morlyasu and S Ikeda, J Inorg Nucl Chem , 38 (1976) 1329 4 J C Veselsky, B Kwlecmska, E Wehrstem and 0 Suschny, Analyst, 113 (1988) 451 5 G H Hleftje and G R Haugen, Anal Chum Acta, 123 (1981) 255 6 J C Robbms, prehmmary draft for dehvely to 30th Annual Conference on Bioassay, AnalytIcal and Envuonmental ChemlstIy, Cmcmnatl, OH, October 10-12, 1984 7 KW Jung, J M IOm, C J &m and J M Lee, J Kor Nucl Sot , 19 (1987) 242