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Table I. Detection Limits and Sensitivities for Two-Color LEI in the T-Furnace with Corresponding Spectroscopic Data
det limit, pg ultimat det limit, pg cont blank, pg sensitivity, fC/pg XI, nm XZ, nm laser energy/pulse XI, pJ laser energy/pulse X p , p J
Mn
Sr
1 0.001
2 0.001 2
1 50 279.48 456.4* 5 5000
500" 460.73 554.34 0.1 1
Sensitivity obtained with strongly attenuated lasers. second step cannot be identified.
The
sensitivities for LEI in flames (3). The sensitivities for flame LEI are given as charge collected per concentration. Assuming an aspiration rate of 6 mL/min and a laser repetition rate of 50 Hz, we equate the 5000 aC/ppb for Mn to 2.5 fC/pg, and the 4000 aC/ppb value for Sr to 2 fC/pg. This comparison gives the sensitivities for the T-furnace measurement to be 20 times more sensitive for Mn and 250 times more sensitive for Sr than flame LEI. Detection limits and sensitivities for two-color LEI in a T-furnace with corresponding spectroscopic data are compiled in Table I. The sensitivity is given as the total amount of charge collected at the plates during the whole time of atomization per picogram of analyte (femtocoulombs per picogram). I t should be pointed out that in the present construction of the T-furnace there is a high temperature gradient between the center of the graphite tube and the detection region. It is also noteworthy that the graphite in the furnace is not pyrolytically coated. These two facts imply that only a very low fraction of the atoms in the sample will reach the detection region because of the atoms sticking to the walls and formation of molecules. Comparing the sensitivity figures obtained here with the ideal case, i.e. 100% ionization of all atoms in the sample and unit charge collection efficiency, gives absolute efficiencies of about for Sr and for Mn. An improvement of the above-mentioned factors (which is now in progress) will increase the number of atoms in the detection region by several orders of magnitude. This will probably give detection limits for the method in the order of 1 fg. The linearity was only tested over 1 order of magnitude, where it was good. Also the reproducibility was good according to these preliminary measurements. Disturbances from electrons emitted from the heated graphite tube were not observed with a negative potential on the outer plate. A positive voltage, however, resulted in a very high noise level which was due to a large dc current carried by thermionically emitted electrons. Blocking of the laser light did not result in any detectable decrease of the noise level.
This strongly proves that the earlier statement considering the thermionic emission of electrons inside the heated graphite tube is correct and shows that this emission is very large. No effects from photoelectric emission due to scattering of laser light were observed in the present measurements. Thermionic electrons caused the main problem when an electrode inside the graphite tube was used for signal collection since the dc current caused by them made it impossible to retain a voltage between the electrode and the graphite tube. Due to this dc current it was only possible to measure a LEI signal during the first few seconds of atomization. This was a sufficiently long time to measure Mn but not long enough for Sr determination because of the slow atomization of that element. No disturbances from the large heating current (500 A) were observed in these experiments and it was not necessary to use the special trigger unit that was used in our earlier works (5-7). In conclusion, we have demonstrated in this work a very high sensitivity of the T-furnace in combination with insensitivity to disturbances from the heating current as well as to the thermally emitted electrons. This suggests that LEI in the T-furnace can become a very attractive method for ultrasensitive trace element analysis of microsamples when the problems in the present construction are overcome, i.e. the temperature gradient between the atomization and detection region is lowered and pyrolytic graphite is used for the construction of the furnace.
ACKNOWLEDGMENT The support of Ingvar Lindgren is greatfully acknowledged as are discussions with Ove Axner. Registry No. Mn, 7439-96-5; Sr, 7440-24-6;graphite, 7782-42-5. LITERATURE CITED (1) Travis, J. C.; Turk, G. C.; Green, R. B. Anal. Chem . 1982, 5 4 , 1006A. (2) Camus, P. Euroanal. V, Rev. Anal. Chem. 1987, 107-115. ( 3 ) Axner, 0.; Magnusson, I.; Petersson, J.; Sjostrom, S. Appl. Spectrosc. 1987, 4 1 , 19. (4) Gonchakov, A. S.;Zorov, N. B.; Kuzyakov, Yu. Ya.; Matveev, 0. I. Anal. Left. 1979, 72, 1037. (5) Magnusson, I.; Axner, 0.; Lindgren, I.; Rubinsztein-Dunlop, H. Appl. Spectfosc. 1986, 4 1 , 968-971. (6) Magnusson, I.; Sjostrom, S.;Lejon, M.; Rubinsztein-Dunlop, H. Spectrochim. Acta, Part B 1987, 428, 713-718. (7) Magnusson, I. Spectrochim. Acta, Part 8 , in press.
Sten Sjostrom* Ingemar Magnusson Mats Lejon Halina Rubinsztein-Dunlop Department of Physics Chalmers University of Technology S-412 96 Goteborg, Sweden RECEIVED for review October 6, 1987. Accepted March 19, 1988. This work has been financed by the Swedish Natural Science Research Council.
Presence of Hydroxylamine in the Phosphoric Acid/Nitric Acid/Hydrogen Peroxide Digestion Procedure for Selenium Determination Sir: In a recent paper in Analytical Chemistry (1) an interesting method for digestion of soil and plant materials for selenium determination was presented. The method included an addition of Mn(I1) to a mixture of phosphoric
acid/nitric acid/hydrogen peroxide, Mn(I1) serving as a covenient indicator of completeness of the digestion as it was oxidized to the purple MnO, ion when oxidation of the sample was complete.
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As the final step of the procedure an addition of hydroxylamine hydrochloride, NH,OH.HCl (in a solution of EDTA) was included in order to minimize any oxidation of diaminonaphthalene (DAN), which served as the complexing agent for Se(IV) in the subsequent fluorometric determination. Although the addition of NHzOH has also been recommended by other workers who use the DAN-fluorometric method (2, 3), it appears to be rather doubtful advice, as this agent is known to be an effective reductant for Se(IV), resulting in the formation of elemental selenium. As such, it has been commonly used in gravimetric methods for selenium and its reduction properties on Se(1V) have been discussed elsewhere ( 4 ) . It is obvious that if NHzOH would reduce any Se(1V) to the elemental state, this fraction would become unavailable for further complex formation with DAN or for determination by other spectrometric techniques as well. In order to elucidate the potential problem, it was decided to examine the effect of hydroxylamine in the present procedure in some detail. The results of the study are presented in this paper. EXPERIMENTAL SECTION For details of the digestion procedure ref 1should be consulted. Experiment 1. Wheat corn (0.200 g) plus 0.150 g of selenourea, (NH,),CSe ( E N Pharmaceuticals, Inc.), was taken through the given procedure, resulting in the predicted formation of Mn04-, indicating complete oxidation. After the addition of 6 M HC1 and the subsequent heating and cooling, 8 mL of the 0.04 M EDTA/10% NH,OH-HCl solution was added, and the development of color due to the formation of elemental selenium was observed (see "Results"). Experiment 2. Two samples of wheat corn, each of 0.300 g, were dissolved according to the recommended procedure, and after final cooling, 1.6 pg of Se(1V) (in 200 pL) was added to each solution. To one of the solutions the recommended 8 mL of EDTA/NH20Hsolution was added immediately after the cooling, whereas for the other solution, this addition was done after the elapse of 1 h. Both solutions were thereafter (1h) transferred to 100-mLvolumetric flasks, which were filled to volume. The solutions were immediately analyzed for selenium by hydride generation atomic absorption spectrometry using an automatic hydride generator (P.S. Analytical, U.K.) and an atomic absorption spectrometer (Perkin-Elmer Model 300). R E S U L T S A N D DISCUSSION Experiment 1. The large amount of selenium present allowed a visual observation of elemental selenium. After the addition of EDTA/NH,OH, the solution became yellow, orange, and at last (within 10 min) definitely red owing to the formation of elemental (red) selenium. This clearly demonstrated that the suspected reaction had taken place. Experiment 2. The absorbance signal for the solution to which hydroxylamine was added just before measuring was
taken as 100%. The signals for the solution to which the hydroxylamine was added 1h before measuring were recorded as 80% of the former signal. This also indicated that the unwanted reaction had, to some extent, taken place. General Discussion. The reduction of Se(IV) to elemental selenium by hydroxylamine is not a very fast reaction, as indicated in experiment 1. Thus, the success of the method seems to depend on an immediate addition of the complexing agent (DAN) after the addition of hydroxylamine. By that means the selenium will probably be completely complexed. This may also explain the lack of interference from hydroxylamine with the DAN-selenite reaction reported earlier (1, 2). Moreover, acidity may also influence the sample solution, since both the precipitation rate and the rate of the complex formation with DAN are pH-dependent. The rate of precipitation of elemental selenium is also probably even slower when trace concentrations of selenium are concerned, as indicated in experiment 2. However, it is recommended that the addition of hydroxylamine should be omitted, thus making the procedure even more attractive. With fluorometric methods the claimed purpose with the addition was to minimize oxidation of DAN by residual HN03. But the amount of H N 0 3 in the final solution is probably very minute and the DAN is always added in quite large excess, so this precaution is perhaps be unnecessary. Any excess of H N 0 3 may also be destroyed by addition of formic acid (3),as also mentioned by the authors. For other determination techniques, such as atomic absorption spectrometry and inductively coupled plasma, the digestion method should be equally well suited; moreover, the presence of hydroxylamine will not serve any purpose with these techniques and can certainly be omitted. The digestion method itself appeared to be very convenient. The use Mn(I1) as a redox indicator seems to be a nice solution to a problem common with many digestion procedures, i.e. to decide when the oxidation of the sample is completed. Registry No. Se, 7782-49-2; "OB, 7697-37-2; H3P04,766438-2; HzOz,7722-84-1;",OH, 7803-49-8; seleno urea, 630-10-4. LITERATURE C I T E D (1) Dong, A.; Rendig, V. V.; Burau, R. G.; Besga, G. S. Anal. Chem. 1887, 59, 2730-2732. (2) Cukor, P.; LOR, P. F. J . Phys. Chem. lQ65, 69, 3232-3239. (3) Reamer, D. C.; Veillon, C. Anal. Chem. lQ83, 55. 1605-1606. (4) Bye, R. Talanfa IQS3, 3 0 , 993-996.
R a g n a r Bye Department of Chemistry Agricultural University of Norway 1432 Aas-NLH, Norway
RECEIVED for review December 29,1987. Accepted March 25, 1988.
Characterization of Unseparated Nucleic Acid Restriction Enzyme Fragments by Electric Birefringence Frequency Dispersion Sir: Electric birefringence of nucleic acids in gels is a common tool for investigating the dynamics of internal motions and the mechanism of electrophoretic transport of the polymers (1-3). Classically, interest has centered on large polymers, but recently several investigators have reported the behavior of small (50-25000 bp (base pair)) monodisperse fragments (4-6). Monodisperse fragments oriented in an electric field usually obey an exponential relaxation relation,
which can be related to the electrophoretic mobility (6, 7). Electric birefringence, and less commonly, relaxation of fluorescence polarization (8-11), are convenient methods for testing such theories. Electric birefringence measurements are most commonly made in the time domain. The sample is oriented in a pulsed electric field, and relaxation constants are evaluated from the time course of the birefringence decay. A few workers have
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