Anal. Chem. 1984, 56, 839-842
that helium will move a pneumatic valve actuator about 2.4 times faster than air. This study also shows that as the valve actuation time becomes smaller, the size of the injection pulse that will hit the HPLC column will decrease. Since injection pulses can cause HPLC column breakdown (I-3), faster injections should lead to longer HPLC column life. Recently we reported a “moving injection technique” for injecting very small samples directly onto microcolumns (5). This technique was based on an electrically actuated valve that had a transit time of 500 ms. Table I shows that a helium piloted actuator can move the valve in 8 ms from load to inject. A moving injection based on helium actuation should inject much smaller samples than reported using the electric actuator. It is difficult to say how fast it is necessary to move the valve to stop deterioration of high-speed, high-pressure HPLC columns. Most piston type HPLC pumps give small pulses due to piston crossover. In this study, the pump gave 4 psi pressure pulses to the column (2000 psi on injection valve, 2.0
839
mL/min flow rate). From Table I it can be seen that the injection pulse will be equal to the pump pulse a t a transit time of 49 ms. It has been suggested (6) that actuation times of less than 100 ms should be used for HPLC columns that are sensitive to pressure pulses. Fast valve switching may also be of advantage in reducing pressure surges in gas chromatography sampling and switching applications.
LITERATURE CITED (1) Dicesare, J. L.; Dong, J. R.; Gant, J. R. Chromatographla 1982, 159, 595-598. (2) Erni, R., Presented at the 7th International Symposium on Column Liquid Chromatography, May 2-6, 1983, Baden-Baden, West Germany. J. Chromatogr. 1983,282,371-383. (3) Lamer, W.; Molnar, I.“Practical Aspects of Modern HPLC”; Moinar, I., Ed.; Walter de Gruyter & Co.: New York, 1982; pp 213-225. (4) Graham, T. Philos. Trans. R . SOC.,London 1846, 136, 573. (5) Harvey, M. C.; Stearns, S. D. J. Chromafogr. Sci. lg83, 21, 473-477. (6) van der Waals, Sj., Varlan, personal communication, 1983.
RECEIVED for review June 13, 1983. Resubmitted December 7, 1983. Accepted January 3, 1984.
Spectrophotometric Determination of Trace Boron in Biological Materials after Alkali Fusion Decomposition Kazuo Yoshino,*’ Makoto Okamoto, and Hidetake Kakihana2 Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Ookayama, Meguro-ku Tokyo 152, J a p a n
Takabumi Nakanishi, Masamitsu Ichihashi, and Yutaka Mishima Department of Dermatology, Kobe University School of Medicine, 7 Kusunoki-cho, Chuo-ku Kobe 650, J a p a n In neutron capture therapy, which utilizes the energy of the lOB(n,c~.)~Li reaction for destruction of malignant tissue, loB must be selectively accumulated in the malignant part before irradiation with neutrons (1-4). For this purpose, it is necessary to determine the loB distribution in the animal bearing the tumor after the loB compound, which is expected to accumulate loB in the malignant tissue, is injected into the animal. Kobayashi and Kanda have reported the prompt y-ray spectrometry method for determining the trace amount of loB in animal tissue (5). Although this method has the advantage of not requiring pretreatment, the necessity of a nuclear reactor as the neutron source restricts the use of this method. In the chemical method for the determination of trace boron in animal tissue, it is necessary to decompose tissue and degrade the boron compounds in i t into determinable boron species. For the decomposition of plant tissue, the dry ashing technique has been used (6, 7). But it seems difficult to decompose animal tissue by this method, and no reliable data have been observed. Ikeuchi and Amano have used the radio frequency combustion system with excited oxygen plasma for the decomposition of animal tissue, and boron was determined by the curcumine method (8). But the plasma ashing method took a relatively long time and the loss of boron could not be avoided. Further, the method had the drawback that some of the boron compound may remain unchanged. Isozaki and Utsumi employed alkali fusion for the decomposition of rocks and determined boron by a spectrophotoPresent address: Department of Chemistry, Faculty of Science, Shinshu University, Asahi, Matsumoto 390, Japan. Present address: Institute of Plasma Physics, Nagoya University, Furo-cho, Chikusa-ku Nagoya 464, Japan,
metric method in which boron was extracted as tetrafluoroborate with methylene blue (9). This method has not been applied to biological materials. In this paper the authors described the application of Isozaki’s method to normal liver and malignant melanoma. The proposed decomposition method is simple and effective enough to degrade the boron compound, which has a B-C bond, and moreover has the advantage that the loss of boron is scarcely observed.
EXPERIMENTAL SECTION Apparatus and Solutions. The spectrophotometerused was a Shimazu Model 50 PL. Deionized water was distilled twice with a quartz distillation apparatus and this distilled water was used throughout the experiments. Standard Boron Solutions. Boric acid, 2.860 g, which had been recrystallized three times from aqueous solution, was dissolved in 1000 mL of water (500 pg of B mL-l). By diluting the solution we prepared 0.1 and 0.2 pg of B mL-’ solutions. Hydrofluoric Acid Solution. Hydrofluoric acid solution (50.1%) for semiconductor use was diluted to give a 5% solution. Sulfuric Acid Solution. Sulfuric acid, 202 g (97% S.S.G.) was diluted to 1024 g with water (2 mol dm-3). Sodium Carbonate Solution. Sodium carbonate anhydrous, 10.6 g (99.97-99.99%, Asahi Glass Co.), was dissolved to give a 1000 mL solution (0.1 mol dm-3). Methylene Blue Solution. Methylene blue for microscopy, 3.739 g (Merck), was dissolved to give a 1000 mL solution, and a 100-mL portion of the solution was diluted to 1000 mL (0.001 mol dm-3). Other solutions were prepared by use of the reagents of analytical-reagent grade. Stock solutions were kept in polypropylene vessels. Decomposition of Biological Materials. To weighed tissue in a platinum crucible, 2 mL of 2 mol dm-3 sodium hydroxide solution and 5 mL of 0.1 mol dm-3sodium carbonatesolution were added. The crucible was gently warmed on a hot plate and with
0003-2700/84/0356-0839$01.50/00 1984 American Chemical Society
840
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
0.9
08 a00 7 -
OG-
0.7
a I
05-
nc a
0.6
bO4a
W
U
z
C O
' 03-
8 05 ' 5:m E
-C
,/'
/&
0.4
0.3 0.2
Flgure 2. Calculated calibration curve for the experiment with 200 mg of wet normal liver (full line) and without tissue (dotted line): the mean absorbance difference with liver at a given amount of boron (Ant- A i ) (e)and that without tissue (A, - A ob) (0).
0.1
550
600 WAVELENGTH
650 nm
Figure 1. Absorption spectra of methylene blue-tetrafluoroborate complex in the case of alkali fusion with normal liver (full line) and without tissue (dotted line). The decomposed amount of normal liver was 200 mg: (a) without addition of boron, (b) with addition of 1 .O pg of boron.
an infrared lamp until dryness. Sodium carbonate anhydrous, 4.947 g, was added to it. The covered crucible was heated with an oxidizing flame which was gradually strengthened until the alkali began to fuse. After the content became a fused liquid, foaming began. The alkali fusion continued about 10 min after foaming stopped. After the crucible was allowed to cool, the splashes were washed from the cover with water; then the water was evaporated to dryness. The content was fused again for about 15 min. In this case the alkali splashes attached to the inside of the wall were fused down by heating the outside of the crucible. The content was transferred to a 100-mLTeflon beaker, to which 35 mL of 2 mol dm-3 sulfuric acid solution was added slowly. Then the solution was transferred to a 100-mL quartz flask and water was added up to 100 mL. This was used as the sample solution. Determination of Boron. The sample solution, 10 mL, was transferred to a polypropyrene separatory funnel with a polypropylene pipet. Into the funnel 3 mL of 5% hydrofluoric acid solution and 7 mL of water were added. After the solution was allowed to stand for more than 1h at about 25 "C, 3 mL of 0.001 mol dm-3 methylene blue solution and 10 mL of 1,2-dichloroethane were added. The funnel was shaken for about 3 min and the 1,2-dichloroethanephase was transferred to another separatory funnel which had 5 mL of silver sulfate solution (9.25 X lo-* mol dm-3). This was shaken for about 3 min. After the mixture was allowed to stand for more than 30 min, the absorbance peak at about 660 nm in the 1,2-dichloroethane phase was measured on the spectrophotometer. The path length of the cell (quartz) was 10 mm, and 1,2-dichloroethane was used as the reference. Calibration Curve. Boron-free tissue was decomposed by the procedures described in the above section, and 10 mL of the obtained sample solution was transferred to a separatory funnel. Boric acid solution (0.1 or 0.2 gg of B mL-') was added t o the funnel to give the boron content from 0.0 to 1.0 pg. Water was added to make the total volume of water and boric acid solution 7 mL. The same procedures as those described in the spectrophotometric measurement were carried out.
RESULTS AND DISCUSSION Calibration Curve. In the case of the residue of 200 mg of wet normal liver after alkali fusion, a slight deviation of the peak absorbance in the spectra of methylene blue-tetrafluoroborate complex was observed as shown in Figure 1. A
similar slight deviation was also observed in the case of melanoma and blood. Then, in order to evaluate the influence of the residue on the calibration curve, the calibration curve for tissue was compared with the curve obtained by the alkali fusion without tissue. In both cases, the curves were found to fit the equation
Ant - Aot
CUZ'
+ bn + c
or
Anb - A,h = UTI'
+ bn + c
where A: and Ad are the absorbances obtained by adding n or 0 p g of boron to the solution made by decomposing boron-free tissue, Anband Aob are those obtained by adding n or 0 pg of boron to the blank solution without tissue, and a, b, and c are constants. In the case where JAot- AobJwas below 0.006, tissue was regarded as boron-free. The calibration curve changed when the reagent and/or the stock solutions were renewed. A typical example of the calibration curve with 200 mg of wet normal liver and the curve without tissue for Aob of 0.212-0.273 is shown in Figure 2. In this case, the s of the percent deviation (PD) of the experimental value (A$ A$)exptl from the corresponding calculated value (A$ - Aob),dcd was 1.4% for 22 measurements, and the mean value of the PD of (A: - AOt)exptl from the corresponding (Anb - AOb)calcd was -1.7% (s = 1.6%) for 13 measurements. In the case of eight measurements of melanoma, with the increase of the dry weight of melanoma from 15 mg to 38 mg, the PD of (Ao: - AOt)exptlfrom (Ao.db- Aob)cdcdand (A0.k - AOt)exptl from (-40 ab - AOb)calcddecrease from 4-3 and +4% to -2 and -l%, respectively. In the case of 8 and 2 1 mg of dry blood, the PD of (A: - AOt)exptl from the corresponding (Anb- AOb)calcd( n = 0.2, 0.4, and 0.8) was between C0.7 and -4.1% for six measurements. Recovery of Added Boron. Since alkali fusion is performed a t a very high temperature, preventing the boron from escaping is a serious problem, especially if boron exists as an organic species in the tissue. It is necessary to fix boron before the alkali fusion. To solve this problem, before the alkali fusion, tissue was heated in a solution of both sodium carbonate and sodium hydroxide and this solution was evaporated to dryness. With evaporation the tissue was partially degraded and homogenized with the alkali. To check the loss of boron during alkali fusion, the recovery of added boron with and without tissue was examined. In the case of alkali fusion without tissue, when boric acid of 5.0, 10.0, and 10.0 pg of
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
841
4 Ot
L
2
4
6
8
1 0 1 2
PH
Figure 3. "6 NMR data at 31.96 MHz for solution of phenylboronic acid (0.05 mol dm-3) and at 28.75 MHz for solution of bQrax (0.05 mol dm-3 in borate). The chemical shift at a given pH is shown by 0 for phenylboronic acid and by 0 for borax. The line width at half height ( Wo,5)is shown by a vertical bar.
boron were added, 4.9,9.8,and 9.8 pg of boron were recovered, respectively; Le., the recovery was 98%. In the case of boric acid with 10.0 pg of boron with 2 g of wet normal liver, the recovery was 98%. In the case of [lOB]-p-boronophenylalanine hydrochloride ('OB-p-bpa HCl) with 2 g of wet normal liver, when 4.7, 4.7, 9.3, and 9.3 pg of boron were added, 4.5, 4.7, 9.6, and 9.4 pg were recovered, respectively. In the case of 1°B-p-bpaHCl with melanoma, when 4.7 (0.16), 9.3 (0.18), 4.7 (0.20),and 2.3 (0.22) pg of boron were added, in the blanket dry weight of melanoma (g), 4.6, 9.2, 4.6, and 2.4 pg were recovered, respectively. In these cases of 1°B-p-bpa HC1, the concentration of boron in 1°B-p-bpa HC1 solution, which was added to the tissue, was determined by this method. The mean recovery of boron of 1°B-p-bpa HC1 was 100% with s of 3% for eight measurements. llB NMR Study. Treatment of boronic acid with sodium hydroxide has been known to effect deboronation. This indicates that when 'OB-p-bpa HCl is treated with sodium hydroxide, phenylalanine and boric acid should be produced. Snyder et al. reported that after 0.5 g of p-boronophenylalanine (p-bpa) was refluxed in 20 mL of 5% sodium hydroxide solution for 48 h, a mixture of p-bpa and phenylalanine was obtained (IO). This suggests that the B-C bond in 'OB-p-bpa HC1 might not have been completely broken on the evaporation of the alkali solution to dryness in the decomposition procedures. Therefore, it is necessary to investigate whether alkali fusion does cut all B-C bonds or not. By use of llB NMR spectrometry, this problem was examined by using phenylboronic acid instead of 1°B-p-bpa HC1, because "B-p-bpa HCl (natural abundance of boron) was not available. Although phenylboronic acid has no alanine part, it is suitable for examination of the B-C bond between boron and benzene. As shown in Figure 3, the chemical shifts and the line width of phenylboronic acid are different from those of boric acid in alkaline solution (11-13). Phenylboronic acid, 61 mg, and boric acid, 31 mg, were decomposed by the present method independently. In this case, the total amount of sodium carbonate was 1 g. After alkali fusion, each content was dissolved into about 10 mL of water. llB NMR Spectra of the obtained solutions are shown in Figure 4. Since, in the case of phenylboronic acid, only one signal of boric acid in alkaline solution was observed, it is clear that phenylboronic acid was completely decomposed by the alkali fusion, and this indicates that the B-C bond of *OB-p-bpaHC1 may be com-
10 5 0 b PP-
f (a) fused b ric Flaure 4. "B NMR mectra of the alkaline 3lutic acid and (b) fused phenylboronic acid at 31.96 MHZ.' The boron concentration of both solutions was 0.05 mol dm3, and "6 abundance of boric acid used was 92.1 % . The reference signal of sodium tetrafluoroborate at 6 0.0 is not shown.
Table I. Assay Results of Boron in Hamster into Which 'OB-p-bpa HCI Was Injected
tissue liver
control
weight, g wet
dry
0.24 0.43
1.08 1.99
boron found, p g in 1 g in of wet sample tissue 0.0 0.0
0.0 0.0
0.1 0.1 0.1 0.3 0.1
0.1 0.0
melanoma control
0.17
liver
0.26 0.51
1.01
0.25
0.49
1.05 2.01
0.2
0.20
1.04
1.0
1.0
0.40
2.06
2.1
1.0
0.23 0.30
1.35 1.74
0.9 1.2
0.7 0.7
injected ( a ) liver injected ( b ) melan orna injected (a) melanoma injected ( b )
0.40
0.95 2.23 2.02
0.1 0.1 0.1 0.1
pletely decomposed by the alkali fusion and that all of the boron in 1°B-p-bpa HCl may be determined by the present method. Assay of Normal Liver and Melanoma. The present method was tested by determining the boron content in normal liver and malanoma of hamster, into which 1°B-p-bpa HCI had been injected. The results are shown in Table I. The boron content in the same tissue of different weight was measured twice. The agreement of the concentration of boron between the two measurements demonstrates that this method is sufficiently precise.
ACKNOWLEDGMENT The authors are grateful to Tadao Kanzaki for helpful advice. Registry No. B, 7440-42-8; log,14798-12-0. LITERATURE CITED (1) Soloway. A. H. "Progress in Boron Chemistry Vol Press: Oxford, 1964; Chapter 4.
1"; Pergamon
042
Anal. Chem. 1904, 56,842-845
(2) Mishima, Y.; Shimakage, T. "Pigment Cell Vol. 2"; S. Karger: Easel, 1976; pp 394-406. (3) Kakihana, H.; Yoshino, K. Gendai Kagaku 1977, 71,42-48. (4) Hatanaka, H. Shikei Shinpo 1978, 22, 142-157. (5) Kobayashi, Y.; Kanda, K. Nucl. Instrum. Methods 1983, 204, 525-531. (6) Powell, T. P.; Mitchell, 0.A. Commun. Soil Sci. Plant Anal. 1979, 10, 1099-1 108. (7) Krug, F. J.; Mortatti, J.; Pessenda, L. C. R.; Zagatto, E. A. G.; Eergamin-F., H. Anal. Chim. Acta 1981, 125, 29-35. (8) Ikeuchi, I.; Amano, T. Chem. Pharm. Bull. 1978, 26, 2619-2623. (9) Isozaki, A.; Utsumi, S. Nippon Kagaku Zasshi 1967, 8 8 , 741-744. (10) Snyder, H. R.; Reedy, A. J.; Lennarz, W. J. J . Am. Chem. Soc. 1958, 80,835-838.
(11) How, M. J.; Kennedy, G. R.; Mooney, E. F. Chem. Commun. 1969, 267-268. (12) Henderson, W. G.;How, M. J.; Kennedy, G. R.; Mooney, E. F. Carbohyd. Res. 1973, 28, 1-12. (13) Yoshino, K.; Kotaka, M.; Okamoto, M.; Kakihana, H. "ACS/CSJ Chernical Congress"; Honolulu, HI, April 1979; American Chemical Society: Washington, DC, 1979; Abstr. No. Physical Chemistry 83.
RECEIVED for review July 11, 1983. Accepted December 5, 1983. This study was supported by a Grant-in-Aid from Minister of Education.
Diffusion Denuder Assembly for Collection and Determination of Gases in Air Ella E. Lewin* and Knud A. Hansen National Agency of Environmental Protection, Air Pollution Laboratory, Riso National Laboratory, DK-4000, Roskilde, Denmark In air pollution studies a widely used collection arrangement consists of an aerosol filter followed by a wet impinger or specially prepared filter for retention of a gaseous pollutant. However, it was shown that in many cases this method can lead to undesirable changes of the sample during collection. For example, an absorption of gaseous ammonia on the collected aerosol can result in partial neutralization of the particulate matter (1). The calculations of Brosset (2) predict production of gaseous hydrochloric acid upon contact of the collected aerosol with SOz, and the experiments of Harker et al. (3) demonstrated production of artifact nitrate under similar circumstances. Some of these undesirable processes can be hindered when gaseous pollutants are removed prior to collection of the aerosol. Such a separation can be achieved when a laminar flow of air passes through a tube coated inside with a suitable absorption agent: Gas molecules diffuse to the walls and are retained there, while aerosol particles pass virtually unaltered because of an order of magnitude lower diffusion coefficient. Durham and Wilson (4) applied a Pb02-coatedtube for separation of SOz from sulfate particles under their studies of photochemical production of sulfate. Using an absorption tube coated with a film of oxalic acid, Ferm (5) showed that ammonia gas can be both retained by and liberated from the collected dust. Lewin and Klockow (6) investigated characteristics of a TCM-coated absorption tube for easy collection and determination of sulfur dioxide. The demand of laminar flow considerably limits the amount of material collected in the tube and on the following filter. In order to overcome this difficulty, Stevens et al. (1) used a battery of 16 parallel tubes arranged in a circle for separating ammonia from particles, which were then collected in a dichotomous sampler. Forrest et al. (7) used a battery of 48 tubes covered with a Na2C0, film for the studies of gaseous nitric acid; particulate nitrate was collected on the filter following the tubes. The total flow through the system was between 10 and 30 L min-I. In the mentioned studies every tube was coated separately by injecting some amount of covering solution into the tube, which was then slowly rotated until the film was dried out. Taking into account the number of tubes involved, it is, of course, a very laborious and timeconsuming process. Therefore, probably, no attempt was made to determine directly the content of the exposed tubes. Also, if every tube was to be extracted separately, the risk of contamination would be high and the amount of water necessary for extraction would counteract the advantage of using so
many tubes. It was our aim to construct a system suitable for direct determination of the gaseous components and for collecting a reasonable volume of air. Also, it was decided to design an apparatus which allowed easy, quick, reproducible, and clean coating and extraction procedures and enable us to use the fairly low extraction volumes for exposed tubes. The present paper describes the construction and features of such a diffusion denuder assembly (DDA), preparation of tubes and samples, and testing of the applicability of this system for collecting ammonia and gaseous nitric acid. Knowledge of concentrations of ammonia and acidic gases in air is a prerequisite for the understanding of processes important for acidification of dry and wet deposition. The system described for collecting and treating gaseous samples can be helpful in obtaining reliable results on content of these important species in air.
EXPERIMENTAL SECTION Construction and Function of the Assembly. As already mentioned, the aim of the design was to use the absorption tubes in connection with collecting aerosols on filters. The tubes were arranged in a way similar to those described by Stevens et al. (1) and Forrest et al. (S), i.e., in a circle and placed in a round Plexiglas protective cover. The assembly, as used during sampling,is shown in Figure 1. To facilitate the connection with the filter, an open part of the Selectron filter holder (B) is glued to the protective cover (C). In our laboratory we employ 47- or 50-mm Selectron filters and appropriate filter holders. Therefore, the assembly consists of 15 quartz glass tubes, each 30 cm and 0.4 cm i.d. The tubes are supported by two silicon rubber plates (A) and at the inlet end they go into a 0.5-cm Teflon plug (D), which also facilitates connection with the vacuum system during the preparation procedure. To prevent the coating film to deliquesce at high humidities, warm air is led into the protective cover to heat the tubes a few degrees above the ambient temperature (7).To speed up the coating and extraction procedures and to limit the extraction volume as far as feasible, an apparatus-shown schematically in Figure 2-was constructed. The coating solution (or water for extraction) is sucked into the tubes of the DDA by connecting them to a vacuum pump, and the coating film is afterward dried out with a gentle flow of clean pressurized air. The apparatus enables alternative use of sucking (valve E) or blowing (valve G). Every tube is connected to the main line through a separatevalve (H). This gives us a possiblity of choosing how many tubes we coat or extract simultaneously, depending on the volume of liquid available. The DDA is connected t o the vacuum pump from its inlet end (D). This is important, as the first few centimeters of the absorption tube should remain un-
0 1984 American Chemical Society 0003-2700/84/0356-0842$01.50/0
