- Email: [email protected]
Feasibility study for the quantification of total protein content by multiple prompt gamma-ray analysis
Applied Radiation and Isotopes 70 (2012) 984–987
Contents lists available at SciVerse ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Feasibility study for the quantification of total protein content by multiple prompt gamma-ray analysis Y. Toh a,n, Y. Murakami b, K. Furutaka a, A. Kimura a, M. Koizumi a, K. Hara a, T. Kin a, S. Nakamura a, H. Harada a a b
Japan Atomic Energy Agency, Nuclear Science and Engineering Directorate, Tokai, Naka, Ibaraki 319-1195, Japan University of Fukui, Research Institute of Nuclear Engineering, 3-9-1 Bunkyo, Fukui City, Fukui 910-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2011 Received in revised form 2 February 2012 Accepted 27 February 2012 Available online 6 March 2012
Protein is an important nutrient in foods. The classical nitrogen analysis method is the Kjeldahl technique, which is time-consuming and inconvenient. As a convenient method to quantify protein content in biological samples, the feasibility of application of multiple prompt gamma-ray analysis (MPGA) to the quantification was studied. Results for protein content are reported for several reference materials and prove the method to be reliable. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Prompt gamma-ray activation analysis Coincidence method Nitrogen Protein Biological samples Standard reference materials
1. Introduction One of the main factors determining the taste of cooked rice is protein content. Reducing protein content in rice grain leads to a better taste for cooked rice. An increase in the amount of total protein content negatively affects the eating quality of rice (Terao et al., 2005). Certain diseases, such as kidney and liver disease, require a low or high protein diet to control protein intake (Fouque and Laville, 2009; Feldman et al., 2010). By using results of nitrogen content, total protein content can be calculated from published conversion factors (Souci et al., 2000) for most foods. The Kjeldahl method for the determination of organic nitrogen is the worldwide standard for the purpose of calculating protein content in foods. However, this method is time-consuming and inconvenient. In addition, results obtained are highly variable. Prompt gamma-ray analysis (PGA) is known as a non-destructive and multielement analytical method, and has been used for the quantification of nitrogen (Anderson and Mackey, 2005). However, it is also a time-consuming method for nitrogen analysis because the neutron capture cross section of nitrogen is small. Furthermore, nitrogen is the largest component of air, and
n
Corresponding author. Tel.: þ81 29 282 6211; fax: þ 81 29 282 5458. E-mail address: [email protected] (Y. Toh).
0969-8043/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2012.02.116
produces background gamma ray. The status of the neutron beam of a neighboring line is subject to change without advance notice. Passive lead and active BGO shields can reduce the background gamma ray from neighboring line to some extent. However, nitrogen emits high-energy prompt gamma rays which are very difficult to shield. Therefore prompt gamma rays from neighboring lines may influence data derived from a detector. The multiple gamma-ray detection method, also known as the coincidence method, is widely used in nuclear spectroscopy. By applying the multiple gamma-ray detection method to prompt gamma-ray analysis, called MPGA (multiple prompt gamma-ray analysis), the interference from hydrogen contained in a sample can be reduced. Therefore, this method can improve the signal-tonoise ratio, and provide a rapid screening method of toxic elements, particularly cadmium (Gardner et al., 2000; Ember et al., 2002, 2004; Oshima et al., 2007; Toh et al., 2008). The MPGA detector system has a high detection efficiency because of the proximity between the detectors and the target (approximately 5 cm) and eight Clover detectors, which have a large active volume, are employed. Therefore, MPGA is not only suitable for a rapid screening method of toxic elements but also a method for the quantification of nitrogen which has a small cross section. In addition, since the coincidence rate is inversely proportional to the square of the distance, multiple gamma-ray detection method can reduce the effect of background gamma rays from neighboring beamline effectively.
Y. Toh et al. / Applied Radiation and Isotopes 70 (2012) 984–987
A method for the quantification of nitrogen by MPGA was developed and evaluated through the analysis of the National Institute of Standards and Technology (NIST) standard reference materials. Protein concentrations in certified reference materials from the National Institute for Environmental Studies (NIES) of Japan were analyzed by MGPA.
2. Experimental The MPGA phase-I detector system was constructed at the cold neutron guide tube C2-3-2 of JRR-3 reactor at Tokai site of Japan Atomic Energy Agency (JAEA) in January 2005. This system consisted of three Clover Ge detectors with BGO Compton suppressors. In March 2007, it was upgraded to the present phase-II detector system, which comprised eight Clover detectors with BGO Compton suppressors. Each Clover detector consists of four individual crystals sharing a common cryostat. The relative efficiency of each Clover detector at 1.33 MeV is approximately 120% compared to that of a 300 300 . NaI detector. The MPGA detector system, which consists of eight large-volume Ge detectors, is placed at a short distance (approximately 5 cm) from the sample. Therefore, a high-speed data acquisition (DAQ) system is required for the MPGA measurements. Digital signal processor (DSP) and field programmable gate array (FPGA)-based modules are used to perform data acquisition (Kimura et al., 2008). The acquired data were saved to a hard disk drive. The MPGA system also contains an automatic sample-changing system, which can accommodate 160 samples and change the samples within 30 s. Air in the MPGA beamline, which is sealed with silicone rubber, is replaced by carbon dioxide. Fig. 1 shows the dependence of background spectra on the flow rate of carbon-dioxide gas. The gamma-ray peaks from air (nitrogen) in beamline almost completely disappeared at a flow rate of 4 L/min. The flow rate was set to 6 L/min for the measurements of nitrogen. For the nitrogen measurements by MPGA, samples (typical size: 1.0 1.5 cm2) were sealed in envelopes (3 4 cm2) of FEP (Fluorinated ethylene propylene) film under ambient conditions. The neutron beam of MPGA beamline had a fluence rate of 1.4 107 cm 2 s 1 and was collimated to 2 3 cm2 with a 6LiF tile aperture. A background spectrum from an empty FEP film was also measured. Measurement times, dependent on the sample size and amount of nitrogen in the sample, were varied from
985
approximately 15 min for the melamine standard and NIES samples (124.6–144.9 mg) to up to 50 min for NIST samples (100.3–119.7 mg) (SRM1571, 1977; SRM1572, 1982; SRM1573, 1976; SRM1577a, 1985). The samples were measured together with a 133Ba source. The peak counts of the 133Ba gamma ray were used for the dead time correction. A He-3 detector was used to monitor the neutron flux. In MPGA, two or more cascade gamma rays emitted in the deexcitation of nuclei are detected simultaneously with several gamma-ray detectors. Events comprising a pair of prompt gamma rays were collected in the experiment. With the collected data, a histogram of gamma–gamma energy correlation, which is a three-dimensional gamma-ray spectrum, is constructed.
3. Results and discussion The excited N-15 nucleus, formed in 14N(n, g)15N reaction, emits a number of gamma rays (1678, 1885, 3678, 5269 and 5533 keV, etc.). The coincident gamma-ray pairs of 1885–3678, 1885–5269 and 1885–1678 keV are the predominant ones, and were used for the quantification. Fig. 2 shows a part of the MPGA gamma-ray spectrum for the analysis of bovine liver (SRM 1577a) acquired for 50 min. The gamma-ray pair of 1885 and 3678 keV, which is the strongest pair of nitrogen, was clearly observed. A calibration curve was obtained by plotting the ratio of the peak count of nitrogen to the peak count of the 133Ba gamma ray versus the known nitrogen weight of 10 melamine samples (10.4, 10.6, 19.8, 19.9, 29.9, 30.6, 49.8, 50.8, 99.5, 100.8 mg), which contain 66.6% nitrogen by mass. Calibrations with high linearity (R2 ¼ 0.9983) and reproducibility were achieved over a broad range (Fig. 3). To verify the accuracy of the calibration curve, concentrations of nitrogen in orchard leaves (SRM1571), citrus leaves (SRM1572), tomato leaves (SRM1573), bovine liver (SRM1577a) were calculated using the calibration curve; Method I. It has been known that element sensitivities are affected by neutron scattering by hydrogen (Mackey et al., 1991; Mackey and Copley, 1993). Neutron scattering from the sample can also enhance background count rates (Anderson and Mackey, 1993). To evaluate the effect of sample size, the results with only two melamine samples (99.5, 100.8 mg) which have almost the same weight as SRMs (100.3– 119.7 mg) are also shown in Table 1; Method II. Precision of nitrogen content effectively is limited by counting statistics.
N N
N
N
N N
50 40 Counts
10 7 10 6 10 4
10 0
78
10 1
ke
0L/min 2L/min 4L/min 6L/min
2
188
5ke
10 0 0
2000
V
10 3 10
20
36
Counts
10 5
30
4000 Energy (keV)
6000
8000
Fig. 1. Background spectra at carbon dioxide gas flow rates of 0, 2, 4, and 6 L/min.
V
Fig. 2. A part of MPGA spectrum for the analysis of bovine liver (NIST SRM 1577a).
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Y. Toh et al. / Applied Radiation and Isotopes 70 (2012) 984–987
On the other hand, the main source of error of hydrogen content is the drying process. Method II gives approximately 2% larger values than these by Method I, which may indicate that neutron scattering by H causes the enhancement. The results by Method II are generally acceptable, except for SRM1572. The nitrogen and hydrogen concentrations for SRM1571 determined by Method II agree well with the certified value or other method within the specified uncertainty limits. Nitrogen mass fractions by Method II for SRM1572, SRM1573 were approximately 20% higher and 10% lower, respectively, than the information values. In the cases of SRM1573, the nitrogen and hydrogen concentrations by two methods found here agree with the information values within 2s. On the other hand, concentration of hydrogen in SRM1572 was in good agreement with other method, however, nitrogen value represents a discrepancy of greater than 3s between the information value and the present measurement. The intensity of the neutron beam was almost homogeneous in the vertical direction. A non-uniform intensity distribution existed in the horizontal direction of the beam. The angle of the beam at which point the edge of the beam had an intensity 110% that of the center. However, the widths of samples (horizontal direction) were almost same, and the position repeatability of the autosampler was less than 71 mm. The method of Monte Carlo simulation by PHITS was used to estimate the effect of neutron scattering and absorption (Iwase et al., 2002). The neutron energy distribution was adopted to the Maxwellian distribution at 50 K (E¼5.5 meV). The cold neutron beam is partially thermalized in the sample, a phenomenon known as up-scattering. Therefore, the simulation took into account the effect of up-scattering. The simulation results of relative hydrogen sensitivity in melamine (4.80%, H), urea (6.71%, H) and Tris–Hydroxymethyl Amino-Methane (THAM; 9.15%, H) are shown in Table 3. The simulation results suggest that
the effect of neutron scattering and absorption is small under the experimental conditions of Method II. Therefore, the effects of nonuniformity of the beam, neutron scattering and absorption cannot explain the difference in the result of SRM1572. Our result of SRM1572 is close to the value (3.1870.28%) by a ratio technique in Rossbach and Hiep (1992). However, the result (2.82%) by a macrocombustion method in Schmitter and Rihs (1989) agrees with the information value. This suggests that the sample of SRM1572 may be insufficiently homogeneous with regard to its nitrogen content. Nitrogen and hydrogen concentrations measured in NIES samples analyzed by Method II are shown in Table 2 along with the calculated protein contents. The indicated error includes the uncertainty (3%) of the effect of neutron scattering and absorption obtained by the simulation. A conversion factor of 6.25 has been used to estimate the protein content of a variety of foods and feeds based on a nitrogen analysis. However, it depends on the amino acid composition of the protein. Therefore, we adopted the specific factors derived in Sosulski and Imafidon (1990) and Fois et al. (1988). The value of protein content in rice has a wide range of variance, 5–10%. The protein content in fresh fish is approximately 17–25% but in dried fish the protein content is over 50%. The quantity of protein is variable and depends on the drying time of the fish. Human hair is composed of approximately 90% protein called keratin. The results in Table 2 are consistent with the expected values. MPGA is accurate enough for measuring the concentration of total protein by Method II. From this study, it has been proven that the MPGA method can be applied to the quantification of protein content in biological samples. The method of analysis can be used not only for the rapid screening method for toxic elements, but also for the quantification of protein content in biological materials. These elements are determined simultaneously, so there is no need to carry out separate analyses.
N counts, normalized to 133Ba counts
0.16 0.14
R2 = 0.9983
4. Conclusions
0.12
The feasibility of MPGA for the measurement of protein content in biological samples has been investigated. The calibration curve of nitrogen generated shows good linearity. However,
0.1 0.08
Table 2 Results of NIES certified reference materials (n ¼3) by Method II.
0.06 0.04
NIES sample
N mass fraction (%)
N to protein factor
Protein (%)
H mass fraction (%)
No.10c rice flour No.11 fish tissue No.13 human hair
1.59 7 0.14 11.4 7 0.7 12.9 7 0.4
5.61 5.82 7.39
8.97 0.8 66.37 4.1 95.37 3.1
7.17 0.5 7.37 0.5 5.47 0.4
0.02 0 0
10
20
30 40 50 60 70 Nitrogen in melamine (mg)
80
Fig. 3. Calibration curve for nitrogen from 7 to 67 mg.
90
Mean 7 s(n¼ 3).
Table 1 Results of NIST SRM samples. Sample
SRM1571 orchard leaves SRM1572 citrus leaves SRM1573 tomato leaves SRM1577a bovine liver
N mass fraction (%)
H mass fraction (%)
Method I
Method II
Certified value (%)
Method I
Method II
Other methods (%)
2.68 7 0.16 3.38 7 0.07 4.43 7 0.30 10.397 0.17
2.74 7 0.17 3.45 7 0.07 4.53 7 0.30 10.61 7 0.17
2.76 7 0.05 (2.86)b (5.0)b (10.7)b
5.58 7 0.21 5.68 7 0.21 4.66 7 0.25 6.60 70.36
5.72 7 0.21 5.82 7 0.21 4.79 7 0.25 6.78 7 0.36
5.84 7 0.26a 5.96 7 0.01a 5.08 70.07a 6.77 7 0.68c
Mean 7 sðn ¼ 3Þ for Methods I and II. a b c
Gladney and States (1987). The values are not certified because they are not based on the results of either a definitive method of known accuracy or two or more independent methods. Rossbach (1991).
Y. Toh et al. / Applied Radiation and Isotopes 70 (2012) 984–987
Table 3 Results of relative hydrogen sensitivity in melamine, urea and THAM by PHITS. Sample
H mass fraction (%)
Sample size (mm)
Sample weight (mg)
Relative H sensitivitya (%)
Melamine-1 Melamine-2 Melamine-3 Urea-1 Urea-2 Urea-3 THAM-1 THAM-2 THAM-3
4.80 4.80 4.80 6.71 6.71 6.71 9.15 9.15 9.15
10 15 0.424 10 20 0.318 10 20 0.477 10 15 0.505 10 20 0.379 10 20 0.568 10 15 0.493 10 20 0.370 10 20 0.554
100 100 150 100 100 150 100 100 150
100.0 100.4 100.0 99.6 100.3 99.4 99.4 100.3 99.1
a
The relative sensitivity normalized to melamine-1.
the correlation coefficient does not give sufficient information to characterize a calibration curve. The results of the simulation indicate that the effects of neutron scattering and absorption are largely eliminated when the same weight of sample is used for analysis. The results obtained with orchard leaves (SRM 1571) by Method II are in good agreement with the certified value. Protein concentration of three NIES certified reference materials were determined in this study. MPGA provides a tool for the rapid and non-destructive quantification of total protein content in biological samples.
Acknowledgements We would like to express the gratitude to Drs. H. Matsue and M. Oshima for their support and the crew of the JRR-3 for providing the neutron beam. This work was supported in part by Grants-in-Aid for Scientific Research (22750077). References Anderson, D., Mackey, E., 1993. Neutron scattering-induced background enhancement in prompt g-ray activation analysis. J. Radioanal. Nucl. Chem. 167, 145–151. doi:10.1007/BF02035474. Anderson, D.L., Mackey, E.A., 2005. Improvements in food analysis by thermal neutron capture prompt gamma-ray spectrometry. J. Radioanal. Nucl. Chem. 263, 683–689. doi:10.1007/s10967-005-0643-7. Ember, P.P., Belgya, T., Molnar, G.L., 2002. Improvement of the capabilities of PGAA by coincidence techniques. Appl. Radiat. Isot. 56 (3), 535–541. Ember, P.P., Belgya, T., Weil, J.L., Molnar, G.L., 2004. A practical test of a gamma– gamma coincidence measurement setup for PGAA. Nucl. Instrum. Methods
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Phys. Res. Sect. B: Beam Interact. Mater. Atoms 213, 406–409. (5th Topical Meeting on Industrial Radiation and Radioisotope Measurement Applications). Feldman, M., Friedman, L.S., Brandt, L.J., 2010. Sleisenger and Fordtran’s Gastrointestinal and Liver Disease: 2 Volume Set: Pathophysiology, Diagnosis, Management. Expert Consult Premium Edition, Saunders. Fois, A., Balestri, P., Calvieri, S., Zampetti, M., Giustini, S., Stefanini, M., Lagomarsini, P., 1988. Trichothiodystrophy without photosensitivity. Eur. J. Pediatr. 147, 439–441. doi:10.1007/BF00496431. Fouque, D., Laville, M., 2009. Low protein diets for chronic kidney disease in non diabetic adults. Cochrane Database Syst. Rev. (3), CD001892. Gardner, R.P., Mayo, C.W., El-Sayyed, E.S., Metwally, W.A., Zheng, Y., Poezart, M., 2000. A feasibility study of a coincidence counting approach for PGNAA applications. Appl. Radiat. Isot. 53 (4–5), 515–526. Gladney, E.S., States, U., 1987. Compilation of elemental concentration data for NBS clinical, biological, geological, and environmental standard reference materials [microform]/E.S. Gladneyy [et al.]. U.S. Department of Commerce, National Bureau of Standards, Gaithersburg, MD. Iwase, H., Nakamura, T., Niita, K., 2002. Development of general-purpose particle and heavy ion transport Monte Carlo code. J. Nucl. Sci. Technol. 39 (11), 1142–1151. Kimura, A., Toh, Y., Koizumi, M., Furutaka, K., Kin, T., Oshima, M., 2008. Performance of a high speed and high density data acquisition system for multiple gamma-ray detection. In: Nuclear Science Symposium Conference Record, NSS ’08, October. IEEE. pp. 2107–2111. Mackey, E., Copley, J., 1993. Scattering and absorption effects in neutron beam activation analysis experiments. J. Radioanal. Nucl. Chem. 167, 127–132. doi:10.1007/BF02035471. Mackey, E.A., Gordon, G.E., Lindstrom, R.M., Anderson, D.L., 1991. Effects of target shape and neutron scattering on element sensitivities for neutron-capture prompt .gamma.-ray activation analysis. Anal. Chem. 63 (3), 288–292. (pMID: 1824014). Oshima, M., Toh, Y., Kimura, A., Ebihara, M., Oura, Y., Itoh, Y., Sawahata, H., Matsuo, M., 2007. Multiple prompt gamma-ray analysis and construction of its beam line. J. Radioanal. Nucl. Chem. 271, 317–321. doi:10.1007/s10967-007-0210-5. Rossbach, M., 1991. Multielement prompt .gamma. cold neutron activation analysis of organic matter. Anal. Chem. 63 (19), 2156–2162. Rossbach, M., Hiep, N.T., 1992. Prompt gamma cold neutron activation analysis applied to biological materials. Fresenius J. Anal. Chem. 344, 59. Schmitter, B.M., Rihs, T., 1989. Evaluation of a macrocombustion method for total nitrogen determination in feedstuffs. J. Agric. Food Chem. 37, 992. Sosulski, F.W., Imafidon, G.I., 1990. Amino acid composition and nitrogen-to-protein conversion factors for animal and plant foods. J. Agric. Food Chem. 38 (6), 1351–1356. Souci, S.W., Fachmann, W., Kraut, H., 2000. Food Composition and Nutrition Tables, sixth ed. Medpharm. SRM1571, 1977. Certificate of Analysis for SRM 1571, Orchard Leaves. SRM1572, 1982. Certificate of Analysis for SRM 1572, Citrus Leaves. SRM1573, 1976. Certificate of Analysis for SRM 1573, Tomato Leaves. SRM1577a, 1985. Certificate of Analysis for SRM 1577a, Bovine Liver. Terao, T., Miura, S., Yanagihara, T., Hirose, T., Nagata, K., Tabuchi, H., Kim, H.Y., Lieffering, M., Okada, M., Kobayashi, K., 2005. Influence of free-air Co2 enrichment (face) on the eating quality of rice. J. Sci. Food Agric. 85 (11), 1861–1868. Toh, Y., Oshima, M., Kimura, A., Koizumi, M., Furutaka, K., Hatsukawa, Y., Goto, J., 2008. Reduction of Compton background from hydrogen in prompt gammaray analysis by multiple photon detection. J. Radioanal. Nucl. Chem. 278, 685–689. doi:10.1007/s10967-008-1505-x.
Contents lists available at SciVerse ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Feasibility study for the quantification of total protein content by multiple prompt gamma-ray analysis Y. Toh a,n, Y. Murakami b, K. Furutaka a, A. Kimura a, M. Koizumi a, K. Hara a, T. Kin a, S. Nakamura a, H. Harada a a b
Japan Atomic Energy Agency, Nuclear Science and Engineering Directorate, Tokai, Naka, Ibaraki 319-1195, Japan University of Fukui, Research Institute of Nuclear Engineering, 3-9-1 Bunkyo, Fukui City, Fukui 910-8507, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 October 2011 Received in revised form 2 February 2012 Accepted 27 February 2012 Available online 6 March 2012
Protein is an important nutrient in foods. The classical nitrogen analysis method is the Kjeldahl technique, which is time-consuming and inconvenient. As a convenient method to quantify protein content in biological samples, the feasibility of application of multiple prompt gamma-ray analysis (MPGA) to the quantification was studied. Results for protein content are reported for several reference materials and prove the method to be reliable. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Prompt gamma-ray activation analysis Coincidence method Nitrogen Protein Biological samples Standard reference materials
1. Introduction One of the main factors determining the taste of cooked rice is protein content. Reducing protein content in rice grain leads to a better taste for cooked rice. An increase in the amount of total protein content negatively affects the eating quality of rice (Terao et al., 2005). Certain diseases, such as kidney and liver disease, require a low or high protein diet to control protein intake (Fouque and Laville, 2009; Feldman et al., 2010). By using results of nitrogen content, total protein content can be calculated from published conversion factors (Souci et al., 2000) for most foods. The Kjeldahl method for the determination of organic nitrogen is the worldwide standard for the purpose of calculating protein content in foods. However, this method is time-consuming and inconvenient. In addition, results obtained are highly variable. Prompt gamma-ray analysis (PGA) is known as a non-destructive and multielement analytical method, and has been used for the quantification of nitrogen (Anderson and Mackey, 2005). However, it is also a time-consuming method for nitrogen analysis because the neutron capture cross section of nitrogen is small. Furthermore, nitrogen is the largest component of air, and
n
Corresponding author. Tel.: þ81 29 282 6211; fax: þ 81 29 282 5458. E-mail address: [email protected] (Y. Toh).
0969-8043/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2012.02.116
produces background gamma ray. The status of the neutron beam of a neighboring line is subject to change without advance notice. Passive lead and active BGO shields can reduce the background gamma ray from neighboring line to some extent. However, nitrogen emits high-energy prompt gamma rays which are very difficult to shield. Therefore prompt gamma rays from neighboring lines may influence data derived from a detector. The multiple gamma-ray detection method, also known as the coincidence method, is widely used in nuclear spectroscopy. By applying the multiple gamma-ray detection method to prompt gamma-ray analysis, called MPGA (multiple prompt gamma-ray analysis), the interference from hydrogen contained in a sample can be reduced. Therefore, this method can improve the signal-tonoise ratio, and provide a rapid screening method of toxic elements, particularly cadmium (Gardner et al., 2000; Ember et al., 2002, 2004; Oshima et al., 2007; Toh et al., 2008). The MPGA detector system has a high detection efficiency because of the proximity between the detectors and the target (approximately 5 cm) and eight Clover detectors, which have a large active volume, are employed. Therefore, MPGA is not only suitable for a rapid screening method of toxic elements but also a method for the quantification of nitrogen which has a small cross section. In addition, since the coincidence rate is inversely proportional to the square of the distance, multiple gamma-ray detection method can reduce the effect of background gamma rays from neighboring beamline effectively.
Y. Toh et al. / Applied Radiation and Isotopes 70 (2012) 984–987
A method for the quantification of nitrogen by MPGA was developed and evaluated through the analysis of the National Institute of Standards and Technology (NIST) standard reference materials. Protein concentrations in certified reference materials from the National Institute for Environmental Studies (NIES) of Japan were analyzed by MGPA.
2. Experimental The MPGA phase-I detector system was constructed at the cold neutron guide tube C2-3-2 of JRR-3 reactor at Tokai site of Japan Atomic Energy Agency (JAEA) in January 2005. This system consisted of three Clover Ge detectors with BGO Compton suppressors. In March 2007, it was upgraded to the present phase-II detector system, which comprised eight Clover detectors with BGO Compton suppressors. Each Clover detector consists of four individual crystals sharing a common cryostat. The relative efficiency of each Clover detector at 1.33 MeV is approximately 120% compared to that of a 300 300 . NaI detector. The MPGA detector system, which consists of eight large-volume Ge detectors, is placed at a short distance (approximately 5 cm) from the sample. Therefore, a high-speed data acquisition (DAQ) system is required for the MPGA measurements. Digital signal processor (DSP) and field programmable gate array (FPGA)-based modules are used to perform data acquisition (Kimura et al., 2008). The acquired data were saved to a hard disk drive. The MPGA system also contains an automatic sample-changing system, which can accommodate 160 samples and change the samples within 30 s. Air in the MPGA beamline, which is sealed with silicone rubber, is replaced by carbon dioxide. Fig. 1 shows the dependence of background spectra on the flow rate of carbon-dioxide gas. The gamma-ray peaks from air (nitrogen) in beamline almost completely disappeared at a flow rate of 4 L/min. The flow rate was set to 6 L/min for the measurements of nitrogen. For the nitrogen measurements by MPGA, samples (typical size: 1.0 1.5 cm2) were sealed in envelopes (3 4 cm2) of FEP (Fluorinated ethylene propylene) film under ambient conditions. The neutron beam of MPGA beamline had a fluence rate of 1.4 107 cm 2 s 1 and was collimated to 2 3 cm2 with a 6LiF tile aperture. A background spectrum from an empty FEP film was also measured. Measurement times, dependent on the sample size and amount of nitrogen in the sample, were varied from
985
approximately 15 min for the melamine standard and NIES samples (124.6–144.9 mg) to up to 50 min for NIST samples (100.3–119.7 mg) (SRM1571, 1977; SRM1572, 1982; SRM1573, 1976; SRM1577a, 1985). The samples were measured together with a 133Ba source. The peak counts of the 133Ba gamma ray were used for the dead time correction. A He-3 detector was used to monitor the neutron flux. In MPGA, two or more cascade gamma rays emitted in the deexcitation of nuclei are detected simultaneously with several gamma-ray detectors. Events comprising a pair of prompt gamma rays were collected in the experiment. With the collected data, a histogram of gamma–gamma energy correlation, which is a three-dimensional gamma-ray spectrum, is constructed.
3. Results and discussion The excited N-15 nucleus, formed in 14N(n, g)15N reaction, emits a number of gamma rays (1678, 1885, 3678, 5269 and 5533 keV, etc.). The coincident gamma-ray pairs of 1885–3678, 1885–5269 and 1885–1678 keV are the predominant ones, and were used for the quantification. Fig. 2 shows a part of the MPGA gamma-ray spectrum for the analysis of bovine liver (SRM 1577a) acquired for 50 min. The gamma-ray pair of 1885 and 3678 keV, which is the strongest pair of nitrogen, was clearly observed. A calibration curve was obtained by plotting the ratio of the peak count of nitrogen to the peak count of the 133Ba gamma ray versus the known nitrogen weight of 10 melamine samples (10.4, 10.6, 19.8, 19.9, 29.9, 30.6, 49.8, 50.8, 99.5, 100.8 mg), which contain 66.6% nitrogen by mass. Calibrations with high linearity (R2 ¼ 0.9983) and reproducibility were achieved over a broad range (Fig. 3). To verify the accuracy of the calibration curve, concentrations of nitrogen in orchard leaves (SRM1571), citrus leaves (SRM1572), tomato leaves (SRM1573), bovine liver (SRM1577a) were calculated using the calibration curve; Method I. It has been known that element sensitivities are affected by neutron scattering by hydrogen (Mackey et al., 1991; Mackey and Copley, 1993). Neutron scattering from the sample can also enhance background count rates (Anderson and Mackey, 1993). To evaluate the effect of sample size, the results with only two melamine samples (99.5, 100.8 mg) which have almost the same weight as SRMs (100.3– 119.7 mg) are also shown in Table 1; Method II. Precision of nitrogen content effectively is limited by counting statistics.
N N
N
N
N N
50 40 Counts
10 7 10 6 10 4
10 0
78
10 1
ke
0L/min 2L/min 4L/min 6L/min
2
188
5ke
10 0 0
2000
V
10 3 10
20
36
Counts
10 5
30
4000 Energy (keV)
6000
8000
Fig. 1. Background spectra at carbon dioxide gas flow rates of 0, 2, 4, and 6 L/min.
V
Fig. 2. A part of MPGA spectrum for the analysis of bovine liver (NIST SRM 1577a).
986
Y. Toh et al. / Applied Radiation and Isotopes 70 (2012) 984–987
On the other hand, the main source of error of hydrogen content is the drying process. Method II gives approximately 2% larger values than these by Method I, which may indicate that neutron scattering by H causes the enhancement. The results by Method II are generally acceptable, except for SRM1572. The nitrogen and hydrogen concentrations for SRM1571 determined by Method II agree well with the certified value or other method within the specified uncertainty limits. Nitrogen mass fractions by Method II for SRM1572, SRM1573 were approximately 20% higher and 10% lower, respectively, than the information values. In the cases of SRM1573, the nitrogen and hydrogen concentrations by two methods found here agree with the information values within 2s. On the other hand, concentration of hydrogen in SRM1572 was in good agreement with other method, however, nitrogen value represents a discrepancy of greater than 3s between the information value and the present measurement. The intensity of the neutron beam was almost homogeneous in the vertical direction. A non-uniform intensity distribution existed in the horizontal direction of the beam. The angle of the beam at which point the edge of the beam had an intensity 110% that of the center. However, the widths of samples (horizontal direction) were almost same, and the position repeatability of the autosampler was less than 71 mm. The method of Monte Carlo simulation by PHITS was used to estimate the effect of neutron scattering and absorption (Iwase et al., 2002). The neutron energy distribution was adopted to the Maxwellian distribution at 50 K (E¼5.5 meV). The cold neutron beam is partially thermalized in the sample, a phenomenon known as up-scattering. Therefore, the simulation took into account the effect of up-scattering. The simulation results of relative hydrogen sensitivity in melamine (4.80%, H), urea (6.71%, H) and Tris–Hydroxymethyl Amino-Methane (THAM; 9.15%, H) are shown in Table 3. The simulation results suggest that
the effect of neutron scattering and absorption is small under the experimental conditions of Method II. Therefore, the effects of nonuniformity of the beam, neutron scattering and absorption cannot explain the difference in the result of SRM1572. Our result of SRM1572 is close to the value (3.1870.28%) by a ratio technique in Rossbach and Hiep (1992). However, the result (2.82%) by a macrocombustion method in Schmitter and Rihs (1989) agrees with the information value. This suggests that the sample of SRM1572 may be insufficiently homogeneous with regard to its nitrogen content. Nitrogen and hydrogen concentrations measured in NIES samples analyzed by Method II are shown in Table 2 along with the calculated protein contents. The indicated error includes the uncertainty (3%) of the effect of neutron scattering and absorption obtained by the simulation. A conversion factor of 6.25 has been used to estimate the protein content of a variety of foods and feeds based on a nitrogen analysis. However, it depends on the amino acid composition of the protein. Therefore, we adopted the specific factors derived in Sosulski and Imafidon (1990) and Fois et al. (1988). The value of protein content in rice has a wide range of variance, 5–10%. The protein content in fresh fish is approximately 17–25% but in dried fish the protein content is over 50%. The quantity of protein is variable and depends on the drying time of the fish. Human hair is composed of approximately 90% protein called keratin. The results in Table 2 are consistent with the expected values. MPGA is accurate enough for measuring the concentration of total protein by Method II. From this study, it has been proven that the MPGA method can be applied to the quantification of protein content in biological samples. The method of analysis can be used not only for the rapid screening method for toxic elements, but also for the quantification of protein content in biological materials. These elements are determined simultaneously, so there is no need to carry out separate analyses.
N counts, normalized to 133Ba counts
0.16 0.14
R2 = 0.9983
4. Conclusions
0.12
The feasibility of MPGA for the measurement of protein content in biological samples has been investigated. The calibration curve of nitrogen generated shows good linearity. However,
0.1 0.08
Table 2 Results of NIES certified reference materials (n ¼3) by Method II.
0.06 0.04
NIES sample
N mass fraction (%)
N to protein factor
Protein (%)
H mass fraction (%)
No.10c rice flour No.11 fish tissue No.13 human hair
1.59 7 0.14 11.4 7 0.7 12.9 7 0.4
5.61 5.82 7.39
8.97 0.8 66.37 4.1 95.37 3.1
7.17 0.5 7.37 0.5 5.47 0.4
0.02 0 0
10
20
30 40 50 60 70 Nitrogen in melamine (mg)
80
Fig. 3. Calibration curve for nitrogen from 7 to 67 mg.
90
Mean 7 s(n¼ 3).
Table 1 Results of NIST SRM samples. Sample
SRM1571 orchard leaves SRM1572 citrus leaves SRM1573 tomato leaves SRM1577a bovine liver
N mass fraction (%)
H mass fraction (%)
Method I
Method II
Certified value (%)
Method I
Method II
Other methods (%)
2.68 7 0.16 3.38 7 0.07 4.43 7 0.30 10.397 0.17
2.74 7 0.17 3.45 7 0.07 4.53 7 0.30 10.61 7 0.17
2.76 7 0.05 (2.86)b (5.0)b (10.7)b
5.58 7 0.21 5.68 7 0.21 4.66 7 0.25 6.60 70.36
5.72 7 0.21 5.82 7 0.21 4.79 7 0.25 6.78 7 0.36
5.84 7 0.26a 5.96 7 0.01a 5.08 70.07a 6.77 7 0.68c
Mean 7 sðn ¼ 3Þ for Methods I and II. a b c
Gladney and States (1987). The values are not certified because they are not based on the results of either a definitive method of known accuracy or two or more independent methods. Rossbach (1991).
Y. Toh et al. / Applied Radiation and Isotopes 70 (2012) 984–987
Table 3 Results of relative hydrogen sensitivity in melamine, urea and THAM by PHITS. Sample
H mass fraction (%)
Sample size (mm)
Sample weight (mg)
Relative H sensitivitya (%)
Melamine-1 Melamine-2 Melamine-3 Urea-1 Urea-2 Urea-3 THAM-1 THAM-2 THAM-3
4.80 4.80 4.80 6.71 6.71 6.71 9.15 9.15 9.15
10 15 0.424 10 20 0.318 10 20 0.477 10 15 0.505 10 20 0.379 10 20 0.568 10 15 0.493 10 20 0.370 10 20 0.554
100 100 150 100 100 150 100 100 150
100.0 100.4 100.0 99.6 100.3 99.4 99.4 100.3 99.1
a
The relative sensitivity normalized to melamine-1.
the correlation coefficient does not give sufficient information to characterize a calibration curve. The results of the simulation indicate that the effects of neutron scattering and absorption are largely eliminated when the same weight of sample is used for analysis. The results obtained with orchard leaves (SRM 1571) by Method II are in good agreement with the certified value. Protein concentration of three NIES certified reference materials were determined in this study. MPGA provides a tool for the rapid and non-destructive quantification of total protein content in biological samples.
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