ANALYTICAL SCIENCES DECEMBER 2013, VOL. 29 1209 2013 © The Japan Society for Analytical Chemistry Notes Determination of the Carbon, Hydrogen and Nitrogen Contents of Alanine and Their Uncertainties Using the Certified Reference Material L-Alanine (NMIJ CRM 6011-a) Nobuyasu ITOH,*† Ayako SATO,**,*** Taichi YAMAZAKI,* Masahiko NUMATA,* and Akiko TAKATSU* * National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305–8563, Japan ** A-Rabbit-Science Japan Co., Ltd., 5-4-21 Nishihashimoto, Midori, Sagamihara, Kanagawa 252–0131, Japan *** School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422–8526, Japan The carbon, hydrogen, and nitrogen (CHN) contents of alanine and their uncertainties were estimated using a CHN analyzer and the certified reference material (CRM) L-alanine. The CHN contents and their uncertainties, as measured using the single-point calibration method, were 40.36 ± 0.20% for C, 7.86 ± 0.13% for H, and 15.66 ± 0.09% for N; the results obtained using the bracket calibration method were also comparable. The method described in this study is reasonable, convenient, and meets the general requirement of having uncertainties ≤ 0.4%. Keywords CHN analyzer, CHN content, uncertainty, certified reference material (CRM), alanine (Received September 30, 2013; Accepted October 7, 2013; Published December 10, 2013) uncertainties were estimated with the CRM L-alanine and a Introduction CHN analyzer. Single-point and bracket calibration methods were applied because the single-point method is generally The elemental contents, such as the carbon, hydrogen, and simpler to perform, but the bracket calibration method is more nitrogen (CHN) contents, provide important, fundamental reliable; the two methods were compared. information regarding organic compounds, and CHN analyzers have been widely used for compound identification.1–3 High accuracy, generally with errors of less than or equal to 0.4%, is Experimental required for these types of analyses.2,3 The accuracy in the determination of the CHN contents depends on the calibrants Chemicals used; the National Institute of Standards and Technology (NIST) CRM L-alanine (NMIJ CRM 6011-a), issued by the National provide three standard reference materials (SRMs) for elemental Metrology Institute of Japan (NMIJ), was used as the calibrant; analysis.4–6 LGC Standards also provides the certified reference the purity was 0.999 kg/kg with an uncertainty of 0.002 kg/kg, material (CRM) acetanilide (LGC4002) for elemental analysis,7 approximated by the half-width of the 95% confidence interval.9 and the Association of Organic Micro Analyses (Japan) supplies The reagent alanine (DL-α-alanine; SP-35; special grade for the 52 types of reference materials.8 elemental analysis of organic compounds) was obtained from Although the analytical results with uncertainties are currently Kishida Chemical (Osaka, Japan). required to represent the confidence of the analytical results, the only CRM for elemental analysis from LGC Standards with a Instrumentation and sample preparation given uncertainty is acetanilide (LGC4002),7 and it cannot be An ultramicro balance SE2 (0.1 μg to 2.1 g range; Sartorius; expected that they will rapidly provide various CRMs for Gottingen, Germany) and a CHN corder MT-5 (Yanaco; Kyoto, elemental analysis. To fill this gap, it is possible to estimate the Japan) analyzer were used in this study. The combustion and CHN contents and their uncertainties of unknown organic reduction temperatures for the CHN corder were set at 950 and compounds by comparing them with the available CRMs (not 550°C, respectively. for elemental analysis). Furthermore, it is possible that Four different amounts of the CRM L-alanine were weighed uncertainties can be simply determined because the analytical (ca. 1.0, 1.5, 2.0, and 2.5 mg), and a calibration curve was procedure simply involves weighing with a balance and constructed. Only one amount of the reagent alanine sample measurement with a CHN analyzer. was weighed (ca. 2.0 mg). All samples were prepared and In this study, the CHN contents of alanine and their analyzed in quintuplicate using a random measurement order. The calculations of the signal intensities to the observed † To whom correspondence should be addressed. masses were performed according to the literature.10 The E-mail: [email protected] relationships (r2) between the element masses and the signal 1210 ANALYTICAL SCIENCES DECEMBER 2013, VOL. 29 Table 1 Theoretical and observed masses of alanine with uncertainties obtained by the single-point calibration method Theoretical mass of Observed mass of Relative combined a Parameter M′y/(M × Rx) / kg/kg Ux / kg/kg element (M × Rx) / μg element (M′y) / μg uncertainty (uy) / kg/kg C 836.5 834.8 0.998 0.0024 0.005 H 163.8 162.6 0.993 0.0080 0.016 N 325.2 323.8 0.996 0.0030 0.006 a. Expanded uncertainty (Ux) was obtained by multiplying the coverage factor (k = 2) to represent the half-width of 95% confidential interval. intensities for C, H, and N were 0.9998, 0.9987, and 0.9996, Analytical results of the alanine analysis with uncertainties using respectively, when their intercepts were set to zero. Thus, their the single-point calibration method linearities were considered to be sufficient, although the data for In the single-point method, which is relatively simple to H were relatively less accurate than that for C and N. Since the implement, the masses of the elements in alanine (M′x) were relative uncertainties originating from the repeatability of the obtained using blank samples for C, H, and N were 0.00026, 0.0013, and 0.0018, respectively, they could be ignored in this study. PR××x MIcalib × x M x′ = , (3) To estimate the uncertainties in the measured weights, each I calib sample was weighed 20 times; the standard deviation (SD) was 0.0000995 mg. The United States Pharmacopeia (USP) advises where Mcalib is the mass of calibrant CRM L-alanine, Icalib the that the minimum experimental mass should exceed 2000 times signal intensity of the calibrant, and Ix the signal intensity of the that of the SD,11 which equals 0.199 mg in this case; thus, 1 mg alanine sample. sample masses were used. The uncertainties in the element masses can be characterized by u(xi) and the sensitivity coefficients. The sensitivity coefficient, [∂f/∂xi], represents how changes in the uncertainty Results and Discussion of the input contribute to changes in the uncertainty of the output. The sensitivity coefficient of each parameter can be CHN contents of the CRM L-alanine and their uncertainties obtained from the partial differentiation of Eq. (3) (details can To obtain the CHN contents of alanine and their uncertainties, be seen in the Supporting Information). The combined not only the analytical results of the alanine analysis using the uncertainty, uCM′x, is defined by L-alanine CRM, but also the mass of the element in the L-alanine 2 CRM (calibrant) and its uncertainty must be considered. To ufCM′xi=∂∑{[ /∂×xu](xi )} . (4) obtain the expanded uncertainty, which represents the half-width of the 95% confidential interval, calculations are commonly The obtained values (M′x) were 834.8 for C, 162.6 for H and performed according to the Guide to the Expression of 323.8 for N; the combined uncertainties (uCM′x) were 2.0 for C, Uncertainty in Measurement.12 The uncertainties from weighing, 1.3 for H and 1.0 for N, as given in Table S1 (Supporting the purity of the CRM L-alanine, the theoretical relative Information) with those of each parameter. molecular mass of alanine, and the repeatability of the CHN The ratios, R′x, of the observed mass, M′x, to the theoretical analysis were considered. mass, (M × Rx), were obtained using The mass of element X (X = C, H or N) in the calibrant, defined as Mx, can be calculated from R′x = M′x/(M × Rx). (5) Mx = M × Cx, (1) Table 1 summarizes the theoretical masses, Mx, the observed masses, M′x, the mass ratios, R′x, and the expanded uncertainties, where M is the mass of the calibrant and C the elemental content Ux, which were obtained by multiplying the uCM′x values by the of element X in the calibrant. If the purity, P, of the CRM is coverage factor (k = 2), which represents the half-width of the taken into consideration, Eq. (1) becomes 95% confidential interval. The R′x values for C, H, and N were 0.998, 0.993, and 0.996, respectively, and the expanded Mx = (M × P × Rx) + ∑(Ci × Rxi) + (C′ × R′x) , (2) uncertainties were 0.005, 0.016, and 0.006, respectively. Although all of the masses determined were smaller than the where (M × P × Rx) is the contribution from the major theoretical values (ratio: 1.000), the theoretical values were component (alanine), ∑(Ci × Rxi) the contribution from the within their respective uncertainties (Ux). identified impurities, and (C′ × R′x) the contribution from only unknown impurities. Rx is the theoretical ratio of each element Analytical results of the alanine analysis with uncertainties using X, Ci the concentration of the identified impurities, Rxi the ratio the bracket calibration method of the identified elemental impurities, C′ the concentration of In the bracket calibration method, which is more reliable, the the unknown impurities, and R′x the ratio of the unknown elemental masses for alanine, M′y, were obtained using elemental impurities. However, because no significant impurities including water were present in the CRM L-alanine,9 PR××xH()MM−×Ly()II− L M y′ = + ML , (6) ()II the ∑(Ci × Rxi) and (C′ × R′x) terms can be ignored for this HL− calibrant.