英文文献翻译-硼作显色剂在分光光度计法中的应用-一项有关氨甲苯酸与硼酸为决定作用的新颖的褪色分光光度计法

英文文献翻译-硼作显色剂在分光光度计法中的应用-一项有关氨甲苯酸与硼酸为决定作用的新颖的褪色分光光度计法 南华大学化学化工学院毕业 英文文献 Journal of the Chinese Chemical Society,2007,54,983-990 983 Application of Boron as the Chromogenic Agent in Spectrophotometry A Novel Fading Spectrophotometric Method for the Determination of Aminomethylbenzoic Acid with Boric Acid Quan-Min Li* ( ) and Tian-Tian Zhang ( ) College of Chemistry and Environmental Science, Henan Normal University; Henan Key Laboratory for Environmental Pollution Control, Xinxiang, Henan 453007, P. R. China It is the first time that boron has been used as a chromogenic agent to determine aminomethylbenzoic acid (PAMBA) by the fading spectrophotometric method in this paper. The study indicates that at pH 10.00theabsorbance of PAMBA decreases when boric acid(BA) is added to the solution. A simple ,rapid, sensitive and reliable novel method based on the product of PAMBA and BA is obtained. The stoichiometric ratio of the product is 1:2. Beer's law is obeyed in the range of PAMBA concentrations of 0.13~38.40μg/mL 4at a wavelength of 242nm(242 is 8.1×10L/mol/cm) The equation of linear regression is A=—0.06635— -50.02914C (×10 M), with a linear correlation coefficient of 0.9974. The detection limit is 0.12μg/mL and RSD 0.11%.The method is successfully applied to the determination of PAMBA in pharmaceutical samples, and average recoveries from pharmaceutical samples and urine samples were between 98.4~02.5%. The other components in these samples do not interfere with the determination by the proposed method. Keywords: Boron; Aminomethylbenzoic acid; Chromogenic agent; Fading spectrophotometric method. INTRODUCTION Boron is an electron lacking element , and has a strong ability of accepting electrons. It is easy for boron to form a polymetric molecule or a steady complex with an electron donor. The complex characteristic of boron is of important meaning for the study of its biochemical effects and nutrition physiology to plants in life science. Up to the present, a number of specific reagents used to determine boron by spetrophotometric methods have been developed based on the colorimetric reaction between 第 1 页 共 1 页 南华大学化学化工学院毕业设计 12-45BA and organic reagents, such as chromotropic acid, Azomethine-H ,curcumin, crystal 5violet, methyl orange (MO), and so on. Furthermore, several new derivatives of these organic 7 8-10 dyes were synthesized and studied for spectrophotometric determination of boron(see Table1). However, there are no reports on the use of boron as the chromogenic reagent to determine the organic pharmaceuticals. This paper firstly described a novel method to determine Aminomethylbenzoic acid (PAMBA) with boron by the fading spectrophotometric method. PAMBA is a kind of antihemorrhagic agent, which has been used for treating all kinds of bleeding, with the advantage of a distinct hemostasis effect on oozing blood. The Chinese 11 Pharmacopoeia (Part II)describes a titrimetric method for determination of PAMBA in tablets and injections. However, this method is only suitable for deter- mining PAMBA of high concentration and ineffective for measuring PAMBA of low content in biological samples. 12Additionally, Wang et al. reported the determination of PAMBA by flow-injection chemiluminescence, with poor selectivity and a narrow linear range(0.02~1.0μg/mL). Ultraviolet spectrophotometry13 was also used for determination of PAMBA in injections, again with a relatively narrow linear range of 4.8 ~ 11.2μg/mL and low sensitivity. The study in this paper shows that absorbance of PAMBA decreases after boric acid (BA) is added in an alkali medium ( λmax=242nm),and the complex (Product I) can be formed by the reaction between BA and PAMBA. Consequently, PAMBA can be determined using this discoloring system. The linear range is 0.13~38.40μg/mL,and the limit of detection is 0.12μg/mL. 第 2 页 共 2 页 南华大学化学化工学院毕业设计 2+2+2+2+2+2+2+3+ The interference of metal ions (Mg ,Ca , Co,Mn,Ni,Zn,Cuand Fe) can be eliminated easily in the proposed method at pH 10.00. At the same time, in order to demonstrate the performance of the described method, the determination of PAMBA was carried out in pharmaceutical samples and biological samples, and satisfactory results were obtained. Other components in those samples don’t interfere with the determination. EXPERIMENT Reagents and Apparatus Unless specially stated, all reagents used were of analytical grade and all solutions were prepared with distilled water. The main solutions were prepared as follows. A stock of standard solution of 400μg/mL of Aminomethyl Benzoic Acid(PAMBA)was prepared by dissolving 0.1000μg PAMBA (Chinese Drugs and Biological Goods Company) in 250 mL distilled water 。(The solution was preserved at 4C without light) and used to prepare working PAMBA standards by suitable dilutions. A standard boron stock solution (0.20M) was prepared by dissolving 6.2025g of boric acid (BA) (Beijing Xinguang Chemical Reagent Plant, Beijing, China) and 7.4560g of KCl (Taishan Chemical Reagent Plant, Guangdong, China) in distilled water heated for a few minutes. This solution was stored in a 500 mL standard flask.14 Buffer solution of pH 10.00 was obtained by mixing 10.70mL solution of 0.10 M NaOH and 50.00 mL solution of 0.05 M NaHCOin 100mL standard flask15 and adjusted by a pH-3C digital pH meter(Shanghai 3 Lei Ci Device Works, Shanghai, China). 第 3 页 共 3 页 南华大学化学化工学院毕业设计 A model 752 ultraviolet-visible spectrophotometer(Xiamen Analytical Instrument Plant, Xiamen,China)was employed for photometric measurements.A TU-1900 ultraviolet-visible spectrophotometer(PGeneral Instrument Plant,Beijing,China)was used for scanning the absorption spectrum. All pH measurements were performed with a pH-3C digital pH meter (Shanghai Le Ci Device Works, Shanghai, China). A model CS-501 super constant temperature instrument(Chongqing Experiment Equipment Plant, Chongqing, China) was used for temperature measurements. A BS 110s electro-analytical balance (Beijing Sartorius Balance Ltd., Beijing, China) was used to weigh the materials. Procedure 1.00mL of 400g/mL of PAMBA was taken into a 25.0mL comparison tube. Sequentially, 2.00mL NaOH-NaHCO buffer solution of pH10.00 was added and then1.50mL of 0.20 M BA 3 was added and the solution was diluted to 12.5mL with distilled water. The mixture was shaken well and the pH of solution was measured with a pH-3C digital pH meter. This solution was made at room temperature, and the absorbance was measured immediately at 242 nm(242nm proved to be the maximal discoloring wavelength of the system by the absorption spectrum of the system with TU-1900 UV spectrophotometer) against the solution of PAMBA and buffer solution of pH 10.00. RESULTS AND DISCUSSION Absorption spectrum According to the procedure, the absorption spectrum of the product produced by the reaction between PAMBA and BA was recorded. As can be seen in Fig. 1, PAMBA can be discolored by boron to a great extent, and the product has a minimal absorption at 242 nm, where BA has no absorption. Though the absorbance of PAMBA has a high absorbance at 242 nm, the product of PAMBA and BA shows a sensitive absorption against the solution of PAMBA and buffer solution. In order to obtain the highest sensitivity, all the following measurements were carried out at 242nm against the solution of PAMBA and buffer solution. 第 4 页 共 4 页 南华大学化学化工学院毕业设计 Fig. 1. Absorption spectrum. B: Absorption spectrum of PAMBA against water. C: Absorption spectrum of BA. D: Absorption spectrum of product against reagent blank. PAMBA (400μg/mL): 1.00 mL; BA (0.20 M): 1.50 mL; temperature: room temperature. Study of composition of product of PAMBA and BA The mole ratio method and the slope ratio method were used to determine the composition of the product formed of BA and PAMBA. The results are shown in Fig. 2 and Fig. 3. As can be seen, the mole ratio of the product is 2:1. -3 Fig. 2. The determination of the complex formation by mole ratio method. VR: BA (2.62×10 M); -3VD:PAMBA (2.62×10 M); VR+VD =1.00 mL; NaOH-NaHCO buffer solution (pH 10.00):2.00 mL; 3 temperature: room temperature. 第 5 页 共 5 页 南华大学化学化工学院毕业设计 . fig.3.The determination of the complex formation by slope ration method. VR: BA;VD:PAMBA;NaOH-Na HCO buffer solution(pH 3 10.00):2.00mL;temperature:Room temperature Discussion of reaction mechanism of PAMBA and BA -5The dissociation constant (Ka) of benzoic acid is 6.2×10. Since the molecular structure of -5PAMBA is similar to that of benzoic acid, Ka of PAMBA is supposed to be near 6.2×10. Because of the smaller Ka, it is difficult for H+ to dissociate from the carboxylic group () of PAMBA in acidic medium. In addition, the amino group (-NH)of PAMBA is 2 protonized in acidic medium. So the lone pairs of electrons of the carboxylic group ( )and the amino group (-NH) of PAMBA lose complexation capacity for the empty 2 +atomic orbit of boron. However, at pH 10.00, H has been completely dissociated from the carboxylic group ()of PAMBA, and the O negative ion with a lone pair of electrons 3+being obtained. The protonated amine salt (-NH) of PAMBA, which is formed in acidic solution, turns back into the amino group (-NH) with a lone pair of electrons. Thereby, in the 2 alkaline solution, the lone pairs of electrons of the carboxylic group()and the amine group(-NH)of PAMBA can complex with the empty atomic orbit of boron in light of the 2 第 6 页 共 6 页 南华大学化学化工学院毕业设计 16literature where boron can chelate with N and O. Based on that the mole ratio of the reaction between BA and PAMBA is2:1, it seems reasonable that the reaction equation is as follows: Effect of pH on the absorbance of Product I The effect of pH on the absorbance of Product I was examined by varying pH from 1.00 to 13.00. As shown in Fig.4,at pH1.00~3.00,the absorbance of Product I (formed from PAMBA and BA) is almost 0, which indicates that under high acidity, it is difficult for BA to react with +PAMBA. The possible reason is that the dissociation of H from the carboxylic group (-COOH) of PAMBA is difficult, and the amino group(-NH) of PAMBA is protonized and it turns into 2 3+protonated amine salt (-NH).So the lone pairs of electrons lose complexation capacity for the empty atomic orbit of boron. Therefore, the discoloration of the system almost doesn’t occur. When pH is above 3.00, the absorbance of Product I descends, corresponding to the growth of +pH. Assumably, when the acidity of solution becomes low the protonated amine salt (-NH)of 3 + PAMBA turns back into amino group (-NH), and the degree of the dissociation of Hfrom the 2 carboxylic group (-COOH) also rises. The higher the pH is, the more effective it is. Then O 16,17negative ion with lone pairs of electrons was formed. In light of the literature ,boron can be chelated with N and O, and then N of the amino group(-NH)and O of the carboxylic 2 group(-COOH) of PAMBA can possibly chelate with boron. Probably absorbance of the product of the reaction between BA and PAMBA is smaller than that of PAMBA at Ph10.00 at 242 nm, which leads to the formation of the discoloring system of PAMBA-BA. Fig.4 shows that the absorbance becomes minimal at pH10.00; in other words, the degree of the reaction is maximal. However, when pH increases to13.00, the absorbance is almost 0.It is likely that a high - 18concentration of hydroxide ion (OH)0.10 M) can chelate with BA to form [B(OH)]-,which 4 第 7 页 共 7 页 南华大学化学化工学院毕业设计 holds back the complexation between BA and the lone pairs of electrons of the carboxylic group (-COOH) and the amino group(-NH)of PAMBA. In order to keep the high sensitivity for 2 determination of PAMBA, the experiment was carried out at pH10.00. Fig. 4. Effect of pH on absorbance of the system. PAMBA (400μg/mL): 1.00 mL; BA (0.20 M): 1.50mL;temperature : room temperature. Effect of amount of BA on the absorbance of Product I In order to study the effect of the amount of BA on the determination of PAMBA, the amount of BA ranging from 0.00 to 3.00mL was submitted to the proposed procedure (see Fig.5).The absorbance descends substantially with the rise of the amount of BA when the amount is below 1.50mL. It is likely that the degree of the reaction between BA and PAMBA is enhanced with the increase in the amount of BA .The degree of the discoloration becomes greatest when the amount of BA is1.50mL.However, when the amount is above 1.50mL, the absorbance rises from-0.715(1.50mL) to-0.654(4.00mL).And then , when the pH of the solution is a bit below or above 10.00, the absorbance of Product I increases obviously (see Fig. 4). Because the increase of the amount of BA results in the decrease of the pH of the solution, then the absorbance of Product I increases when the amount of BA is more than 1.50mL, as can be seen in Fig.5. It accords with the result obtained from the section of “Efect of pH on the absorbance of Product I(see Fig. 4).In order to obtain the highest sensitivity, 1.50mL of BA was selected in the later experiments. 第 8 页 共 8 页 南华大学化学化工学院毕业设计 Effect of amount of buffer solution on the absorbance of Product I The effect of amount of buffer solution on the determination of PAMBA was studied according to the procedure , keeping pH at10.00.Fig.5shows that the absorbance gets to the minimum when the amount of buffer solution is2.00mL. Fig. 5. Effect of amount of BA and buffer solution. PAMBA (400g/mL): 1.00 mL; temperature:room temperature; B: NaOH-NaHCO 3buffer solution (pH 10.00): 2.00 mL; C: BA (0.20 M):1.50mL When the amount is above 2.00mL, there is little increase in absorbance, from -0.750 (2.00mL) to -0.661 (4.00mL). The possible reason is that when the amount of buffer solution added rose from 2.00mL to 4.00mL, the concentration of buffer solution increased. Thereby the -concentration of OH in the solution has a little increase, as does the pH of the solution, so then - 418the ability of OHto chelate with BA to form[B(OH)]-isenhanced,which results in that the complexation of boron and PAMBA is interfered to some extent. It accords with the result of “effect of pH on the absorbance of Product I (seeFig.4).So when the amount of buffer solution is above2.00mL,the absorbance of Product I begins to increase. In order to keep the high sensitivity of thedetermination,2.00mL of buffer solution was chosen. Effect of standing time on the absorbance of Product I The absorbance of Product I was measured at room temperature every 5 min, and the values obtained from 0 min to 2 h can be seen in Table 2. It is found that the reaction between BA and PAMBA takes place immediately when the solutions are mixed at pH 10.00, and the speed of the reaction is very rapid at room temperature. The absorbance of Product I remains the same from 0 min to 25 min, which shows the high 第 9 页 共 9 页 南华大学化学化工学院毕业设计 stability of the system. Hence, the experiments were carried out at room temperature, and the absorbance of Product I was measure dimmediately after the solution was made. Study of potential interference A systematic study of the potential influence of common ions and urea was carried out on the determination of PAMBA. The tolerance limits were defined with an error less than 5% in the analysis. As can be seen from Table 3, a certain amount of metal ions are allowed to exist when the determination of PAMBA is carried out. When the concentrations of metal ions are beyond the tolerance limits, the absorbance of the solution increases to some extent. The possible reason is that the hydroxide micelle of these metal ions can be formed atpH10.00,which makes the absorbance of the solution increase because of light dispersion. In addition, the tolerance limits 2+3+ of Cu and Feare relatively lower. It is likely that their solubility product constant (Ksp) are -19-39smaller, 2.6×10 and 3×10 , respectively, which indicates that it is much easier for the formation of their hydroxide micelles. When the proposed method is applied to the determination of PAMBA in real samples, if these metal ions mentioned above coexist with it, they can be pre-separated from the solution by the formation of precipitated hydroxide in alkali solution. According to the Ksp of these metal ions, it can be calculated that their concentration left in the solution is very far from their tolerance limits, which indicates that the determination of PAMBA is free from the interference of these metal ions. Therefore the selectivity of this proposed method is enhanced greatly. It also can be seen form Table 3 that the tolerance limit of urea is 7.7 mg/mL, which indicates that the proposed method should be applied to the determination of PAMBA in urine samples free from interference. Table2. The absorbance of Product I at different times (room temperature) 时间 (分钟) 0 5-25 30~35 40~50 55 1 h 2 h 吸光度 -0.690 -0.690 -0.695 -0.703 -0.711 -0.720 -0.732 Table3. Effect of foreign ions on the determination of PAMBA with BA 第 10 页 共 10 页 南华大学化学化工学院毕业设计 Tolerance Foreign ions Added as limit g/mL) ++3-2-K,Na ,NO ,Cl-,SO KClNaCl, NaNO,NaSO 1.2(mg/mL) 4,324 2-CO NaCO 180 323 2+2+2+Mg ,Ca ,Co Mg(NO),CaCl ,CoSO 40 3224 2+Mn MnSO 32 4 2+Ni NiSO 12.80 4 2+Zn ZnSO 8.80 4 2+Cu CuSO 1.44 4 3+Fe NHFe(SO) 1.20 442 Urea CO(NH) 7.7(mg/mL) 22 Calibration curve -5-5-5-5-5According to the procedure,0.08×10, 0.21×10, 0.42×10, 1.27×10, 2.11×10, -5 -5-5-5-53.39×10, 4.23×10 ,12.70×10 ,21.17×10 and25.40×10 M of PAMBA solution were prepared, respectively, then the absorbance of the discoloring system was measured under the optimum conditions. Absorbance has been plotted as function of the concentration of PAMBA -50.02914C (×10(see Fig. 6). A linear regression equation is attained as A=-0.06635— M) with a linearly dependent coefficient of 0.9974, and 242 is 8.1×104 L/mol/cm. The linear range of PAMBA is 0.13~38.40 μg/mL). Determination of reproducibility and detection limit According to the procedure, the solution of the system PAMBA-BA was determined 11 times (n=11) with a RSD of 0.11,.Then a reagent blank solution was measured 11 times (n=11). The standard deviation of the reagent blank σ)is 0.000802. Therefore, the limit of detection of this proposed method evaluated by calibration curve (3 σ/k) is 0.12 μg/mL)。 第 11 页 共 11 页 南华大学化学化工学院毕业设计 Fig. 6. Calibration curve. PAMBA (400 μg/mL): 1.00 mL; NaOH-NaHCO3 buffer solution (pH10.00): 2.00 mL; BA (0.20 M): 1.50 mL; temperature: room temperature. Table4. Analysis of PAMBA in pharmaceutical samples (n=5, t,4=2.78) 0.05 The Certified Present Method Sample Added Recovery Sample Found(μg/mL) RSD(,) (mg/10 mL) (mg/10 mL) Content(μg/mL) (μg/mL) (,) 050901 100.31 99.76 ?0.43 7.70 3.20 3.24?0.04 1.01 10 1.3 () 050902 100.74 101.98 ?0.32 7.74 6.40 6.45?0.20 2.51 100.8 050903 100.40 100.92?0.36 7.71 9.60 9.45?0.13 1.12 98.4 050915 99.86 98.45 ?0.51 5.75 6.40 6.34?0.12 1.57 99.1 050916 99.90 98.77 ?0.64 9.59 6.40 6.56 ?0.17 2.10 102.5 SAMPLE ANALYSIS Sample preparation Prior to analysis, 10 mL of the certified sample of the PAMBA injection (The Pharmacy Group Co., Ltd of Yangzhou Zhongbao) was accurately taken into a 250mL standard flask and then diluted to the mark with distilled water. Subsequently, the solution was mixed well and preserved without light at 4?. Analysis of PAMBA in pharmaceutical samples 第 12 页 共 12 页 南华大学化学化工学院毕业设计 According to the procedure, different concentrations of pharmaceutical sample solution were measured and the results agree well with the certified reference values (see Table 4). In addition, the results are satisfactory, with low RSD and high recovery. As the sample in the experiment is acompound prescription, it also shows that other components in the pharmaceutical sample do not affect the determination of PAMBA in the discoloring system, and the results are satisfactory. Analysis of recovery of PAMBA from urine samples Three urine samples were prepared for the analysis of recovery of PAMBA with the proposed method, and the results are shown in Table 5. These results have high accuracy and good recovery, which indicates that the proposed method can be successfully applied to recover PAMBA in urine samples. CONCLUSION It is the first time that the determination of PAMBA was carried out using boron as chromogenic agent by the spectrophotometric method. The experiment was carried out at pH 10.00 NaOH-NaHCO buffer solution at room 3 temperature, and the amounts of BA and buffter solution are 1.50 mL and 2.00mL, respectively. The linear range of PAMBA is 0.14~38.40μg/mL with the limit of detection0.12μg/mL. When boron is used as chromogenic reagent, it is economic and easily obtained. A simple, sensitive, rapid and reliable method of the direct determination of PAMBA is developed. The determination of PAMBA with the proposed method is free from the interference of metal ions and urea. The new reagent dose not need to be synthesized; expensive apparatus are not necessary. The proposed method can be successfully applied to the determination of PAMBA in pharmaceutical samples with satisfactory results, and average recovery from pharmaceutical and urine samples is between 98.4~102.5%. 第 13 页 共 13 页 南华大学化学化工学院毕业设计 Table5. Analysis of recovery of PAMBA from urine samples (n=5, t,4=2.78) 0.05 Urea Added Found Recovery RSD, Sample (μg/mL) (μg/mL) (,) 1 3.20 3.16?0.07 1.68 98.8 2 6.40 6.51?0.17 2.06 101.7 3 9.60 9.50?0.16 1.34 99.0 In this study, boron can complex with the lone pairs of electrons of the carboxylic group ( )and the amino group (-NH)of PAMBA. Presumably boron can react with other 2 compounds with either of these two functional groups. Then the application of boron as the chromogenic agent in spectrophotometry may be developed to a wider scope. Therefore the determination of this kind of organic pharmaceuticals using boron as the chromogenic agent by the spectrophotometric method has important practical value. Received November 2, 2006. 第 14 页 共 14 页 南华大学化学化工学院毕业设计 990 J. Chin. Chem. Soc., Vol. 54, No. 4, 2007 Li and Zhang REFERENCES 1.Kuemmel, D. F.; Mellon, M. G.Anal. Chem.1957,29(3),378. 2.Carrero,P.;Malave,A.;Rojas,E.;Rondon,C.;PetitdePena,Y.;Burguera,J.L.;Burguera,M.Talanta 2005,68,374. 3. VanStaden,J.F.;Merwe,T.A.Analyst2000,125(11),2094. 4. Korenaga, T.; Motomizu, S.; Toei, K.Analyst1978,103,745. 5.Thangavel,S.;Dhavile,S.M.;Dash,K.;Chaurasia,S.C.Anal.Chim.Acta2004,502,265. 6. Garcia, I. L.; Cordoba, M. H.; Concepcion, S. P. Analyst 1985,110,1259. 7.VanStaden,J.F.;Tsanwani,M. Talanta2002 , 58 ,1103. 8. Li,Z.J.;Cui,Z.W.;Tang,J.FoodChem.2006 ,94,310. 9.Akhmad, S.; Koji, O.; Mitsuko, O.; Shoji, M. Talanta2005 ,66,136. 10. Li, Z. J.; Song, Q. J.; Cui, Z. W.; Wei, Q.; Zheng, J.Talanta 2005,65,1307. 11.Committee of Chinese Pharmacopoeia,ChinesePharmacopoeia (Part II);China Chemical Industry Press: Beijing,1995;p801. 12.Wang,Z.P.; Zhang, Z. J.; Fu, Z. F.; Luo, W. Z.; Zhang, X. 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Law,K.Y.J.Photoch.Photobio.A1997,107,115. 18.Inorganic Chemistry Studio of Beijing Normal University, Huazhong Normal University and Nanjing 第 15 页 共 15 页 南华大学化学化工学院毕业设计 文献翻译 硼作显色剂在分光光度计法中的应用 一项有关氨甲苯酸与硼酸为决定作用的新颖的褪色分光光度计法 作者:李全民、张甜甜 河南师范大学化学与环境科学学院，河南环境污染控制重点实验室 中国河南新乡，邮编453007 此篇论文首次将硼用作显色剂来氨甲苯酸,通过褪色分光光度法得以实现。该文指出,当PH值为10.00往氨甲苯酸溶液加入硼酸时,其吸光度降低。一个简便迅速敏感而可靠的新式方法是基于氨甲苯酸和硼酸的产物上进行的。该产物的化学计量学中的比率是1:2.比尔定律使用于氨甲苯酸浓度为0.13 ~ 438.40μg/mL、波长为242nm(εis 8.1×10 L/mol/cm)的溶液中。反应式的线性242 -5 回归方程为A=-0.06635-0.02914C (×10M),其相关系数为0.9974.检测限为0.12μg/mL 相对偏差,RSD,0.11%。此法成功应用于药物样品的检测,来自药物样品和尿样的平均回收率为98.4 ~ 102.5%.样品中的其他成分不干扰所提及方法的测定。 关键词:硼酸，氨甲苯酸，显色剂，褪色分光光度计法 简 介 硼是一种缺电子元素，有较强的吸电子能力。硼很容易形成一个多聚体分子或复杂稳定的电子供体。硼所具有的复杂性质对于研究生命科学中的生化作用和营养生理学有重要意义。 截至目前，许多特定显色剂通过分光光度法来检测硼已经日趋成熟，这些方 12-45法基于硼酸和有机试剂的显色反应，比如铬酸、二乙基偶氮、姜黄色素、结 578-10晶紫、甲基橙等等。而且，这些合成的有机染色剂衍生物用来研究硼的分光光度法测定(见1) 然而目前没有以硼作显色剂来检测有机药物的报导。这篇论文首次描述了一 第 16 页 共 16 页 南华大学化学化工学院毕业设计 种新方法来检测氨甲苯酸和硼，即褪色分光光度法。 氨甲苯酸一种抗凝血剂，用来治疗各种出血，尤其在渗血上有显著疗效。中 11国药典(第二部分)中说，用滴定法测定片剂和注射剂中氨甲苯酸的含量。但是这个方法只适用于高浓度或生物样品中低含量而对检测氨甲苯酸无干扰的情况。 12另外，Wang et al.中报导，用流动注射剂荧光法测定，选择性差而且线性范 13围窄(0.02~1.0μg/mL)。紫外分光光度法也是用来检测注射剂中的氨甲苯酸含量，其次线性相关范围窄4.8 ~ 11.2 μg/mL和低选择性。 文章研究表明，在碱性介质(λmax=242nm)中加入了硼酸后氨甲苯酸的吸光度下降，而且其复合体(产物I)能够形成于硼酸和氨甲苯酸的反应之间。因此，氨甲苯酸可以用这种褪色体系来检出。其线性范围在0.13~38.40μg/mL之间，检测限为0.12μg/mL。 表1 有机试剂测定硼酸及其产物 2+2+2+2+2+2+2+3+有影响作用的金属离子有Mg , Ca , Co , Mn , Ni , Zn ,Cu 和Fe ，在所给中(PH=10.00)很容易估算出。同时，为了证实所给方案得出结论的可信性，氨甲苯酸的测定是在药物样品和生物样品中进行，得到了令人满意的结果。样品中的其他组分不影响检测。 实验 第 17 页 共 17 页 南华大学化学化工学院毕业设计 试剂与仪器 除非特别说明，所用试剂都是阶段分析试剂，所有溶剂都是蒸馏水。给出溶液主要有一下几种。已配制好的浓度为400μg/mL的标准氨甲苯酸溶液，溶解了0.1000g氨甲苯酸(中国药品与生物制剂公司)于250毫升蒸馏水中(溶液保存在4摄氏度无光环境中)，用以稀释成标准氨甲苯酸溶液。标准硼酸溶液(0.20 M)是用6.2025g硼酸(北京新光化学试剂厂)和7.4560g氯化钾(广东台山化学制剂厂)在蒸馏水中加热几分钟。此溶液用500毫升长颈烧瓶保存。Ph为10的缓冲溶液用10.70毫升0.10摩尔每升的NaOH和50.00毫升0.05摩尔每升的NaHCO3 混合于100毫升的标准烧瓶，用一个pH-3C数字ph计加以调节(上海雷词设备制造商)。 一型号为725的紫外可见分光光度计(厦门分析仪器厂)用来光学测量，TU-1900紫外可见分光光度计(北京PG仪器厂)扫描吸收光谱。所有ph测量仪都以pH-3C数控仪(上海雷词设备)。型号为CS-501精密衡量温度设备(重庆实验设备厂)用来测量温度。BS 110s电子分析平衡仪(北京电子天平有限公司)用以称量物重。 实验步骤 浓度为400μg/mL的氨甲苯酸1.00mL加入25.0mL对照管，继之加入ph=10.00的NaOH-NaHCO缓冲溶液，然后加入1.5毫升0.20摩尔每升的硼酸溶液，再用3 蒸馏水稀释至12.5毫升。将混合物充分摇匀，用pH-3C数控仪测量其ph值。该溶液在室温下制备，迅速在242 nm(该波长下证明是用TU-1900紫外光谱仪体系测量时具有最大褪色波长)测定吸光度，用ph为10.00含氨甲苯酸的NaOH-NaHCO作对照溶液。 3 结果与讨论 吸收光谱 根据实验步骤，产品记录的吸收光谱产生于氨甲苯酸和硼酸反应物。我们能够从图1中可以看出，很大程度上氨甲苯酸可以被硼酸褪色，而且产物在242nm有最小吸收，此时硼酸没有吸收峰。虽然氨甲苯酸在242nm处有个高的吸收峰，但氨甲苯酸和硼酸的产物相对于氨甲苯酸和缓冲溶液有更为敏感的吸收。为了得 第 18 页 共 18 页 南华大学化学化工学院毕业设计 到更高的敏感度，以下测量都是在242nm处相对氨甲苯酸溶液和缓冲溶液而进行。 图1 吸收光谱 B:氨甲苯酸对于水的吸收光谱。C:硼酸的吸收光谱。D:产物相对空 白试剂的吸收光谱。氨甲苯酸(浓度400μg/mL):1.00mL;硼酸(0.20mL/mol):1.50 mL; 温度:室温。 氨甲苯酸和硼酸产物的成分研究 摩尔比率方法斜度比率方法是曾经用来测定硼酸与氨甲苯酸产物成分。结果如图2和图3中所示。我们看到，产物的摩尔比率是2:1。 -3-3图2 复合物用摩尔比率法测定结果.VR: 硼酸(2.62×10 M); VD:氨甲苯酸 (2.62 ×10 M); VR+VD 1.00 mL; NaOH-NaHCO缓冲液(PH=10.00):2.00Ml;温度:室温。 3 第 19 页 共 19 页 南华大学化学化工学院毕业设计 图 3 用斜度比率法测定复合物。VR: 硼酸; VD: 氨甲苯酸; NaOH-NaHCO缓冲液(PH=10.00) 温度:室温。 3 氨甲苯酸与硼酸反应机理讨论 -5苯甲酸的解离常数(Ka)是6.2×10。由于氨甲苯酸的分子结构和苯甲酸类 -5似，故氨甲苯酸的解离常数(Ka)也可认为是6.2×10。 )很难解离出由于较小的解离常数，酸性介质中氨甲苯酸的羧基( H+,而且氨甲苯酸的氨基(-NH)在酸性介质中。由于硼的原子空轨道使得羧基的2 +孤对电子和氨甲苯酸的氨基失去配合能力。然而在PH=10.00时，H可以从氨甲苯酸的羧基中完全解离，从而形成带孤对电子的氧负离子。在酸性溶液中质子化形成的氨甲苯酸铵盐，反过来作用于带一对孤对电子的氨基。因此在碱性溶液中， 16羧基的孤对电子和氨甲苯酸的氨基能够因硼的空原子轨道而复合，鉴于文献里硼能够和N与O原子螯合。基于硼酸和氨甲苯酸的摩尔比率为2:1，以下的反应式就是合理的: 第 20 页 共 20 页 南华大学化学化工学院毕业设计 PH在产物I的吸光度上的影响 对产物I吸光度的PH值影响范围在1.00到13.00。 如图表4所示，当PH值在1.00~3.00时产物I(氨甲苯酸和硼酸形成)的吸光度几乎为0，这表明在强酸下硼酸很难与氨甲苯酸反应。原因可能是氨甲苯 +酸的羧基很难电离出H，氨甲苯酸的氨基被质子化后给出质子而形成铵盐 3+(-NH)。由于硼可以的空轨道使孤对电子失去配合能力。因此体系的褪色几乎就不会发生。当PH值大于3.00时，产物I的吸光度下降，这与PH值的增加相称。 3+大概当溶液的酸性降低时氨甲苯酸的质子化的铵盐(-NH)反过来作用于氨基 +(-NH)，同时羧基(-COOH)上H电离度也增加。PH值越大，其效用越大。然后形2 16、17成具有孤对电子的氧负离子。文献指出，硼能够和N、O原子螯合，接着氨基(-NH)上的N原子和氨甲苯酸的羧基(-COOH)上的O原子和硼螯合。硼酸与氨2 甲苯酸的产物的吸光度可能小于PH=10.00波长为242nm时的氨甲苯酸，这也导致氨甲苯酸—硼酸体系的褪色。图表4显示在PH=10.00时吸光度变得最小。换句话说，反应以最大程度进行。但是当PH增加至13.00时，吸光度接近于0.这 -18可能是高浓度的氢氧根离子(OH) (0.10 M)能够和硼酸螯合成[B(OH)]-,这就4抑制了羧基孤对电子和硼酸以及氨甲苯酸的氨基(-NH) 间的化合。为了保持测2 定氨甲苯酸的高敏感性，实验在PH=10.00下进行。 图4 PH值对体系吸光度的影响。氨甲苯酸(400 g/mL):1.00mL;硼酸(0.20 M):1.50mL; 温度:室温。 第 21 页 共 21 页 南华大学化学化工学院毕业设计 硼酸的量对产物I吸光度的影响 为了研究硼酸的量对氨甲苯酸测定的影响，硼酸的量从0.00到3.00不等，用来进行实验步骤(见图5)。当硼酸的量在1.50mL下时，随着其量的增加吸光度下降。这也许是硼酸也氨甲苯酸的反应程度伴随硼酸的增加而加强。当硼酸的量为1.50mL时褪色程度达到最大。然而当量高于1.50mL时吸光度从-0.715(1.50mL)增至-0.654(4.00mL)。再当溶液的PH比10.00高一点或低一点时，产物I的吸光度明显增加(见图4)。因为硼酸的量增加而导致溶液PH降低，然后当硼酸的量多于1.50mL时产物I的吸光度增加。从图5中可以看出，以上结论与“PH对产物I吸光度影响”(见图4)的部分结果相一致。为了得到高灵敏度的测定，一下实验的硼酸量选择为1.50mL。 缓冲液的量对产物I吸光度的影响 缓冲液量对氨甲苯酸测定的影响是根据实验步骤而进行研究的，维持PH=10.00。图5表明当缓冲液的量为2.00mL时吸光度达到最小值。 图5 硼酸与缓冲液的量的影响。 氨甲苯酸(400μg/mL):1.00mL; 温度:室温;B: NaOH-NaHCO缓冲液(PH=10.00):2.00mL;C:硼酸(0.20 M):1.50 mL. 3 当该量超过2.00mL时吸光度只有很细微的增加，从-0.750(2.00mL)到-0.661(4.00mL)。原因可能是当缓冲液的量从2.00mL增加至4.00mL时缓冲液的浓度增加了。因此溶液中的OH-浓度有了少许增加，溶液的PH也增加了，所以OH-与硼酸的螯合能力增强了。这也就导致了某些程度上干扰了硼酸与氨甲苯酸的结合。这与“PH对产物I吸光度的影响”(见图4)相一致。所以当缓冲液 第 22 页 共 22 页 南华大学化学化工学院毕业设计 的量多于2.00mL时，产物I的吸光度开始增加。为了保持测定的高灵敏度，我们选择缓冲液的量为2.00mL。 滞留时间对产物I吸光度的影响 产物I的吸光度在室温下每隔5分钟测定一次，其值从0到2小时得出，如表2所示。 表2. 产物I在不同时间段的吸光度(室温)。 时间(分钟) 0 5-25 30~35 40~50 55 1 h 2 h 吸光度 -0.690 -0.690 -0.695 -0.703 -0.711 -0.720 -0.732 我们发现，当溶液在PH=10.00条件下混合时硼酸和氨甲苯酸的反应迅速发生，室温下的反应速度非常快。产物I的吸光度在开始到25分钟时保持相同，这表明体系的较强稳定性。 因此实验在室温下进行，当溶液制备好后立刻测定产物I的吸光度。 潜在性干扰因素研究 普通离子和尿素潜在性影响的系统研究开展于氨甲苯酸的测定。耐受量定义为分析误差小于 5%)。 表3.外界离子对氨甲苯酸和硼酸的测定。 外界离子 加入形式 耐受限 (μg/mL) ++3--2-K,Na,NO,Cl,SO KClNaCl, NaNO,NaSO 1.2(mg/mL) 4, 324 2-CO NaCO 180 323 2+2+2+Mg ,Ca ,Co Mg(NO),CaCl,CoSO 40 3224 2+Mn MnSO 32 4 2+Ni NiSO 12.80 4 2+Zn ZnSO 8.80 4 2+ Cu CuSO?1.44 4 3+Fe NHFe(SO) 1.20 44 2 Urea CO(NH) 7.7(mg/mL) 22 我们从表3中看到，当测定氨甲苯酸时是允许一定量的金属离子的存在。在 第 23 页 共 23 页 南华大学化学化工学院毕业设计 当金属离子浓度在耐受量以内时，某些程度上溶液的吸光度有所增加。原因可能是这些金属离子的氢氧根胶粒在PH=10.00可以形成，由于分散系的作用使得溶 2+3+液的吸光度增加，而且Cu 和Fe 耐受限非常低。这或许是因为他们的可溶性 19-39产物的溶解度(Ksp)很小，分别为2.6×10- 和 3×10，这意味着很容易形成他们的氢氧根胶粒。当在现实样品中运用所给方法测定氨甲苯酸时，假如有所提及的金属离子共存，他们可以通过在碱性溶液中形成沉淀而提前分离。根据这些金属离子的Ksp，可以从残留的浓度估算出他们的浓度远低于其耐受量，这表明氨甲苯酸的测量不受这些金属离子的干扰。因此所给方案的选择性是非常高的。 我们也可以从表3中得出尿素的耐受值为7.7 mg/mL，表明用所给方法测定尿素中的氨甲苯酸时是不受干扰的。 标准曲线图 -5 -5 -5依据实验步骤，我们准备了浓度为0.08×10, 0.21×10,0.42×10 ,1.27 -5-5-5-5-5-5×10 ,2.11×10 ,3.39×10 , 4.23×10 ,12.70×10 ,21.17×10 和25.40 -5×10 M的氨甲苯酸溶液，在最适条件下分别测量褪色体系的吸光度。吸光度作为氨甲苯酸的浓度函数在坐标上标出(见图6)。得到的线性回归方程为A= -5 0.06635,0.02914C (×10-M)，随之而得的线性系数为0.9974， ε为 8.1242 ×104 L/mol/cm。氨甲苯酸的线性范围是0.13~38.40μg/mL。 可再生的检测与检测限 依实验步骤，氨甲苯酸-硼酸溶液体系需要在相对标准偏差(RSD)为0.11,下测定11次(n=11)。空白试剂的标准偏差(σ)为 0.000802。因此，该方案的检测限通过标准曲线(3σ/k)估计为0.12μg/mL 第 24 页 共 24 页 南华大学化学化工学院毕业设计 图6 标准曲线。氨甲苯酸(400μg/mL):1.00 mL;NaOH-NaHCO 缓冲溶液(pH=10.00)2.00 mL; 3 硼酸 (0.20 M): 1.50 mL;温度:室温。 表4. 药物药品中氨甲苯酸的分析(n=5, t,4=2.78) 0.05 参考量 方法合格量样品含量 添加量 测量值 相对标准回收率 样品编号 (mg/10 mL) (mg/10mL) (μg/mL) (μg/mL) (μg/mL) 偏差(,) (,) 050901 100.31 99.76?0.43 7.703 3.20 3.24?0.04 1.01 101.3() 050902 100.74 101.98?0.32 7.74 6.40 6.45?0.20 2.51 100.8 050903 100.40 100.92?0.36 7.71 9.60 9.45?0.13 1.12 98.4 050915 99.86 98.45?0.51 5.75 6.40 6.34?0.12 1.57 99.1 050916 99.90 98.77?0.64 9.59 6.40 6.56?0.17 2.10 102.5 试样分析 样品准备 分析前，准确量取10mL的氨甲苯酸注射液(扬州中宝医药有限公司)作参照样品，小心移入250mL标准烧瓶，用蒸馏水稀释至刻度。接下来将溶液混合均匀，保存在无光、4摄氏度条件下。 第 25 页 共 25 页 南华大学化学化工学院毕业设计 药物样品中的氨甲苯酸分析 根据实验步骤测定了不同浓度下的药物样品，所得结果与参考值十分吻合。(见表4)。而且在较低的相对标准偏差和较高的回收率下取得了满意的结果。实验中的样品是处方药，也说明药物样品中其他成分在褪色体系中不影响氨甲苯酸的测定，结果是令人满意的。 尿素样品中氨甲苯酸回收率的分析 此方案中准备了三组尿素样品来分析氨甲苯酸的回收率，结果如表5所示。这些结果具有精确度高、重复性好，这意味着该方法运用于尿素样品中氨甲苯酸回收率的测定是比较成功的。 第 26 页 共 26 页 南华大学化学化工学院毕业设计 表5 尿素样品中氨甲苯酸回收率分析 (n=5, t,4=2.78) 0.05 添加量 测量值 相对标准 尿素样品 回收率(,) (μg/mL) (μg/mL) 偏差 , 1 3.20 3.16?0.07 1.68 98.8 2 6.40 6.51?0.17 2.06 101.7 3 9.60 9.50?0.16 1.34 99.0 总结 这是首次通过分光光度法将硼作为显色剂来测定氨甲苯酸的含量。 实验在PH=10.00的NaOH-NaHCO缓冲溶液、室温下进行，硼酸和缓冲溶液3 的量分别为1.50mL 、2.00mL。氨甲苯酸的线性回归范围在0.14~38.40μg/mL，其检测限为0.12μg/mL。当硼酸用作显色剂时既经济又容易得到。一个简单、灵敏、迅速而又可靠的方法用以直接测定氨甲苯酸含量发展了起来。所给方法测定氨甲苯酸不受金属离子和尿素干扰。该新试剂不必合成，不需要昂贵的实验仪器。该方法成功应用于药物样品中氨甲苯酸的测定，并取得满意结果，其药物和尿素样品的平均回收率在98.4~102.5%间。 在此论文中，硼能够和带孤对电子的羧基()与氨甲苯酸的氨基(-NH)2结合。据推测，硼能够和这两个官能团中的任何一个反应。然后硼在分光光度法中作为显色剂的应用也许有较大的范围。因此这种通过分光光度法用硼作显色剂的有机药物的测定具有重要的实用价值。 2006年11月2号收录。 第 27 页 共 27 页 南华大学化学化工学院毕业设计 参考文献: 1.Kuemmel, D. 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