Journal of the Chinese Chemical Society, 2007, 54,

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1 Journal of the Chinese Chemical Society, 2007, 54, Application of Boron as the Chromogenic Agent in Spectrophotometry I. 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 , 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 the absorbance 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 ~ g/ml at a wavelength of 242 nm ( 242 is L/mol/cm). The equation of linear regression is A = C ( 10-5 M), with a linear correlation coefficient of The detection limit is 0.12 g/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 ~ 102.5%. 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. INTRDUCTIN 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 BA and organic reagents, such as chromotropic acid, 1 Azomethine-H, 2-4 curcumin, 5 crystal violet, 5 methyl orange (M), 7 and so on. Furthermore, several new derivatives of these organic dyes were synthesized and studied for spectrophotometric determination of boron 8-10 (see Table 1). 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 Pharmacopoeia (Part II) 11 describes a titrimetric method for determination of PAMBA in tablets and injections. However, this method is only suitable for determining PAMBA of high concentration and ineffective for measuring PAMBA of low content in biological samples. Additionally, Wang et al. 12 reported the determination of PAMBA by flow-injection chemiluminescence, with poor selectivity and a narrow linear range (0.02~1.0 g/ml). Ultraviolet spectrophotometry 13 was also used for determination of PAMBA in injections, again with a relatively narrow linear range of 4.8 ~ 11.2 g/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 ( max = 242 nm), 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 0.13~38.40 g/ml, * Corresponding author. Tel: ; Fax: ; mercury6068@hotmail.com

2 984 J. Chin. Chem. Soc., Vol. 54, No. 4, 2007 Li and Zhang Table 1. The organic reagents to determine B and their products rganic reagents Product Reference _ Chromotropic acid H H 3 S 3 S S 3 H H B H B H H H 3 S S or 3 H 1 Azomethine-H H N CH 3 S 3 S N H CH 2- or 3 S 3 S N CH (2,3,4-Trihydroxybenzylidene- amino)-8-hydroxynaphthalene- 3,6-disul-fonic acid (one of the derivatives of Azomethine-H) B/ 2 10 H H N CH H H N CH H and the limit of detection is 0.12 g/ml. The interference of metal ions (Mg 2+,Ca 2+,Co 2+,Mn 2+,Ni 2+,Zn 2+,Cu 2+ and Fe 3+ ) can be eliminated easily in the proposed method at ph 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. ther components in those samples don t interfere with the determination. EXPERIMENTAL 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 400 g/ml of Aminomethylbenzoic Acid (PAMBA) was prepared by dissolving g PAMBA (Chinese Drugs and Biological Goods Company) in 250 ml distilled water (The solution was preserved at 4 C without light) and used to prepare working PAMBA standards by suitable dilutions. A standard boron stock solution (0.20 M) was prepared by dissolving g of boric acid (BA) (Beijing Xinguang Chemical Reagent Plant, Beijing, China) and g 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 was obtained by mixing ml solution of 0.10 M NaH and ml solution of 0.05 M NaHC 3 in 100 ml standard flask 15 and adjusted by a ph-3c digital ph meter (Shanghai Lei Ci Device Works, Shanghai, China). 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 Lei 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.

3 Determination of Aminomethylbenzoic Acid J. Chin. Chem. Soc., Vol. 54, No. 4, Procedure 1.00 ml of 400 g/ml of PAMBA was taken into a 25.0 ml comparison tube. Sequentially, 2.00 ml NaH- NaHC 3 buffer solution of ph was added and then 1.50 ml of 0.20 M BA was added and the solution was diluted to 12.5 ml 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 (242 nm 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 NaH-NaHC 3 buffer solution of ph 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 242 nm against the solution of PAMBA and buffer solution. 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. Discussion of reaction mechanism of PAMBA and BA The dissociation constant (Ka) of benzoic acid is Since the molecular structure of PAMBA is similar RESULTS AND DISCUSSIN Fig. 2. The determination of the complex formation by mole ratio method. V R : BA ( M); V D : PAMBA ( M); V R V D 1.00 ml; NaH-NaHC 3 buffer solution (ph 10.00): 2.00 ml; temperature: room temperature. 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 (400 g/ml): 1.00 ml; BA (0.20 M): 1.50 ml; temperature: room temperature. Fig. 3. The determination of the complex formation by slope ration method. V R : BA; V D : PAMBA; NaH-NaHC 3 buffer solution (ph 10.00): 2.00 ml; temperature: room temperature.

4 986 J. Chin. Chem. Soc., Vol. 54, No. 4, 2007 Li and Zhang to that of benzoic acid, Ka of PAMBA is supposed to be near Because of the smaller Ka, it is difficult for H + to dissociate from the carboxylic group ( C H ) of PAMBA in acidic medium. In addition, the amino group (-NH 2 ) of PAMBA is protonized in acidic medium. So the lone pairs of electrons of the carboxylic group ( C H ) and the amino group (-NH 2 ) of PAMBA lose complexation capacity for the empty atomic orbit of boron. However, at ph 10.00, H + has been completely dissociated from the carboxylic group ( C H) of PAMBA, and the negative ion with a lone pair of electrons being obtained. The protonated amine salt (-NH + 3 ) of PAMBA, which is formed in acidic solution, turns back into the amino group (-NH 2 ) with a lone pair of electrons. Thereby, in the alkaline solution, the lone pairs of electrons of the carboxylic group ( C - ) and the amino group (-NH 2 ) of PAMBA can complex with the empty atomic orbit of boron in light of the literature 16 where boron can chelate with N and. Based on that the mole ratio of the reaction between BA and PAMBA is 2:1, 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 As shown in Fig. 4, at ph , 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 (-CH) of PAMBA is difficult, and the amino group (-NH 2 ) of PAMBA is protonized and it turns into protonated amine salt (-NH 3 + ) in acidic medium. 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 3 + ) of PAMBA turns back into amino group (-NH 2 ), and the degree of the dissociation of H + from the carboxylic group (-CH) also rises. The higher the ph is, the more effective it is. Then negative ion with lone pairs of electrons was formed. In light of the literature, 16,17 boron can be chelated with N and, and then N of the amino group (-NH 2 ) and of the carboxylic group (-CH) 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 ph at 242 nm, which leads to the formation of the discoloring system of PAMBA-BA. Fig. 4 shows that the absorbance becomes minimal at ph 10.00; in other words, the degree of the reaction is maximal. However, when ph increases to 13.00, the absorbance is almost 0. It is likely that a high concentration of hydroxide ion (H - ) (0.10 M) can chelate with BA to form [B(H) 4 ] -, 18 which holds back the complexation between BA and the lone pairs of electrons of the carboxylic group (-CH) and the amino group (-NH 2 ) of PAMBA. In order to keep the high sensitivity for determination of PAMBA, the experiment was carried out Product I Fig. 4. Effect of ph on absorbance of the system. PAMBA (400 g/ml): 1.00 ml; BA (0.20 M): 1.50 ml; temperature: room temperature.

5 Determination of Aminomethylbenzoic Acid J. Chin. Chem. Soc., Vol. 54, No. 4, at ph 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 3.00 ml 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 1.50 ml. 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 is 1.50 ml. However, when the amount is above 1.50 ml, the absorbance rises from (1.50 ml) to (4.00 ml). 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 1.50 ml, as can be seen in Fig. 5. It accords with the result obtained from the section of Effect of ph on the absorbance of Product I (see Fig. 4). In order to obtain the highest sensitivity, 1.50 ml of BA was selected in the later experiments. Fig. 5. Effect of amount of BA and buffer solution. PAMBA (400 g/ml): 1.00 ml; temperature: room temperature; B: NaH-NaHC 3 buffer solution (ph 10.00): 2.00 ml; C: BA (0.20 M): 1.50 ml. 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 at Fig. 5 shows that the absorbance gets to the minimum when the amount of buffer solution is 2.00 ml. When the amount is above 2.00 ml, there is little increase in absorbance, from (2.00 ml) to (4.00 ml). The possible reason is that when the amount of buffer solution added rose from 2.00 ml to 4.00 ml, the concentration of buffer solution increased. Thereby the concentration of H - in the solution has a little increase, as does the ph of the solution, so then the ability of H - to chelate with BA to form [B(H) 4 ] -18 is enhanced, 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 (see Fig. 4). So when the amount of buffer solution is above 2.00 ml, the absorbance of Product I begins to increase. In order to keep the high sensitivity of the determination, 2.00 ml 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 stability of the system. Hence, the experiments were carried out at room temperature, and the absorbance of Product I was measured immediately 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 at ph 10.00, which makes the absorbance of the solution increase because of light dispersion. In addition, the toler-

6 988 J. Chin. Chem. Soc., Vol. 54, No. 4, 2007 Li and Zhang Table 2. The absorbance of Product I at different times (room temperature) Time (min) (h) 2 (h) Absorbance Table 3. Effect of foreign ions on the determination of PAMBA with BA Foreign ions Added as Tolerance limit K +,Na +,N - 3,Cl - 2-,S 4 KCl, NaCl, NaN 3,Na 2 S (mg/ml) 2- C 3 Na 2 C Mg 2+,Ca 2+,Co 2+ Mg(N 3 ) 2,CaCl 2,CoS 4 40 Mn 2+ MnS 4 32 Ni 2+ NiS Zn 2+ ZnS Cu 2+ CuS Fe 3+ NH 4 Fe(S 4 ) Urea C(NH 2 ) (mg/ml) ance limits of Cu 2+ and Fe 3+ are relatively lower. It is likely that their solubility product constant (Ksp) are smaller, and , 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. Calibration curve According to the procedure, , , , , , , , , and Mof 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 (see Fig. 6). A linear regression equation is attained as A C ( 10-5 M) with a linearly dependent coefficient of , and 242 is L/mol/cm. The linear range of PAMBA is g/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 Then a reagent blank solution was measured 11 times (n 11). The standard deviation of the reagent blank ( ) is Therefore, the limit of detection of this proposed method evaluated by calibration curve (3 /k) is 0.12 g/ml. Fig. 6. Calibration curve. PAMBA (400 g/ml): 1.00 ml; NaH-NaHC 3 buffer solution (ph 10.00): 2.00 ml; BA (0.20 M): 1.50 ml; temperature: room temperature.

7 Determination of Aminomethylbenzoic Acid J. Chin. Chem. Soc., Vol. 54, No. 4, Table 4. Analysis of PAMBA in pharmaceutical samples (n 5, t 0.05,4 2.78) Sample The Certified (mg/10 ml) Present Method (mg/10 ml) Sample Content Added Found RSD ( ) Recovery ( ) 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 250 ml standard flask and then diluted to the mark with distilled water. Subsequently, the solution was mixed well and preserved without light at 4 C. Analysis of PAMBA in pharmaceutical samples According to the procedure, different concentrations of pharmaceutical sample solutions 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 a compound 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. CNCLUSIN 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 NaH- NaHC 3 buffer solution at room temperature, and the Table 5. Analysis of recovery of PAMBA from urine samples (n 5, t 0.05,4 2.78) Urea Sample Added Found amounts of BA and buffter solution are 1.50 ml and 2.00 ml, respectively. The linear range of PAMBA is g/ml with the limit of detection 0.12 g/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%. In this study, boron can complex with the lone pairs of electrons of the carboxylic group ( C H) and the amino group (-NH 2 ) of PAMBA. Presumably boron can react with other 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, RSD ( ) Recovery ( )

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