Ionic liquids (ILs),salts that are liquids at or approaching room temperature,are normally composed of relatively large organic cations (e.g. alkylammonium,alkyllimidazolium,alkylpyridinium etc.) and inorganic (e.g. [Br]-,[PF6]-,[BF4]- etc.) or organic anions (e.g. [(CF3SO2)2N]-,[CF3CO2]- etc.). Compared to normal organic solvents,ILs have prominent physicochemical properties,such as negligible vapor pressure,nonflammable,good thermal stability,good conductivity and miscibility with water and organic solvents. Utilizing these characteristic properties,applications of ILs in the fields involving extraction,synthesis,catalysis,electrochemistry,environment protection and analytical chemistry are becoming widely used as new ‘‘green chemistry’’ solvents [1, 2]. More recently,the attention of ILs employed in chromatographic analysis have been growing rapidly [3, 4]. ILs are primarily used as gas chromatography (GC) stationary phases,high performance liquid chromatography (HPLC) mobile phase additives and stationary phases,as well as capillary electrophoresis (CE) running electrolytes.
An extensive effort has been made in elucidating the effects of ionic liquids on the retention behavior and the separation as mentioned in the following reports. Recently,Yu et al.  reported that the addition of ILs to a mobile phase had an advantageous effect on the separation of morpholinium in a HPLC system. The determination and separation of alkyl sulfonates  were investigated by reversed-phase HPLC using 1-ethyl-3-methylimidazolium salts as new mobile phase additives. The separation of naphazoline nitrate  was studied using ILs as mobile phase additives. The determination of a group of heterocyclic aromatic amines  was achieved using a group of alkylimidazolium-based ILs as mobile phase additives in HPLC with electrochemical detection. The o-amino benzoic acid (OABA),m-amino benzoic acid (MABA) and p-amino benzoic acid (PABA) are commonly used in compositions or as intermediates in pharmaceutical products. Similarly,the compendial limit of PABA,which is a degradation impurity in Hydrochloric Acid Procaine Injection is not allowed to exceed 1.2% using thin layer chromatography (TLC) in the 1995 China Pharmacopoeia. Since the method is by visual inspection comparing with the sizes of pigmentation and the depths of color,it is not easy to estimate levels,especially near or at the specification limit. Presently,the reported methods for analysis of aromatic carboxylic acids are mainly by HPLC ,CE ,ion exclusion chromatography  and liquid chromatography- mass spectrometry .
In this study,we developed a method for the separation and determination of three amino benzoic acid isomers by HPLC with ILs as mobile phase additives,and investigated the factors that impacted the separation of the three amino benzoic acids and the retention behavior in HPLC. The optimized chromatographic conditions were selected,and then a method for simultaneous determination of isomers of amino benzoic acid was established. This method was employed in the determination of PABA in the pharmaceutical,Bromine Mitag Procaine Injection.2. Experimental
HPLC separation was performed on an Agilent 1200 system (Agilent Technologies,USA) equipped with a quaternary pump- G1311A,an autosample injector Model ALS-G1329A,a thermostatted (30 ℃) column compartment Model TCC-G1316A,a diodearray multiple wavelength UV/vis detector Model DAD-G1315D and a degasser system Model Degasser-G1322A. A Model PHSF-3F pH meter (China) was used for pH measurements. A Millipore Milli-Q water purification system (Millipore,Bedford,MA,USA) was used to deionize distilled water. A Model DOA-P504-BN pump (IDEX,USA) and 0.22 mm membrane filter (China) were used to filter mobile phases and sample solutions.
The studied amino benzoic acids were OABA,MABA and PABA,purchased from Bailingwei Chemicals (China). Ionic liquids (purity ≥ 99.0%) were obtained from Shanghai Chengjie Chemical Ltd. (China). The ILs were 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIm][BF4]),1-butyl-3-methylimidazolium tetrafluoroborate ([BMIm][BF4]),1-hexyl-3-methylimidazolium tetrafluoroborate ([HMIm][BF4]). Methanol (HPLC grade) was obtained from Shanghai Xingke Biochemistry Co.,Ltd. (China). Acetic acid and NaOH were supplied by Shanghai Chemical Reagent Factory (China). Bromine Mitag Procaine Injection was marketed drug. The pH of ionic liquid solutions was adjusted with acetic acid.
Solutions were prepared using 18.2 MΩ/cm water. Standard stock solutions at a concentration 500 mg/L were prepared: accurately weighed OABA,MABA,and PABA at 0.0250 g,respectively,dissolved with 20% methanol solution and diluted to 50 mL,then preserved in refrigerator. Working standard solutions from each respective stock solution were prepared on a daily basis as required.
All separations were performed on a ZORBAX ODS column (250 mm × 4.6 mm i.d.,5 μm). The mobile phase was 0.5 mmol/L [BMIm][BF4] solution (pH 3.0 adjusted with acetic acid)/methanol (40:60,v/v) without need of gradient elution. UV/vis detection (245 nm) was employed. Flow rate was 1.0 mL/min. Injection volume was 20 mL. Column temperature was 30 ℃. The chromatographic system control,data acquisition and data analysis were performed using the Agilent Rev.B.04.01 workstation (Agilent,US). In order to eliminate the reagents absorbed on the chromatographic column,the chromatographic column should be flushed using at least 30 mL water-methanol (95:5,v/v) eluent after analysis everyday.3. Results and discussion
Ionic liquids were used as mobile phase additives that possess exceptional properties. In reversed phase HPLC,the silanol groups attract the polar groups of a solute molecule through specific electrostatic interactions,while the alkyl groups attract the alkyl groups of a solute molecule through hydrophobic interactions. Such behavioral interactions between the analytes and the stationary phase can be interfered (disrupted) when ionic liquids are used as mobile phase additives,since the addition of ionic liquids to mobile phase leads to competition between cations of the ionic liquid and polar groups of the analytes for the polar silanols groups of the alkyl silica surface,and thus can reduce the retention of acids and cause bases to become more retained [13, 14]. In our study,[BMIm][BF4] was added to the mobile phase to separate three amino benzoic acids. All these organic substances have different affinities to silanols groups. Thus,all the employed solutes were apt to migrate along the chromatographic column due to the mixed retention mechanism.
The maximum absorbances of the isomers of amino benzoic acid each had a different response by UV detection. Further observation and comparison determined that all three amino benzoic acids had ultraviolet absorbance and high sensitivity when the detection wavelength was 245 nm. Therefore,the wavelength of 245 nm was selected for the detection of amino benzoic acids in the following investigations.
The effects of varying the concentration of [BMIm][BF4] from 0.5 mmol/L to 5 mmol/L on the determination of amino benzoic acids were investigated. In Fig. 1,the results showed that the retention time of amino benzoic acids increased as the concentration of [BMIm][BF4] solution was gradually reduced. The retention of three amino benzoic acids was improved when the concentration of [BMIm][BF4] solution was 1.0 mmol/L,but the resolution of amino benzoic acids was worse than at a concentration of 0.5 mmol/L. Consequently,the appropriate concentration of the [BMIm][BF4] solution was 0.5 mmol/L.
|Fig. 1. Chromatograms of separation of the amino benzoic acids with mobile phases containing different concentrations of [BMIm][BF4]. Concentrations of [BMIm][BF4]: (a) 5.0 mmol/L; (b) 3.0 mmol/L; (c) 1.0 mmol/L; (d) 0.5 mmol/L. Chromatographic conditions: mobile phase, [BMIm][BF4] solution (pH 3.0)/ methanol (60:40, v/v); ZORBAX ODS column (250 mm × 4.6 mm i.d.); UV/vis detection, 245 nm; flow rate, 1.0 mL/min; column temperature, 30 ℃; inject volume, 20 mL. Peaks: 1, PABA; 2, MABA; 3, OABA.|
The effects of the methanol concentration on the retention of amino benzoic acids were also investigated,when the volume fractions of methanol were 40%,50%,60% and 70% with 0.5 mmol/L [BMIm][BF4] solution as the mobile phase. As a general trend,the retention times of three amino benzoic acids decrease with increasing the concentration of methanol. But the separation of amino benzoic acids was poor with an increasing concentration of methanol. Upon comprehensive consideration of the resolution and retention times,the optimum content of methanol was selected at 60%.
The effect of the pH of the [BMIm][BF4] solution on the separation of amino benzoic acids was investigated by changing the pH value using 0.5 mmol/L [BMIm][BF4] solution/methanol (40:60,v/v) as the mobile phase. The separation of amino benzoic acids was improved when the pH value increased from 2.5 to 3.0,but when the pH was increased to 3.5,separation was inferior to pH 3.0. The complete separation of amino benzoic acids was accomplished within four min,and the retention times were better at the pH of 3.0. Therefore,the pH values of the following ILs solutions were all adjusted to 3.0.
In order to study the effects of alkyl groups on the imidazolium ring of ILs on the separation of amino benzoic acids,experiments on the addition of the same concentrations (0.5 mmol/L) of three ILs with the same counterion ([BF4]-) with 60% methanol were conducted at pH 3.0. When the alkyl chain was increased from [EMIm]+,[BMIm]+ to [HMIm]+,the retention times of the amino benzoic acids were shifted,but (only slightly) little. In consideration of the retention and resolution of amino benzoic acids,[BMIm][BF4] was suitable as the mobile phase additive.
For a better understanding of the benefits of using [BMIm][BF4] as a mobile phase additive,a comparison of mobile phases with and without ILs on the separation of amino benzoic acid was carried out experimentally. The results were shown in Fig. 2. Obviously,the separation of amino benzoic acids using [BMIm][BF4] as a HPLC mobile phase additive resulted in better results with the separation times shortened,and good chromatographic peak shapes compared to the mobile phase without [BMIm][BF4].
|Fig. 2. Chromatograms of the amino benzoic acids using (a) water (pH 3.0 adjust with acetic acid)/methanol (40/60, v/v) and (b) aqueous 0.5 mmol/L [BMIm][BF4] solution (pH 3.0 adjust with acetic acid)/methanol (40:60, v/v) as the mobile phases. Chromatographic conditions as in Fig. 1. Peaks: 1, PABA; 2, MABA; 3, OABA.|
Consequently,the addition of ionic liquid to the mobile phase of reversed phase chromatography decreases the chromatographic retention times of solute molecules . Part of the ionic liquids coat the surface of the stationary phase,on which they suppress free silanols and improve the shape of peaks and although ionic liquids interact greatly with free silanols,ion pairings would have effects in these separations. The delocalization of the charge on the imidazolium cation would produce a lower association constant between analytes and ionic liquids. We surmise that there would be also an ion pairing mechanism when ionic liquids are used as mobile phase additives.
Aliquots of each of standard stock solution were diluted with water to make up a series of accurate standard solutions of concentrations on 2,10,20,60,120 mg/L. Under the optimum chromatographic conditions,all experiments of the standard solutions mentioned above were carried out in triplicate at each level. Linear regression equations were obtained from the relationship between integrated peak area and control solution concentration (mg/L). Detection limit was calculated with a signalto- noise ratio of 3. Relative standard deviations of peak area on five replicates were obtained from successive injections of the standard solutions. The results are listed in Table 1.
The recovery of the method was evaluated by standard addition methodology. Two mL samples of Bromine Mitag Procaine Injection were removed and added to OABA,MABA and PABA standard solutions,respectively,diluted to 10 mL,then filtered through a 0.22 mm filter. The diluents were determined using the established conditions for six times. Recoveries of each of the three amino benzoic acids are listed in Table 2.
This method was applied to determine the content of PABA in Bromine Mitag Procaine Injection samples. Two mL samples of Bromine Mitag Procaine Injection were taken and diluted to 10 mL. The diluent,filtered through 0.22 mm membrane filter,was used for the determination with the established chromatographic conditions. Chromatograms are shown in Fig. 3. The measured average concentration of PABA in the standard diluent was 1.17 mg/L,the content in the marketed sample was 5.85 mg/L and RSD was 0.64%. These results showed good repeatability.
|Fig. 3. Chromatograms of blank (a), contrast (b) and marketed sample (c). Chromatographic conditions: ZORBAX ODS column (250 mm × 4.6 mm i.d.); mobile phase, 0.5 mmol/L [BMIm][BF4] solution (pH 3.0)/methanol (40/60, v/v). UV/vis detection, 245 nm; flow rate, 1.0 mL/min, column temperature, 30 ℃; inject volume, 20 mL. Peaks: 1, PABA; 2, MABA; 3, OABA; N, unknown components in the marketed sample.|
This research produced (or generated) a rapid,efficient and reliable method for separation and determination of isomers of amino benzoic acids by reverse phase HPLC using an ionic liquid as the mobile phase additive. The method improved the separation of amino benzoic acids by HPLC. The results indicated that,using ILs as HPLC mobile phase additives for separation of amino benzoic acids,could significantly inhibit chromatographic peak tailing caused by the mobile phase of organic-water without ILs,and the separation of analytes was improved through the interaction between the ionic liquid and analytes. This method could achieve good separation on amino benzoic acids even at a concentration of the [BMIm][BF4] solution at only 0.5 mmol/L,and it lessened the damage to column and equipment correspondingly. The simple,accurate and reliable method can be applied in the analysis of relevant pharmaceutical formulations.
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