1. Results Initial chromatographic conditions [Figure 2a caption] for the separation of the degradation products of aspirin were problematic due to the long run time and broad peaks. To correct these problems, the effect of acid eluent, pH, and concentration on retention time was studied using mobile phases of sulfuric acid, carbonic acid, perchloric acid, phosphoric acid, and boric acid, a pH range of 3-4, and a concentration range of 0.5 M to 0.05 mM. References [1] K. Ohta, K. Tanaka, P.R. haddad, J. Chromatogr. A. 782 (1997) 33-40. [2] F.E. Blondino, P.R. Byron, J. Pharm. Biomed. Anal. 13 (1995) 111-119. Optimized Separation of Aspirin and its Degradation Products by Ion Exclusion Chromatography Christine L. Kirkpatrick, Fotouh R. Mansour, Neil D. Danielson* Department of Chemistry and Biochemistry Miami University, Oxford, OH, USA *danielnd@muohio.edu 1 Introduction The separation of a mixture of aliphatic and aromatic acids has presented a problem in ion exclusion chromatography (IEC) due to the long retention times of aromatic acids caused by the π-π interaction of these analytes with the aromatic rings of the stationary phase [1]. The degradation of aspirin is a suitable sample to address this problem because its degradation products are acetic acid, an aliphatic acid, and salicylic acid, an aromatic acid. Stability indicating assay methods for aspirin have been of importance in the pharmaceutical industry for quality control of the raw material and of the finished product. Stability indicating methods for aspirin are also important for product development such as a change in packaging material. Methods other than IEC have been employed to study the stability of aspirin, including reversed phase LC [2]. However, these methods did not detect the aliphatic degradation product, acetic acid. Also, the economic, environmental, and simplicity advantages of IEC make this method competitive with previously developed methods. Method Two optimization methods were attempted in this study. The first consisted of the creation of calibration curves to optimize eluent, pH, and concentration of the mobile phase separately. The second method employed Design Expert 8 © StatEase professional optimization software, where concentration, pH, flow rate and injection volume were optimized together. These parameters were optimized for aspirin based on four responses: resolution of system peak from acetic acid, resolution of acetic acid from aspirin, resolution of aspirin from salicylic acid, and run time. To find a starting place, the first run consisted of water as the eluent. As shown in Figure 1, water was too strong of an eluent, as the peaks were overlapped and separation could not be achieved. To correct for this overlap, weaker mobile phases such as dilute acid eluents were used. Conclusion Separation of the degradation products of aspirin has been achieved by ion exclusion chromatography. Due to optimization of mobile phase concentration, pH, flow rate, and injection volume, a run time of 6 minutes was achieved for the separation of the degradation products of aspirin. This decrease in run time for aspirin is significant when compared to reported values of 60 plus minutes. Considering the economic, environmental, and versatility benefits of IEC, and seeing the success of the optimized separation of the degradation products of aspirin, IEC can be considered a competitive method for these types of separations. Figure 3: Optimization study of the effect of flow rate and injection volume on the desirability of the experiment. Design Expert 8© StatEase. Column: Tosohaas TSK-gel SP-5PW column (7.5 mm i.d.× 7.5 cm) packed with the PMA-based strongly acidic cation-exchange resin in the H + -form (particle size: 10 μm). From Figure 3, it was found that the optimal conditions for the separation of the degradation products of aspirin were using 0.35 mM sulfuric acid at pH 3.00, injection volume of 100 μL, and flow rate of 1.00 mL/min. At these optimal conditions, resolutions of system peak from acetic acid, acetic acid from aspirin, and aspirin from salicylic acid were found to be 2.2, 2.0, and 3.3, respectively, and the run time was 7.54 minutes. The optimal separation conditions from these studies, as shown in Figures 2b-d, were found to be a mobile phase of 0.5 mM sulfuric acid at pH 3.77, where a balance was established between having a short run time while preserving baseline resolution. A second optimization study was then performed using professional optimization software, yielding the results shown in Figure 3. Figure 4: Chromatogram of the separation of aspirin at optimal conditions based on Figures 2b-d. Column: Tosohaas TSK-gel SP-5PW column (7.5 mm i.d.× 7.5 cm) packed with the PMA-based strongly acidic cation-exchange resin in the H + -form (particle size: 10 μm). As shown in Figure 4, optimized conditions based on Figures 2b-d led to a chromatogram with a significantly shorter run time, sharper peaks, and better resolution than the initial chromatogram in Figure 2a. While both optimization methods can be deemed successful due to the short run time of the optimized conditions, each method has its strengths and weaknesses. The benefits of using the first optimization method employing calibration curves were that fewer runs were needed to find the optimized conditions, and a slightly shorter overall run time was achieved. However, all the variables had to be analyzed separately. Optimization using StatEase required more runs, but the variables were optimized all together. This is most likely the reason for the variation in optimization conditions between the two methods. Also, optimization with StatEase gave slightly sharper peaks than the first optimization method. Further optimization will be done to complete this work with the professional optimization software. (a) (b) (c) (d) Figure 1: Initial chromatogram. Conditions: water eluent, Flow rate 1.0 mL/min, Injection volume 50 μL. Column: Tosohaas TSK-gel SP-5PW column (7.5 mm i.d.× 7.5 cm) packed with the PMA-based strongly acidic cation-exchange resin in the H + -form (particle size: 10 μm). (a) Figure 2: (a) Initial chromatogram. Conditions: 0.5 mM H 2 SO 4, pH 3.1, Flow rate 1.0 mL/min, Injection volume 50 μL. (b) Effect of acid eluent on retention time. Mobile Phases: (1) sulfuric acid, (2) carbonic acid, (3) perchloric acid, (4) phosphoric acid, and (5) boric acid. Conditions: 0.5mM acid eluent, pH 3.77, Flow rate 1.0 mL/min, Injection volume 50 μL. (c) Effect of pH on retention time. Conditions: 0.5 mM H 2 SO 4, Flow rate 1.0 mL/min, Injection volume 50 μL. (d) Effect of concentration on retention time. Conditions: pH 3.77, Flow rate 1.0 mL/min, Injection volume 50 μL. Separation was achieved for all Figures 2a-d using a Tosohaas TSK-gel SP-5PW column (7.5 mm i.d.× 7.5 cm) packed with the PMA-based strongly acidic cation-exchange resin in the H + -form (particle size: 10 μm).