Complex LC Method Development Using Method Development System and LC Simulation Software Koudi Zhu , 1 Matthias Pursch, 2 Binghe Gu 3 1 Analytical Technology Center, 1897 Building, The Dow Chemical Company, Midland, MI 48667; 2 Dow Stade Produkt. GmbH&Co OHG, Analytical Technologies, Stade, BN, Germany 21677; 3 Analytical Sciences, 1897 Building, The Dow Chemical Company, Midland, MI 48667 The development of a liquid chromatographic (LC) method for complex samples is a time-consuming, sometimes trial-and error process. Newly developed analytical tools, such as Agilent LC method development system and LC simulation software from Advanced Chemistry Development (ACD/Labs), have the potential to significantly improve the productivity of the “slow” method development process. This study explored the capabilities of the method development system and the simulation software by developing a universal LC method for resolution of a total of 26 peaks in raw material and in process samples. With the capability of the multiple solvent selection valve and multiple column compartment of the method development system, four analytical columns, mobile phases with different acid additives, three different gradient conditions and two different column temperatures were screened. The method scouting software in the system has the capability of flushing the system, equilibrating column automatically when column and other LC conditions change. It greatly increased the flexibility of LC column and mobile phase screening. The data generated were transferred to a computer on which ACD/LC simulator software was installed. The peaks were identified according to individual standard retention time match and UV spectra. The simulation software transferred the chromatography data into a table which contains the retention times and peak widths of all peaks of interest. The LC simulation software then built a model to obtain resolution maps. Several method conditions were generated that have potential to separate all the peaks of interest. In addition, manual optimization can be performed to attain tailor-made separation by using the resolution map. The manually optimized LC condition provided by ACD/LC Simulator software was tested. The experimental run matched the predicted run very well and separated all 26 peaks of interest in 14 minutes. Abstract Conclusions References Acknowledgments 1290 Infinity LC/MSD Method Development System ACD/Labs’ LC Simulation Software Model Compound Selection Column Screening Data Collection Mobile Phase Selection Data Transfer ACD/LC Simulator Mode and Method Suitability Table LC Simulation for Method Optimization Dow Chemical Company Analytical Technology Center Leadership Dow Chemical Company Technology Renewal & Development Program Grant Von Wald for ACD Software discussion JD Darland for providing samples LC method development system from Agilent Technologies is an excellent tool for automation of column and mobile phase screening. ACD/LC Simulator software is very helpful in optimizing LC methods for complex samples that contain multiple critical band pairs. A combined use of LC method development system and ACD/LC Simulator software was successfully demonstrated for the development of a universal LC method for in-process and starting material analysis. Snyder gradient flow rate scaling principle: Keep t G x F be constant. By changing flow rate to 1.5 mL/min, baseline resolution of all 26 peaks was achieved in 14 min. • Challenges: AP new process impurities (AP dimers) out of spec; production yield sacrificed for quality; data provided on raw material COA insufficient for process improvement purpose; resolution for critical pair not rugged in the product release method • Solution: Develop a universal LC method that can separate all 26 peaks of interest Choose to use phosphate buffer to stabilize mobile phase pH in an attempt to attain a rugged method used in QC lab A: 5% ACN / 95% 20 mM phosphate buffer, pH at 2.12 B: 70% ACN / 30% 20 mM phosphate buffer, pH at 2.12 1. M Pursch, R Edam, etc.“Rapid Method Development for Industrial LC Separations Using Automated Screening of Stationary Phases and Solvents”, LC-GC 2010, Vol. 28 (4), pg 44–50 2. www.agilent.com 3. www.acdlabs.com/lcsimulator Multiple solvent selection valve & Multiple column compartment 1. Column Screening 2. Mobile Phase Screening 3. Gradient Screening 4. Temperature Screening Method Scouting Software 1. System Flushing 2. Column Equilibration 3. Column Storage 4. Auto Sequence Generation Experimental chromatogram using optimized method by ACD/LC Simulator Predicted chromatogram by ACD/LC Simulator Chromatogram overlay above shows excellent retention time match of most peak of interests in experimental chromatogram using the optimized method and chromatogram predicted by ACD/LC Simulator. Accurate Peak identification & integration are essential in generating successful LC methods 3-D resolution map Visualized chromatogram shows separation when method condition changes without running samples Selected