Speeding Up Pharmaceutical UHPLC Method Development with an Integrated, Ultrafast, Automated Method Scouting Solution
The Application Notebook
In HPLC method development, screening of various para-meters such as stationary phase, eluents, and temperatures is conducted to find optimal resolution. However, method development can be a time consuming and inefficient process. UHPLC technology can be applied to significantly shorten both the analysis and development times. Here we describe an integrated and ultrafast automated method scouting solution that provides fast and efficient method development processes.
In HPLC method development, screening of various para-meters such as stationary phase, eluents, and temperatures is conducted to find optimal resolution. However, method development can be a time consuming and inefficient process. UHPLC technology can be applied to significantly shorten both the analysis and development times. Here we describe an integrated and ultrafast automated method scouting solution that provides fast and efficient method development processes.
Instrumentation
The UltiMate® 3000 RSLC method scouting system (see Figure 1) consists of a binary gradient pump with a maximum pressure of 800 bar at flow rates of up to 5 mL/min. The autosampler is compatible with well plates and standard sample formats. A powerful 100 Hz diode array detector was used for spectral screening of the test compounds. The thermostatted column compartment has two 6-position, 7-port valves that allow for automatic and intuitive scouting of columns and eluents, and provides industry-leading temperature flexibility. A 10-position, 11-port solvent selection valve was applied to select between various buffers.
Figure 1
Method Scouting Results
The separation of various diuretic compounds by reversed-phase chromatography was conducted on six columns with 10 buffers, two organic modifiers, and at two different temperatures. By reducing column internal diameter, column length, and particle size, and by increasing linear flow rate, the analysis time was shortened by almost a factor of 3 compared to 3 μm particle packed columns (compared to 5 μm particle packed columns, a theoretical speed-up of at least 6 times can be achieved). A total of 240 LC runs were finished in 18 h. The ability to use variables for instrument methods in the Chromeleon® Chromatography Management software significantly reduces the number of methods to be created. The parameter values (e.g., column, temperature) are set directly in the sequence table. The resulting data was processed with Chromeleon Chromatography Management software. Methods that meet predefined criteria (e.g., resolution and peak shape) were automatically selected by a database query. In order to select the best method, the software created a chart that displays the analysis time and minimal resolution as shown in Figure 2a. The best LC method is illustrated in Figure 2b.
Figure 2a, b
Conclusions
The UltiMate RSLC method scouting system and Acclaim RSLC columns are excellent tools for speeding up method development. Chromeleon post processing highlights the optimal methods in seconds, eliminating the need to manually assess all data. This turnkey solution can improve method development productivity by at least a factor of six.
Dionex Corporation
1228 Titan Way, P.O. Box 3603, Sunnyvale, CA 94088-3603
tel. (408)737-0700, fax (408)730-9403
Website: www.dionex.com
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