Alliance 2695 Separations Module: Optimization and Performance with 4.6 mm i.d. Intelligent Speed (IS) Columns

This application brief focuses on optimization and performance of the Alliance 2695 Separations Module for use with IS Columns.

The Waters Intelligent Speed (IS) line of columns have a 20 mm packed bed and optimized hardware that can be run at higher flow rates and lower backpressures without sacrificing resolution, reducing run times by up to 90%. This technical note focuses on optimization and performance of the Alliance 2695 Separations Module for use with IS Columns. A series of fast gradients were run at different flow rates to determine the effect on the reproducibility of retention times and peak areas. Peak capacities for the columns at the different flow rates were also calculated. Additional information on Waters instruments with IS Columns can be found in Technical Note 720000722EN titled: Waters 2996 Photodiode Array Detector: Optimization for Intelligent Speed Columns.

Excellent run to run reproducibility achieved for fast gradients with no modifications to instrument configuration

A series of gradients varying from 0.6 to 4.0 minutes going from 0 to 50% ACN at flow rates varying from 1.0 to 4.0 mL/minute were run. Figure 1 shows the retention time reproducibility that was achieved for six replicate injections under each gradient condition. Excellent retention time reproducibility was achieved for a wide range of fast, steep gradients. Only at 4 mL/min with 1 minute and faster gradients did the retention time reproducibility exceed 0.5 %RSD. Figure 2 shows the peak area reproducibility for the same conditions. All of the gradients resulted in %RSD values for the peak area that were well below instrument specification (<0.5 %RSD), indicating no compromise in system performance when using fast, steep gradients.

Higher flow rates produce the best peak capacities

Peak capacity is a function of the peak width, which is in turn a function of the analyte’s linear velocity, and band broadening effects. According to the van Deemter equation, as the flow rate increases, there will reach a point where band broadening effects are minimized and the narrowest peaks will be achieved. For IS 4.6 mm i.d. columns, this point is between 3 and 4 mL/min, under most conditions. Table 1 lists the peak capacities achieved for various gradients. The optimal peak capacity for each gradient is listed in bold.

Shorter run and re-equilibration times

IS Columns allow for much faster separations, Figure 3, increasing sample throughput and decreasing solvent consumption per analysis. Additionally, the higher flow rates and shorter columns allow for much faster post gradient re-equilibration times. Recommended reequilibration times are 3 times system volume and 5 times column volume. For a standard 4.6 x 100 mm column on the Alliance 2695 Separations Module at 1 mL/min, re-equilibration time is 10.3 minutes. For an IS Column, 4.6 x 20 mm at 3 mL/min, re-equilibration time is only 1.2 minutes, resulting in much faster injection to injection cycle times.

The Alliance 2695 Separations Module delivers excellent retention time and area reproducibility for fast gradient separations (0 to 50% ACN over 0.6 to 4.0 minutes) without instrument modifications. The best peak capacities for the 4.6 mm i.d. IS Columns are achieved between 3 and 4 mL/minute. Run times and the reequilibration times are greatly reduced with IS Columns (re-equilibration times reduced 8.6 times as compared to 100 mm columns), allowing for increased sample throughput and increased productivity.