Increase sample throughput by using 2D-based ACQUITY UPLC system combined with parallel column regeneration technology

Dan Root and Pete Claise

<br> target system using ACQUITY UPLC ^® technology-based 2D and binding parallel column regeneration techniques to increase sample throughput.

Background <br>A typical gradient LC analysis involves the following general steps:
â–  Sampling and injection â–  Gradient separation â–  Regeneration (flushing column)
â–  Rebalance (turn the column back to initial conditions)

These sequential operations pass the sample and gradient through a system and column of a specific capacity. The time required to perform these steps is related to the flow rate set by the analysis. Minimize total analysis time by increasing flow rate, using shorter columns (reducing volume), and reducing re-equilibration time. However, the adjustable range of these parameters is limited, especially the latter two parameters, which may affect chromatographic performance when they reach a certain level. Parallel column regeneration provides another way to reduce overall analysis time

The lab is always faced with the challenge of analyzing more samples in less time, and now there is finally a tool that can easily increase sample throughput while ensuring high quality chromatographic results – the ACQUITY UPLC system based on 2D technology.

In parallel column regeneration techniques, sample analysis is alternated between two identical columns with the same flow path. Samples are injected, separated, and regenerated on one column, while another column is rebalanced to allow more samples to be analyzed in a given time. The advantage of this method in terms of time is shown in Figure 1.

Solution <br>The AQUITY UPLC system based on 2D technology consists of two binary solvent managers (BSM), one with a flow-through design of the sample manager and the other with two six-way two-position valves. Column manager. When configuring parallel column regeneration, the BSMs of the two pumps must be the same. The piping connection diagram for this configuration is shown in Figure 2. In this configuration, the alpha pump runs the analytical gradient and the beta pump regenerates the column.

UPLC bioanalytical methods for plasma benzodiazepines alprazolam were analyzed using standard mode UPLC and compared to the parallel column regeneration mode of the ACQUITY UPLC system based on 2D technology. The separation conditions are optimized to effectively eliminate matrix effects associated with plasma protein precipitation in the target analyte in the shortest possible run time. The same column with the same batch number was used in the experiment. The MRM results showed that the chromatographic results obtained under various conditions were similar, as shown in Figure 3a.

The cycle time between the 5.5 min injections required for standard mode operation is easily reduced to 4.1 min without any adjustments to the instrument method. Extrapolation to 24 h for comparison, using this method in parallel column mode increased the amount of sample analysis by 30%, as shown in Figure 3b. The actually increased flux in this method is the result of overlapping the method equilibration time with the next sample injection and analysis. However, the amount of time saved will depend on the degree of overlap possible in the analytical method.

Summary <br>The 2D-based ACQUITY UPLC system combined with parallel column regeneration technology provides a simple tool for increasing sample throughput while ensuring high quality chromatographic results. In this way, performance improvements can be achieved without any changes to the chromatographic parameters of the analytical method. The 2D-based ACQUITY UPLC system meets a wide range of application needs with flexible configurations to meet the challenges of science and business.

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