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How can I achieve shorter cycle times with the Agilent 1200 Series RRLC system?

By Angelika Gratzfeld-Huesgen
Agilent Application Chemist

When you want a flexible system that can run both traditional LC methods and ultrafast RRLC methods, the Agilent 1200 Series Rapid Resolution LC (RRLC) is the system of choice. When you need to maximize sample throughput, you can easily optimize the system for fast LC, and here we discuss some of the ways to increase speed and reduce cycle times.

Selecting a column

One way to achieve shorter cycle times is to choose a short column with good selectivity. The best column dimension depends on whether the detection system is a mass spectrometer (MS) or an ultraviolet (UV) detector. For MS applications, columns with 2.1 mm internal diameter (id) and flow rates up to 1.5 mL/min are good choices because you generally do not need a flow splitter before the MS. For UV detection, 3.0 mm id columns are ideal because you can obtain the highest linear velocities. You can also reach high linear velocities with 4.6 mm id columns, but the maximum flow rate is limited to 5 mL/min.

Figure 1. You can achieve best performance by optimizing the Agilent binary pump SL for columns of different internal diameters. (Click here to see this image larger.)

Figure 2. The setup shown here for alternating column regeneration dramatically decreases the time from injection to injection. (Click here to see this image larger.)

Selecting the optimum pump configuration

When you want to reduce cycle times, it is important to choose the best pump configuration for the analysis. For 4.6 mm and 3.0 mm id columns, you should use the Agilent 1200 Series binary pump SL in its standard-delay-volume configuration (Figure 1). For 2.1 mm id columns, you should use the low-delay-volume configuration.¹ In addition, for 2.1 mm id columns, you should install the low dispersion kit, which provides the lowest extra-column volume. The tubing to the MS should be as short as possible and of small internal diameter, as is delivered with the low dispersion kit. For UV detection, you should also use the short mixer (part number 5067-1565).

Use of alternating column regeneration

You can realize even shorter cycle times by using alternating column regeneration, as shown in Figure 2. With this setup, regeneration of the previously used column takes place during the analysis on the second column, which saves significant time.²

Typically, columns of the same chemistry and the same batch provide a retention time precision that allows you to process data using the same calibration table (Figure 3). You can also achieve stability over several thousand runs if you take precautions against blocking of frits due to particles in the sample or algae or bacteria in the mobile phase.

Figure 3. Comparison of UV chromatograms from an analysis that used alternating column regeneration shows excellent reproducibility across two Agilent columns from the same batch. (Click here to see this image larger.)

Figure 4. Reduction of the delay volume shortens the analysis time and improves the chromatography, especially for the peaks that are eluted first. (Click here to see this image larger.)

Optimizing the autosampler

To increase sample throughput, the autosampler for the Agilent 1200 Series RRLC enables overlapped injections (OI) and/or automatic delay volume reduction (ADVR). The latter means the injection valve is switched out of the flow path after the sample has reached the top of the column. ADVR reduces the delay volume significantly (see Figure 4), and is particularly beneficial at lower flow rates.

Gaining speed while maintaining flexibility

The Agilent 1200 Series RRLC is an adaptable system that allows you to choose between conventional LC and the latest ultrafast RRLC analyses. For fast LC, the system offers a variety of ways to reduce cycle time, so you can choose the specific configuration that best meets your needs.

If you want to learn more about the Agilent 1200 Series RRLC, order the new edition of the Rapid Resolution LC compendium CD. This free CD provides technical literature, a cost-savings calculator, and a method translator to convert conventional LC to RRLC methods.

References

1. Agilent 1200 Series Rapid Resolution LC System Manual, Agilent publication number G1312-90300.
2. “Achieving fastest analyses with the Agilent 1200 Series Rapid Resolution LC system and 2.1-mm id columns,” Agilent publication number 5989-4502EN.