31
Table 1: Original and recalculated gradient profi les for the three methods in Figure 2. Calculations were performed using the ACE Translation Tool [2]. Note real experimentally determined column volumes were used in calculations [3].
Improving sample throughput for gradient LC methods
Improving sample throughput for isocratic LC methods
Figure 1 gives a simple overview of how sample throughput for an isocratic LC method can be increased using two options. The original separation, using a 150 x 4.6, 5 µm column has a run time of 35 minutes, with a back pressure of 66 bar. By translating the method to a UHPLC column (50 x 3.0 mm, 1.7 µm) on a UHPLC instrument, the run time is reduced to 4 minutes with a moderate pressure of 558 bar. This equates to a >8 times increase in sample throughput and a >88% reduction in the runtime. However, if UHPLC instrumentation is not available,
sample throughput could still be more than doubled by using the existing HPLC instrument with a shorter HPLC column and smaller particle size (100 x 3.0 mm, 3 µm). In this case, sample throughput is >2 times better than the original method with run time reduced by 60% to 14 minutes at a reasonable 215 bar. From a solvent perspective, analysing 100 samples (excluding equilibration times, cleaning, shutdown methods etc.) would require 3,500 mL with the original method, 500 mL for the UHPLC method and 994 mL for the modifi ed rapid HPLC option.
Figure 2A uses a 150 x 4.6 mm, 5 µm column and shows a gradient analysis of non-steroidal anti-infl ammatory drugs. The post-gradient re-equilibration time from the gradient table is 20 minutes (or ~13 column volumes). It is possible to translate the gradient method to a new UHPLC format, or a modifi ed rapid HPLC format to understand the impact on sample throughput. Using similar principles as in Figure 1, along with a software translation tool [2], it is possible to quantitatively translate the gradient method to the two new column formats. For gradient methods, it is also necessary to scale the gradient profi le and correct for differences in instrument dwell volume to ensure the same gradient separation and resolution is obtained with the new column formats. Table 1 shows the original and recalculated gradient times for the separation on each column format.
The original HPLC separation in Figure 2, using a 150 x 4.6 mm, 5 µm column, has a run time of 34 minutes, but a total cycle time of 54 minutes due to gradient re-equilibration, with PMAX
of 64 bar. Translating this to the UHPLC format gives a 3.6 minute run time
Figure 2: Increasing sample throughput of an existing gradient HPLC method (A) using UHPLC (B) and modifi ed rapid HPLC (C) options. Mobile phase: A: 0.1% formic acid (aq), B: 0.1% formic acid in MeCN, Gradient: 35-65%B, Injection volume: (A) 5 µL, (B) 0.7 µL, (C) 1.4 µL, Detection: UV (254 nm), Sample: 1. Sulindac, 2. Bendrofl umethiazide, 3. Ketoprofen, 4. Ibuprofen, 5. Diclofenac, 6. Indomethacin, 7. Mefenamic acid, 8. Meclofenamic acid. Note: post-gradient equilibration times are not shown but are detailed in Table 1.
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