Achieving Ultrafast Liquid Chromatography - Pharmaceutical Technology

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Achieving Ultrafast Liquid Chromatography
The authors review methods for reducing analysis time and increasing throughput that are reliable and maintain data integrity.


Pharmaceutical Technology


Carryover. Carryover has gained a great deal of attention in recent years with the widespread adoption and increased sensitivity of current detectors, especially mass spectrometers. As run times get faster, many people try to accelerate them by using advanced injection routines. Taking advantage of these routines is acceptable as long as cross contamination is reduced. For example, with a flow-through injection mechanism (needle-in-the-flow path), one may take the sample loop off line to begin preparing the next sample. On the other hand, if the loop is not sufficiently flushed or cleaned before it is removed, carryover and poor injection relative standard deviations (RSDs) will result.

Reduced carryover is an important factor for UFLC, but combating sample carryover may increase analysis cycle time if additional autosampler needle rinsing is required. Rinsing, cleaning, and flushing takes time, so it is important to choose a design that also minimizes the effects of carryover in other ways.

Gradient. Gradient accuracy and precision also influence system reproducibility. If the gradient controller is not sufficiently precise, the gradient curve varies slightly from run to run. It also may cause distorted peak shapes or resolution fluctuation with closely eluting peak pairs, resulting in poor integration.

Technique. No HPLC–UFLC system compensates for poor technique. As users push the limits of their systems, they must bear chromatography basics in mind. Cutting back on postgradient column equilibration time to shorten the analysis cycle affects system performance. Using a small or inefficient mixer for mobile-phase mixing can result in poor data quality. Small-particle-size columns and tubing entail the need for rigor in mobile phase and sample preparation to prevent clogging. Filtering both the mobile phase and samples is no longer an option, but a requirement. A good quality sample sealing mechanism (e.g., vial septa, microtiter plate covers) is critical to achieving optimum autosampler performance for injection accuracy and carryover prevention. A little time spent reviewing the basics goes a long way toward ensuring the collection of reliable data and results in an HPLC–UFLC system that performs well and reliably. The following examples illustrate these concepts.


Table I: Retention time and peak-area reproducibility of xanthine derivatives.
Table I shows the system reproducibility of the xanthine separation. The retention-time RSD performance demonstrates that the system's pumps deliver a consistent and reproducible gradient. This feature allows the user to create a specific retention-time window in which to look for this particular peak, thus aiding in identification. The peak-area RSD shows that the autosampler provides a reproducible injection and also suggests that carryover is not a problem.


Table II: Retention time and peak-area reproducibility of paraben mixture.
Figure 4 and Table II show a paraben separation completed in less than 1 min. The gradient in this separation is from 5% to 95% B in just over 20 s. Note that it is possible to maintain data integrity while achieving this kind of performance.


Figure 4: Paraben separation completed in ,<1 min. Column: Shimadzu Shim-pack XR-ODS column (50 mmL 3.0mm, 2.2 μm), Mobile phase: A–H2O; B–acetonitrile; B: 5% (0.00 min); B: 95% (0.36 min); B: 95% (0.48 min); B: 5% (0.49 min); stop (0.80 min); Flow rate: 1.2 mL/min; Temperature: 40 C; Detection: UV 254 nm.
Flexibility. Today's HPLC systems are used for many applications, including UFLC, traditional HPLC, QA–QC work, mass-spectrometry front end, multidimensional chromatography, on-line sample cleanup, and setup as column-switching systems for method development. Some of these techniques may require a special system to achieve the optimum performance for the method (e.g., a bioinert system).


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