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 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.
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 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 I: Retention time and peak-area reproducibility of xanthine derivatives.
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.
Table II: Retention time and peak-area reproducibility of paraben mixture.
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
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.