One must consider not only the stability of the analyte, but that of the packing material as well. Much progress on this front
has been made in recent years, resulting in longer column life and more selectivity. As long as the packing material is stable
under the elevated temperature, this technique is quite effective for getting higher resolution and higher speed without making
many changes to existing equipment.
In successful elevated-temperature HPLC, the column environment is critical because a temperature gradient in the column can
distort the peak shape. A forced-air column oven is required to support this application. These ovens are powerful enough
to eliminate the need for a column preheater, which would increase the delay volume of the system and negate many advantages
gained from elevated temperatures. These preheaters or loops of tubing are necessary for typical block-heating-type ovens.
Demands for faster analysis times will multiply as researchers attempt to increase sample throughput and maintain data quality.
Once the separation is optimized for the highest possible speed and resolution, it is necessary to consider the rest of the
HPLC system and its contribution to the total analysis time. Engineers are developing HPLC equipment to work in conjunction
with high-speed columns with the goal of faster analysis in mind.
The first part of this article dealt with columns, and many examples of fast analyses are available in the literature. Figure
2 shows a comparison between a typical isocratic separation performed on a 5-B5m particle and a mid-size (2.2-μm) particle.
Figure 2 shows that the run time was shortened by a factor of 10 with no loss in resolution. Mid-size silica gel-based material
reduces column-flow resistance compared with that of a sub-2 μm particle column, thereby allowing high-speed analysis with
conventional HPLC systems. Since these HPLC columns achieve a good balance between separation efficiency and pressure, resolution
performance is maintained as in a general-purpose column (4.6 mmi.d. × 150 mm, 5 μm), and analysis time is greatly shortened.
Figure 2: Comparison of Shim-pack VP-ODS with conventional 5-μm particle column and the Shim-pack XR-ODS with 2.2-μm particles.
Chromatographic conditions: Mobile phase: water–acetonitrile (40/60, v/v); XR-ODS: Flow rate: 2.0 mL/min, Temperature: 60
°C; VP-ODS: 1.0 mL/min, 50 °C; Detection: absorbance at 210 nm; Peaks: fluorene (1), phenanthrene (2), anthracene (3), fluoranthene
(4), pyrene (5), chrysene (6), benzo(b)fluoranthene (7), benzo(k)fluoranthene (8), benzo(g,h,i)perylene (9). A Shimadzu Prominence
high-performance liquid chromatography system was used.
High-throughput analysis requires reducing the total cycle time, which is defined as the separation, injection interval,
and column equilibration. New autosamplers provide some of the fastest injection cycles (10 s per 10-μL injection) and allow
users to successfully suppress carryover by careful evaluation of all contact materials in the flow path. When seeking short
run times for an assay, the system speed becomes even more critical. Figure 3 shows a separation of different xanthine derivatives
completed in 1.2 min using a conventional Shimadzu Prominence HPLC unit optimized for UFLC by reducing the tubing diameter
and eliminating as much system-delay volume as possible. At such run times, autosampler speed begins to play an important
Figure 3: Ultrafast liquid chromatography (UFLC) chromatogram of xanthine derivatives (10 μg/mL): xanthine (1), theobromine
(2), theophylline (3), caffeine (4). Instrument: Shimadzu Prominence UFLC. Column: Shim-pack XR-ODS (3.0 mmI.D. × 50 mmL,
2.2 μm); Mobile Phase: 20 mM (sodium) phosphate buffer (pH 2.6) for A and acetonitrile for B (70/30), B concentration 10%
initial to 85% at 1.2 min.
Although speed is important, one also must consider data integrity. Many discussions about UFLC focus on data reproducibility.
These discussions arise because the specialized systems designed to tolerate the high pressures that sub-2-μm particle columns
endure may exhibit decreased system reproducibility, whether resulting from premature consumable wear or concessions made
in the design to accommodate the required pressure tolerance. A few possible causes of poor reproducibility merit examination.
The peak widths seen in fast HPLC are extremely narrow. Narrow peak widths require a detector capable of sampling at a speed
that ensures enough points to achieve proper integration. If the sampling rate is too slow, one may miss the apex of the peak
or get sporadic noise in the baseline that contributes to poor peak integration. Many available systems are capable of this
sort of sampling if the user sets them up properly.