Prominence UFLC has been designed to achieve Ultra-Fast HPLC by utilizing the Prominence hardware series. In this respect, we are successful in demonstrating high speed and separation performance without resorting to high pressure, while providing high reliability and expandability not available with other fast LC systems.
Smaller particle columns such as sub-2m columns generate higher pressures but also produce high separation efficiency. Sub-2m columns often require specialized instruments that can operate under high pressure to demonstrate high separation efficiency. Our Shim-pack XR-ODS column was developed with the intention of creating a new high-speed analysis column at much lower pressure than experienced with sub-2m columns. The Prominence UFLC has been optimized to enable highspeed analysis using the Shim-pack XR-ODS.
Shim-pack XR-ODS for High Speed and High Separation Efficiency
The Shim-pack XR-ODS, which is packed for use in reverse-phase chromatography, has 2.2m diameter particles of totally porous high-purity silica gel with chemically bonded octadecylsilane (ODS) as the base material. Generally, columns using smaller particles provide good separation when using shorter length columns and help to minimize the reduction in column efficiency even if the mobile phase flow rate is high. This makes them well suited to high-speed analysis. However, since column pressure increases inversely proportional to the square of the diameter of the packing material particle, special HPLC instruments that have resistance to high pressure are required.
The specialized instruments that allow use of sub-
2m columns often sacrifice operability, reliability and
application flexibility. The design of the
XR-ODS is based on an in-depth evaluation of a variety
of factors such as separation performance, durability and
column pressure. Use of 2.2 m silica gel-based material
reduces column flow resistance, allowing high-speed
analysis, even with systems comprised of conventional
HPLC hardware. Since the XR-ODS achieves a good
balance between separation efficiency and pressure,
resolution performance is maintained as in a generalpurpose
column (4.6mmi.d. x 150mm, 5m) while
greatly shortening analysis time (Figure 1). Moreover,
column pressure is maintained at 30 MPa or less in
most analytical conditions without using a specialized
system for extremely high pressure. We reported the
development concept and some features and applications
of the Shim-pack XR-ODS columns in our 2006 spring
issue of LC WorldTalk . We hope this article will aid in the
understanding of Shim-pack XR-ODS columns.
See Figure 1
Prominence UFLC responds to increased customer requests for ultra-fast analysis. Based on the existing Prominence components, improvements in flow path pressure (autosampler), high-speed gradient (controller and solvent delivery units), and reduction of peak broadening in flow paths (autosampler and detector cell) have been implemented. The maximum pressure of the SIL-20AHT/20ACHT autosampler has been raised to 35 MPa to correspond with Shim-pack XR-ODS pressure tolerances. Basic specifications, including injection volume accuracy, precision, carryover and durability, are the same as those of the SIL-20A/20AC.
High-throughput analysis requires shortening of the total cycle time, which is defined as the separation, the injection interval and the column equili bration. The SIL-20AHT /20ACHT autosamplers boast the worlds fastest injection (10 sec, 10 L injection time), as well as suppressed carryover achieved by special surface coating of the needle and the improved needle seal. Figure 2 shows the analysis of 7 alkyl phenones, demonstrating ultra-fast gradient analysis with a 32-second cycle time. A fast autosampler and standard features such as automatic purging allow users to shorten the overall analysis time and experience truly fast LC analysis.
See Figure 2
The UFLC modules have many design improvements to consumable parts such as check valves, pistons, and the autosampler rotor seal. These improvements allow UFLC components to operate reliably as a conventional or UFLC system, whereas with other systems currently on the market, the extremely high backpressure may contribute to more frequent replacement of consumable parts.
Steep gradients for performing short cycle time analyses and heat generated by high pressure in the column may produce poor reproducibility of retention time and peak area. Responsive tracking of a quickly changing flow rate is essential in highspeed gradient analysis. The Prominence UFLC features excellent solvent delivery flow performance with its pumping resolution of 3.7 nL using micro stroke technology (10 L/stroke) and superior solvent delivery flow control (response of 0.1 second) to provide excellent retention time repeatability. Moreover, at higher flow rates, columns such as the XR-ODS, with an internal diameter of 2 mm to 3 mm, are most appropriate as they can better handle the increased mobile phase flow rate and minimize wear on instrument components, such as pump pistons and seals, autosampler rotor, etc.
As the columns internal diameter becomes smalle r, it is necessary to decrease the sample injection volume in proportion to the column cross-sectional area (about 2 L to 5 L injection volume with 3 mm internal diameter column). With the Prominence UFLC, the SIL- 20AHT/20ACHT uses a high-performance measuring pump and enhanced air tightness of flow lines to achieve peak area repeatability (RSD) of 0.3% or less, even with injections of 2 L to 5 L.
Carryover has gained a great deal of attention in recent years, especially with todays sensitive triple quad mass spectrometers. For ultra-fast LC, reduced carryover is a very important factor, but combating sample carryover may increase analysis cycle time if additional autosampler needle rinsing is required. The Prominence UFLC SIL- 20AHT/20ACHT autosamplers successfully reduce carryover using the same proven techniques of the SIL- 20A/AC models. These include inhibiting the adsorption of samples to needle and seal surfaces, and an option for two different rinse solvents. Based on Shimadzu test parameters, carryover for the SIL-20AHT/10ACHT is typically 0.005% or less.
Figure 3 shows a chromatogram and reproducibility of retention time and peak area of 7 alkyl phenones under steep gradient conditions and a 1-minute run time. Prominence UFLC provided excellent reproducibility on all compounds with a 2l injection. When using instruments designed for high-pressure operation, it is conceivable that there may be problems with the durability of maintenance parts, ease of maintenance and quality of analysis. In the case of a high-pressure autosampler, the longevity of the high-pressure valve seal is shortened. Prominence UFLC uses Shimadzus own proprietary high-precision 6-port valve, which achieves 0.3% RSD or less area repeatability in 100,000 cycle endurance tests (based on Shimadzu test parameters) as shown in Table 1 . As shown, Prominence UFL C conclusively results in significantly faster analysis with high reliability.
Prominence UFLC can be used for a broad range of applications, including conventional HPLC, or set up as a column switching system or a semi-preparative system. The wide flow rate range (100nL-10mL/min) and high delivery resolution of the pump (3.7nL), combined with the reproducibility of the autosampler, offer superior performance for both UFLC and conventional LC, so usage is not restricted to UFLC analyses.
Furthermore, Prominence UFLC excels as a front end for MS and MS/MS. Figure 4 shows high-speed, accurate MSn analysis of Opioid Antagonist and its metabolites utilizing the LCMS-IT-TOF (Shimadzu). With the auto MSn function, the IT-TOF with Prominence UFLC produced MS/MS data with high accuracy in a 2-minute cycle time. High-speed/high-accurate MSn offers identification of target compounds in the field of impurity analysis, biomarker search and metabolite profiling.
Prominence UFLC with XR-ODS columns enables users to shorten analysis time drastically and easily without extremely high pressure, while maintaining the high separation efficiency of conventional columns and system performance features such as reproducibility, carryover and durability. Prominence UFLC is applicable for a wide range of customers as a highly reliable, and flexible, fast LC system that does not require specialized components.
Q: Is h igh pressure required for performing fast HPLC?
A: The short answer is no. High pressure is not the goal; it is simply a consequence of using small particle (sub-2m) HPLC columns. The use of these types of columns subjects hardware to greater stress due to increased resistance to flow and, subsequently, dramatically increases the back pressure, potentially sacrificing basic performance due to design constraints brought on by the need for a high pressure tolerance.
Q: Does high pressure influence consumable parts and maintenance?
A: Pushing any system to the extremes of its performance envelope, but particularly specialized systems running at higher pressures, increases stress on instrument components such as pump pistons and seals and the autosampler rotor and stator. This results in shorter consumable life and less durability even when using such specialized (more expensive) components.
Q: How does high pressure influence peak shape?
A: Under high-pressure conditions, frictional heating occurs in the column, causing a temperature gradient within the columns cross section. This means the mass transfer between the mobile phase and the packing material will be different across that area, possibly resulting in peak distortion. In such situations, any benefits to using high pressure are lost.
Q: What is the goal?
A: The goal of Fast LC is increased sample throughput while maintaining data quality, meaning how many samples can be analyzed per day, per hour, or even per minute. In order to achieve such high throughput, not only must the run time of a single analysis be shortened, but the total cycle time of the injection sequence and run time needs to be optimized. Specialized inject-ahead routines or overlapping injections can shorten cycle times but often compromise an autosamplers carryover performance and should be avoided. Is waiting 45 seconds for the next injection of your 30-second analysis high throughput?
Q: W hat is the solution?
A: The ultimate answer lies in understanding what constitutes the chromatographic separation and optimizing those parameters while maintaining the reliability and precision demanded in the laboratory. Ideally, it would include HPLC hardware that, when coupled with an appropriate column packing, can perform precisely and reliably under a variety of conditions, like the Prominence Series.
Q: Why is Prominence the solution?
A: Simply statedExcellent design,
engineering and quality. The
Prominence Series includes the
proven micro-stroke solvent delivery
mechanism for rapid, precise and
reproducible gradient formation. The
Prominence autosampler is capable
ort injection cycle times and
excellent carryover performance
without sacrificing precision. Stable
and accurate temperature control is
critical to produce good data under
UFLC conditions and can be found
in the Prominence forced-air ovens,
large enough to allow for flexible
column configurations like column
switching. Fast response time and
high sensitivity coupled with a
temperature-controlled, low delay
volume flow cell are incorporated in
the Prominence UV and PDA detectors.
In other words, a good HPLC system
should be engineered and capable of
Fast HPLC from the start; a specialty
system with extreme pressure
tolerances, and the design sacrifices
incorporated because of it, should not
be required. Regardless, there should
be no compromises in the quality of
the datanamely reproducibility, peak
shape, and quantitative accuracy.