Traditionally, the HPLC series of meetings has attracted most of the global leaders in separation science. Thus, each meeting is a tempting venue for new product introductions. This year, at HPLC 2011, held in Budapest, Hungary, June 19–21, 2011, Agilent (Santa Clara, CA) and Waters (Milford, MA) both introduced two premium-performance instruments to take advantage of unexpected advances in column technology.
As discussed in the technical review, “The World of Separation Science: Advanced Column Technology Forces Next-Generation Instrumentation at HPLC 2011,” at www.americanlaboratory.com, new core/shell column technology provides column efficiency that requires reengineering of the instruments to be compatible. However, this advance appears to be practical for a very limited niche.
Band broadening, peak width, and dispersion in chromatography arise from many sources. The contribution of each source is given in Eq. (1):
σtot = (σcol2 + σex2)0.5 (1)
where σtot is the total band broadening, σcol is the band broadening of the column, and σex is the extracolumn band broadening. σex can be further expanded into a laundry list of components that include tubing, fittings, detector cell, and detector electronics.
The peculiar nature of this equation is that the largest terms quickly dominate and mask the smaller contributions. Thus, to characterize the columns with an expected σcol of 3 μL, instruments must have a smaller σex.
Prof. Georges Guiochon (University of Tennessee, Knoxville) compared several instruments, including native versions of the Waters ACQUITY® and Agilent 1290 Infinity®. Extracolumn band broadening (σex) was too large for both. Reducing the σex to this range requires reducing the length and diameter of interconnecting tubing. Both new instruments use 80-μm (0.003-in.) i.d. tubing for transfer lines. Throughports on fittings need to have exactly the same diameter and be coaxial. Conventional UV detector flow cells (1 × 10 mm = 8 μL) are too large, even without a heat exchanger.
The Agilent 1290 Ultra-Low Dispersion (ULD) replaces the 120-μm capillaries of the Infinity 1290 with 80-μm tubes from sampler to detector. This reduces σex from 8.6 μL to 3.0 μL. For low k′ peaks, the improvement in resolution and peak height can be significant: a 60% increase in plate count. Resolution and peak height increase by about 20%. The price is a fourfold increase in system backpressure (without column), up to 1500 psi at 1 mL/min for water. Thus, instrument pressure can be comparable to the backpressure of the column. Flow-induced heating of the mobile phase may be an issue as well.
Agilent also introduced the new High Dynamic Range (HDR) flow cell for the Infinity 1260 and 1290 DAD (diode array detector). This uses total internal reflection in a noncoated fused-silica fiber to provide a 60-mm pathlength. Increasing the pathlength by six times increases the signal six times. Agilent previously offered a 60-mm-long cell, but the cell volume was 78 μL. The new cell has a 4-μL dispersion volume and is useful with “sub-2” columns. What can you expect? Plug the numbers into Eq. (1), and the predicted peak width is σtot = (32 + 32 + 42)0.5 = 5.8 μL. This is still much less than a native 1290.
Another advantage of the new HDR is that it also has a 2.7-mm flow cell for strongly absorbing peaks. Splitting the flow between the two cells (2.7 mm and 60 mm long) in parallel provides two signals that can be combined using a transfer function to produce a chromatogram with a linear dynamic range of 106 and a practical upper absorbance of 8 AU/cm. This greatly simplifies trace analysis, even in high-purity samples.
The new ACQUITY I Class from Waters also reduced the cell volume to 500 nL from 8 μL, both with a conventional 10-mm pathlength. Two sample flow paths are offered for injection. The fixed-loop option offers the lowest dispersion and delay volume. The flow-through needle design provides more flexibility and automation. Since the needle is continuously washed during the run, carryover is greatly reduced. This was confirmed during development of an assay of fluticasone propionate in the low-pg/mL range. No detectable carryover was found for a blank run following a 5-ng/mL standard. The pressure rating of the I Class has been increased to 18,000 psi. To support this, special silica particles have been developed to withstand the higher pressure. The pore size of the particle is 100 Å. Surface chemistry includes C18, C8, pentafluorophenyl (PFP), and cyano (CN) phases. Waters also pointed out that MS detection is much more compatible with narrow, low-volume peaks.
Quant Technologies LLC (Blaine, MN) introduced the NQAD HPLC detector, which uses condensation nucleation to extend detection sensitivity of evaporative light scattering detectors (ELSDs). Both start with nebulization of the column effluent. With evaporative light scattering detection, the analyte dries and the aerosol scatters light. With the NQAD, the aerosol is cooled in the presence of a gas vapor (usually water) just below the dewpoint. Since they are larger, analyte aerosol particles are preferential nucleation sites for condensation, producing a light scattering fog that is detected photometrically. Therefore, with the NQAD, the condensation reaction amplifies the signal that one would get from ELSD. Typical lower detection limits are subnanogram. Dynamic range is 104 to 105, with the linear portion of 103 to 104. Quant offers detectors optimized for ultrahigh-performance liquid chromatography (UHPLC) and supercritical fluid chromatography (SFC).
PostNova Analytics (Landsberg, Germany) was the only vendor of field flow fractionation (FFF) instruments present. The company featured the CF2000 Centrifugal FFF, which is most useful for analytical-scale assay of 7-nm molecules up to 40-μm particles in nearly all solvents. Run times are typically about 1 hr. The engineers at PostNova have developed a finely balanced rotating channel cartridge complete with a low-dispersion inlet and outlet junction between the rotating separation chamber and stationary pumps, injector, and detector.
For preparative applications, PostNova introduced the SF200 G Gravitational Splitt FFF, which is used for fractionating particles in the range of 1–300 μm. Particles are detected with the PN3000 XPT, which utilizes a charge-coupled device (CCD) to measure the size of individual particles. In addition to reporting the population distribution, the XPT also can measure particle shape and color intensity.
Several firms introduced columns using core/shell technology with particle size larger than 2.5 μm. These are designed to extend the separation speed and resolution of non-UHPLC instruments. For example, Thermo Fisher Scientific (Waltham, MA) presented the Accucore™ line of core/shell columns built on a 2.6-μm particle with a 1.6-μm solid core. The company reports that its Core Enhanced Technology™ provides reduced plate heights of 2.0 at linear velocity as fast as 9 mm/sec. Columns are generally 2.1 mm i.d. × 100 mm long. Surface chemistries include C-18, aQ, RP-MS, phenyl-hexyl, PFP, and hydrophilic interaction liquid chromatography (HILIC). Stationary phase selectivity is presented with spider plots covering nine parameters. Column lifetimes were evaluated for isocratic (>4000 injections) and gradient (>6000 injections) elution. It will be interesting to see if Core Enhanced Technology can be extended to sub-2-μm particles.
Credits and future
With 36 editions, the HPLC series is well recognized as the premier meeting focused on liquid-phase separations, particularly on the laboratory scale. The scientific committee should be congratulated for sharing the spotlight with less utilized techniques such as high-performance capillary electrophoresis (HPCE), SFC, and FFF. After all, these are complementary technologies that help applications where HPLC may not work as well. Meeting Chair Prof. Attila Felinger (University of Pécs, Hungary) deserves special thanks for managing the details that made HPLC 2011 an outstanding success.
The next two meetings will be held October 8–11, 2011 in Dalian, China, and June 16–21, 2012 in Anaheim, CA. Please monitor www.CASSS.org for the latest news.
Dr. Stevenson is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: firstname.lastname@example.org.