One of the new stationary phases appears in the Acclaim® column, which was introduced by Xiaodong Liu, Ph.D., group leader for HPLC consumables development, R & D, Dionex (www.dionex.com).
The Dionex Acclaim system scores by being applicable to the simultaneous separation of basic, acidic, and neutral pharmaceuticals, said Dr. Liu. It belongs to the mixed mode class of hydrophobic/ion-exchange materials and, as Dr Liu pointed out, there are various ways of mixing the components.
The Acclaim Mixed-Mode WAX-1 column has “tips” of ion-exchange material on “chains” of reverse-phase material. The selectivity of the column is adjustable by varying the ionic strength, pH, or organic content of the mobile phase so a mixture (1,2,3) that elutes in the order 1,2,3 under one set of conditions can elute in the order 3,2,1 in other conditions, if so required.
The Sigma-Aldrich (www.sigma-aldrich.com/supelco) Supelco® Ascentis® Express series of columns provides an alternative to small-particle HPLC technology, with its associated higher back pressures.
“In recent years, the driving force in HPLC has been for faster method development and analysis,” commented David Bell, Ph.D., manager of applications and technical services at Supelco, introducing the Ascentis Express system, which is based upon Fused-Core™ particle technology.
The particles within the system consist of a 1.7-micrometer diameter solid core surrounded by a 0.5-micrometer porous shell, making up a particle of 2.7-micrometer overall diameter. This structure gives a much shorter diffusion path and a better peak shape compared to fully porous particles on the same diameter, according to the company.
Together with a tight particle size distribution and high packing density, this means that Ascentis Express columns have an efficiency comparable to the sub-2 micrometer particle column and twice that of the 3-micrometer particle column, Dr. Bell said.
Since back pressure is proportional to the inverse square of particle size, the Ascentis Express column generates half the back pressure of the sub-2 micrometer column, which allows the use of longer columns for higher resolution and faster flow rates for higher throughput without compromising efficiency, explained Dr. Bell.
Ascentis Express is capable of resolving D6-benzene (where the hydrogen atoms have been replaced by deuterium or heavy hydrogen) from ordinary benzene which is, Dr. Bell said, “quite a feat.” This was done by coupling together four Ascentis Express columns to create one with dimensions of 55 cm by 4.6 mm.
Using moderate operating conditions of 1mL/min flow rate, 50º C column temperature, and an isocratic mobile-phase composition of acetonitrile/water, efficiencies of greater than 100,000 were measured on all peaks in a test mix of five compounds. Benzene and deuterobenzene were baseline resolved, and the column backpressure was 7,000 psi as measured on a commercial LC system.
Ascentis Express is also good for analyzing peptide fragments and, therefore for protein identification, because it gives more peaks and higher sensitivity. The Ascentis Express columns can be used in most conventional HPLC setups, thereby upgrading them to UPLC status, according to Dr. Bell. Due to the high efficiencies at low back pressures, Ascentis Express can provide high-resolution chromatography that was previously unattainable on commercial LC systems.
“With instruments capable of operating up to 15,000 psi, a quarter of a million plates per column may be possible,” concluded Wayne K. Way, market segment manager HPLC at Supelco. Plates, or theoretical plates, is an HPLC term referring to column performance in terms of separation power.
Hydrophilic interaction liquid chromatography (HILIC) is another separation technology increasing in importance in applications involving polar compounds. Carl Sanchez, research scientist, analytical support and development, R&D, at Phenomenex (www.phenomenex.com), discussed a structured approach to method development and optimization in HILIC. It was shown that in HILIC, a technique mainly applicable to polar compounds, selectivity is significantly different than in reverse phase.