Narrow the Options
Phase-optimized liquid chromatography (POPLC™), developed by Bischoff Chromatography (www.bischoffchrom.com), offers “a completely new approach” to liquid chromatographic separation optimization, according to Stefan Lamotte, Ph.D., vision manager for columns and stationary phase.
In this method, “the mobile phase remains constant,” he says, and the stationary phase is optimized. “This is completely opposite to the way optimization had traditionally been done, where the stationary phase remains constant and the optimization of the mobile phase is the focus of interest.”
The method is based on the PRISMA model (developed by S. Nyiredy of the Research Institute for Medicinal Plants, Hungary) for optimizing the mobile-phase composition in thin layer chromatography. With the help of the model, users can create any combination of phases by combining multiple and varying parts of phases A, B, and C. In his example, Dr. Lamotte may use three parts of C18, three parts phenyl, and one part polar-embedded C18 for a separation that cannot be done by using one of the stationary phases on their own. Varying the segments yields different results. In total, Dr. Lamotte says, there are about 800 different stationary phases available on the market today, so “it is difficult for users to find the right column for their separations.”
Bischoff’s POPLC software streamlines the process by allowing researchers to collect the retention times for all analytes on five different stationary phases that are orthogonal in their selectivity and enter those retention times, column length, and void time in the software. The software then calculates all variations that can be done with those five stationary phases, showing the best resolution set and the optimal resolution set, identifying column length and void time, for example. “The number of combinations can be very high,” Dr. Lamotte says. In an example with five different stationary phases of maximum column length of 250 mm that can be split in segments as short as 10 mm, the shortest segments had 142,500 possible combinations.
When using POPLC to separate 33 compounds in a municipal waste water sample using LC/MS/MS, the separation took 20 minutes and had a 5% deviation between the prediction and the actual measurement, according to Dr. Lamotte. “In comparison, the former gradient method required 45 minutes.”
In most cases, gradient elution is not needed, thus lowering separation costs. There is a constant background under isocratic conditions, which increases the use of detection technologies that cannot be applied with gradients (like RI or conductivity), improves ionization in MS detection, and allows the use of UV wavelengths less than 200 nm. Additionally, the column length is adaptable, and much of the mobile phase can be recycled.