Superficially porous (Fused-Core, Core-shell) particle technologies have gained acceptance in general high-performance liquid chromatography (HPLC) and ultra-high pressure liquid chromatography (UHPLC) practice over the past several years due to improved efficiency relative to comparably sized fully porous particles. The Fused-Core option has also been shown to be a superior approach toward improving column efficiency as compared to smaller porous particle (sub-2 µm, UHPLC) technologies owing to the lessened backpressure penalties that are paid for the efficiency gains. Ascentis® Express HPLC columns were initially introduced that employed Fused-Core particles with an overall 2.7 µm diameter. These columns provide efficiencies equal to sub-2 µm particles at much lower backpressures as well as superior efficiencies when compared to fully porous 3 µm phases. There are still instances, however, where the backpressure generated by a 2.7 µm particle may preclude their use and there are also situations based on available equipment or regulatory guidance where larger particles are preferred. For these reasons, a new generation of 5 µm Fused-Core columns has been developed.
In this seminar we will provide an introduction to the new line of Ascentis Express 5 µm HPLC columns and explore several scenarios practicing analytical chemists might encounter:
• Desire to develop method on the more efficient 2.7 µm column, but need to be able to transfer to different location or lab
1. Can I easily transfer methods from 2.7 µm to 5 µm?
2. Can I easily transfer methods from 5 µm to 2.7 µm?
• Wish to transfer methods based on fully porous column technologies to Fused-Core
1. Can this be done easily?
2. What gains should I expect? What can be done to optimize the gains?
3. Can I transfer both 3 µm and 5 µm fully porous particle method to Fused-Core?
Core-type particles are competing strongly with small porous particles to improve the speed and resolution of HPLC and UHPLC experiments. The pioneering Ascentis Express column with Fused-Core® 2.7µm particles has exploded in popularity because it operates more ruggedly at much lower pressure than current sub-2µm porous particles, yet delivers the same ultra-high performance. This unique performance has been largely attributed to very narrow particle size distribution. Fused-Core® design advantages have also become popular for LC-MS because Ascentis Express columns surpass performance of columns with 3µm porous particles and operate ruggedly at higher velocities and similar pressures.
With new, narrow-distribution 5µm Fused-Core® particles, the same design advantages can now be realized over traditional 5µm and 3µm porous particles that remain very popular for HPLC columns. An Ascentis Express 5µm column brings 3µm performance and extreme ruggedness at 5µm pressures to your laboratory. Like Ascentis Express 2.7µm particles, the 5µm particles show flatter van Deemter plots than same-size porous particles and allow separation speed to be maximized with minimal loss of resolution. Extremely high plates per pressure are observed. The core-type 5µm design should replace porous 5µm columns in routine HPLC applications with traditional instruments, and should also compete with porous 3µm columns in many LC-MS applications. Performance will be compared to 5µm and 3µm particle columns, and examples of method transfer will be shown. Ascentis Express 5µm will be available in the same phase modifications as the original Ascentis Express 2.7µm ultra-high performance column. Highly stable columns are available in various IDs and lengths up to 25cm.
HPLC columns featuring 2.7 µm Fused-Core (superficially porous) particles with 90 Å pores demonstrate very fast separations of small molecules because of high efficiency and a flat van Deemter plot. These particles rival the efficiency of sub-2 µm totally porous UHPLC particles, but show only about one-half the backpressure. Fused-Core 2.7 µm particles with wider (160 Å) pores have been optimized for the rapid separation of peptides and small proteins. The higher efficiency and lower pressure drop of Fused-Core particles allows preparation of longer columns with very large numbers of theoretical plates. This dramatically increases the peak capacity of the column system, which facilitates qualitative and quantitative HPLC and LC-MS analysis.Read more >
With recent advances in HPLC columns and LC/MS hardware, it is possible to increase the throughput of bioanalytical assays without sacrificing quality. By using fused-core columns, it is possible to decrease the run time from ~4 min to 1 min or less, without the use of UHPLC hardware. High quality methods at high flow rates (1-3 mL/min) using non-ballistic gradients as short as 20 seconds were developed that provide comparable or better performance for accuracy, precision, sensitivity, and specificity than traditional slower LC methods. Data will be presented that show that these assays meet regulatory requirements for bioanalytical work. Limitations in the ultimate speed possible for these assays will also be discussed.Read more >
A comprehensive two-dimensional liquid chromatograph (LC × LC) was constructed from commercially available conventional HPLC equipments. This system utilizes two independently configurable 2nd dimension binary pumping systems to deliver independent flow rates, gradient profiles and mobile phase compositions to dual Fused-Core secondary columns. Very fast gradient separations (30 seconds total cycle time) were achieved at ambient temperature without excessive backpressure and without compromising optimal 1st dimension sampling rates by using superficially porous stationary phases. A practical approach to optimize the various inter-related instrumental parameters will also be presented.Read more >
Increasing case loads and budget and staffing cuts in forensic laboratories continue to motivate the development of higher throughput methods, particularly for confirmatory analysis of regulated intoxicants. In this work, we have focused on the development of rapid LC/MS/MS methods for the determination of nine opiates including two glucuronide metabolites, and 16 benzodiazepines, including two amino- metabolites. Here we aim to analyze both the parent compounds and important polar metabolites in a single analysis. To this end we have compared the retention of the target compounds on two different reversed-phase HPLC stationary phases: a conventional C18 type phase, and a perfluorinated phenyl (PFP or F5) phase built upon the increasingly popular Fused-Core particle morphology. We see that the F5 phase not only generally exhibits higher retention than the C18 type phase, but also exhibits very different selectivity such that the nine opiates can be nearly completely resolved in under four minutes. We find that the mixture of 16 benzodiazepines cannot be completely resolved in a reasonable (i.e., less than 20 min.) time, however we have developed a separation with no more than three overlapping peaks in an analysis time of five minutes.
DEVELOPMENT OF RAPID LC/MS/MS-BASED METHODS FOR CONFIRMATORY ANALYSIS OF OPIATES AND BENZODIAZEPINES
SPENCER BONNERUP, D. CHRISTOPHER HARMES, TOMAS LISKUTIN, JONNA BERRY, AND DWIGHT R. STOLL
Department of Chemistry
Gustavus Adolphus College
800 West College Avenue
St. Peter, MN 56082
The Fused-Core particle technology behind Ascentis Express allows for twice the speed of traditional columns at half of the backpressure of sub 2 micron columns. Ascentis Express columns help you to turn any HPLC into a high powered workhorse like an UHPLC instrumentRead more >