Dal Nogare Session Highlights Approaches for Difficult Separations, Improved Efficiency, Better Sustainability, and Career Success

The Dal Nogare Award is presented each year at Pittcon. The award was established in honor of Dr. Stephen Dal Nogare, one of the founders of The Chromatography Forum of the Delaware Valley (CFDV) and a well-known gas chromatographer whose book on the subject was one of the best known to teach the science of GC to a whole generation of separation scientists. Dal Nogare passed away in 1968 and in 1972 the award was established to recognize the work of important separation scientists, based on their contributions to the fundamental understanding of the chromatographic process. Since its founding, the Dal Nogare Award has been given to more than 50 individuals who have distinguished themselves in the field of separation science.

Because the Covid-19 pandemic of 2020–2023 disrupted yearly face-to-face events, the Dal Nogare Award Symposium at Pittcon 2023 included both the 2021 and 2022 winners. The 2021 awardee, recently retired Prof. Apryll Stalcup of Dublin City University in Ireland, was given a “virtual” honor last year but presented her award speech at Pittcon 2023. The 2022 winner, Prof. Luis A. Colón of the State University of New York at Buffalo was presented with his award session in person at Pittcon 2023, by session chair Mary Ellen McNally of FMC Corporation, who is the Dal Nogare chair for the CFDV.

Colón: Using HPLC and SFC to Separate Photoswitchable Isomeric Compounds

Colón led off the session thanking his colleagues and students for their help along the way to receiving the Dal Nogare Award and briefly discussing those areas of chromatography and surface chemistry where his major contributions have been made. In his award talk, “Separation of Diarylethene-based Photoswitchable Isomeric Compounds by HPLC and Supercritical Fluid Chromatography,” Colón discussed his work analyzing diarlyethenes (DAEs), which can switch between two states in response to light. DAE-based photoswitches have good thermal stability and fatigue resistance and are used in diverse areas including drug delivery, chemical sensing, data storage and processing, environmental cleanup, switchable catalysis, and smart functional materials. The analysis of these photoactive materials will facilitate their further development for particular applications.

DAEs can be found as isomers with their rings either open or closed, giving four possible conformations, only one of which is photoactive. In solution, the isomers can switch between conformations but in solid state the conformation is fixed. In his lecture, Colón reported the isomeric separation of a DAE-based photoswitchable compound using high performance liquid chromatography (HPLC) or supercritical-fluid chromatography (SFC), or both. SFC has the advantage of having faster analysis times while maintaining sufficient baseline resolution for the separated compounds and consuming much less organic solvent.

Preparative HPLC was initially used to purify providing fractionated samples to study the isomers individually. A total amount of 13 mg of an isomer of interest was fractionated from a solution of 0.4 mg/mL of the isomeric mixture. However, SFC did allow reduction in the amount of solvent required as compared to preparative LC. Future work on photoswitchable compounds will be performed using SFC, because the mobile phase is predominantly carbon dioxide with a small amount of methanol, which gives shorter run times, good resolution, and a significantly lower consumptions of organic solvent.

The separated isomers were characterized by UV-visible spectroscopy using spectra obtained using a diode array detector and using mass spectrometry to confirm the isomeric nature of the compounds.

Stalcup: Lessons from a Life Enriched by Separations

Stalcup’s lecture, entitled “Lessons from a Life Enriched by Separations,” presented a biographical sketch of her life and the lessons learned, first as a “military brat” and then as an educated scientist whose career path from graduate school to an academic environment led her to cross many disciplines and to make many contributions to separation science as she went.

Students in the audience should pay heed to lessons she learned while finding her way through her career. One reoccurring theme to her life’s journey was “don’t be afraid to move.” She went on to describe the research areas and the many scientific colleagues where she picked up knowledge that she could take to her next opportunity to learn. Her first job in a commercial contract laboratory gave her insight into solving nasty environmental problems using a wide variety of separation and spectroscopic techniques. Lessons learned there were “don’t assume your boss knows everything” and “one must learn when to leave the party.”

In her work at Georgetown University and the National Institute of Standards and Technology (NIST), she learned “how to interpret and tell the story of ambiguous data” and “never miss your opportunity to expand your tool box.” While working in the area of chiral separations, she learned “how to mentor and run a research group” which gave her a head start when she moved to the University of Hawaii Manoa where she became proficient in capillary electrophoresis (CE) applied to chiral separations. There she learned that "having good students in important to success.”

In her 1996 move to the University of Cincinnati, Stalcup ventured into the use of ionic liquids in CE and the application of multimodal stationary phases such as a chiral phases combined with reversed-phase liquid chromatography (RPLC).

Although she is now retired, Stalcup believes that preparative LC still has a strong future, as does the use of separation science in process analytical technology. And she still believes that one should “not be afraid to move,” as demonstrated by her experience delving into many different separation science disciplines in varied contexts and working with many different influential colleagues, through which she learned a lot and had great success.

Kennedy: Pushing Limits of Liquid Chromatography Speed and Efficiency

The laboratory of Prof. Robert Kennedy of the University of Michigan has been exploring the limits of liquid chromatography (LC) in terms of speed of analysis and efficiency. In today’s world, especially in the area of proteomics or lipidomics, where one can run into tens of thousands of compounds in typical sample mixtures, high resolution separations are increasingly desirable. The increase in peak capacity can result in improved peak identity. Mass spectrometry is a powerful technique to help resolve analytical information, but ultrahigh-pressure LC (UHPLC) can increase peak capacity by switching to from traditional 3.5-μm particles to 1.7-μm particles, especially using longer columns that require higher pressure. Such a decrease in particle size can result in an increase in peak capacity. For example, going from a 3.5-μm to 1.7-μm particles results in an increase in peak capacity from 75 to 250.

Pioneering work in the reduction of particle size with great increase in pressure was carried out by the Jorgenson group at the University of North Carolina in the early 2000s (1). To operate at such high pressure, special pumps, gradient delivery systems, special fittings, and special column packing techniques are required. Kennedy’s laboratory started out at pressures up to 35,000 psi. Using 1.7-μm particles, sonication during slurry packing and up to 100 cm columns enabled a peak capacity of over 1000 to be achieved for a single-dimension LC analysis. Using 1.1-μm particles from Waters Corporation enabled faster runs. For example, they demonstrated a peak capacity of 350 in 40 min using 1.1-μm particles; but to achieve the same peak capacity using 1.7-μm particles required 120 min. For higher peak capacities for lipids, they employed two-dimensional LC using capillary columns, combining hydrophilic interaction LC (HILIC) and reversed-phase LC, and achieved peak capacities of 1900. Exploration of higher pressures revealed that improved column fittings would be required to use pressures greater than 35,000 psi.

One area for high-throughput chromatography that needs to be addressed is decreasing the overall injection cycle time. Most autosamplers have a cycle time of 15–30 s, which is too slow for achieving true high-throughput analysis. Using a 96-well plate combined with a segmented flow approach, a throughput of 1 s per sample was achieved, corresponding to a little more than 1.5 min for a 96-well plate. Despite this fast cycle time, carryover from sample-to-sample was still very small. The next step in throughput studies will be to switch to capillary columns (0.3-mm i.d) where 5 s cycle times have already been demonstrated. That work is still in progress.

Olesik: Improving Performance and Sustainability Using Carbon Dioxide as a Solvent in LC

Prof. Susan Olesik of The Ohio State University presented an interesting lecture entitled “Improved Liquid Chromatography and Mass Spectrometry Using Enhanced Fluidity Liquid Solvents.”

Enhanced-fluidity liquid chromatography (EFLC) involves the addition of liquefied carbon dioxide to conventional liquid mobile phases. The addition of liquid carbon dioxide enhances diffusivity and decreases viscosity while maintaining mixture polarity, typically resulting in reduced time of analysis. In her lecture, Olesik described how EFLC can improve separations of proteins and peptides under a range of retention mechanisms including hydrophobic interaction-, ion exchange- and reversed phase-chromatography. In addition to improving the time of analysis and using these separation mechanisms, the value of these solvents for also enhancing mass spectrometric analysis was illustrated. She found that CO₂ acts as an enhancing agent to improve the relative ionization efficiency of all ions to give a better signal and more fragmentation. This study was conducted through an ion internal energy measurement to determine a relative ionization efficiency. The presence of CO₂ especially sharpens the chromatographic bands and decreases pressure drop across the column, allowing for the use of longer columns for enhanced resolution.

Olesik also presented the HPLC environmental assessment tool (HPLC-EAT), through which she evaluated the method greenness score for various solvents, including a variety of alcohols as well as acetonitrile and water. Laboratories across the world are seeking to improve environmental sustainability, and as part of that, chemists are moving toward conducting an overall environmental evaluation of chromatographic methods as they are being developed. Many environmental assessment tools for analytical methods have been reported in the literature, but one has yet to be universally accepted as the best tool. However, it is worth noting that when EFLC is employed, the “green” properties of CO2 are an additional benefit—on top of the method performance gains that can be achieved with this technique.

Weber: Electroosmotic Perfusion for Sampling in the Brain

Prof. Steve Weber of the University of Pittsburgh rounded out the Dal Nogare award symposium with his presentation on “electroosmotic perfusion for sampling in the brain.” Deviating from the other lectures, his presentation was not an evaluation of a chromatographic system per se. Instead, he presented work in which he and his team have been treating brain tissue as a slab gel and conducting electrophoresis to measure mobilities and zeta potentials; “Why not do this?” he asked, given that electroosmotic flow is available in brain tissue. He described his experiments as measurements that were relatively fast, at a minute or less in the tissue, although frequently 5 to 10 min were needed to reach a steady state once the probe was placed into the brain itself. The size of the probe had to be considered; if it were too big, and it disrupted the brain, function could be lost. Current devices have small (90 μm outside diameter) probes for source and sink with a conical terminus (like a pencil) to minimize tissue trauma upon insertion.

It was fascinating to hear in the talk that a former MD–PhD student turned neurosurgeon had studied matrix-assisted laser desorption/ionization–mass spectrometry (MALDI-MS) to learn how to effectively push fluids into the brain after surgery. Weber’s research also investigated the push-pull of a fluid passed through tissue via a fluorescent microscope. The initial concentration of the substrate was measured before it entered the tissue, and the resultant product concentration was then determined after the fluid exited. Again, MALDI-MS was used to understand the composition of the exit fluid. Simulations have provided insight into conditions to use for analysis, specifically to determine the rate of degradation of peptides in the extracellular space.

Reference

1. Jarkovich A. D.; Mellors S.; and Jorgenson, J. W. “The Use of Micrometer-Sized Particles in Ultrahigh Pressure Liquid Chromatography,” LCGC N. Amer. 2003, 21, 600–610.