At the end of March, Lab Manager held a “Product Showcase” webinar on “New Chemistries and Improvements in Liquid Chromatography (LC) Columns” featuring a panel of experts from leading providers of LC columns and equipment. It attracted a large international audience from various fields with diverse backgrounds and varying levels of expertise and provided them with a unique opportunity to interact with the panelists in real time and obtain their insights and advice. All panelists gave a brief presentation to outline their companies’ advances in LC column technology, and attendees posed questions following the talks. The talks included topics such as what type of LC columns can best fit specific applications and workflows, what considerations should take precedence when selecting high-performance liquid chromatography (HPLC) columns, and recent trends in and the long-term outlook for LC columns. Below are some questions that were posed to the panel by the attendees and the panelists’ feedback. The webinar panelists were:
- Philip J. Koerner, Ph.D., Senior Technical Manager, Phenomenex
- Jason Link, Ph.D., Product Manager HPLC, Agilent Technologies
- Xiaodong Liu, Ph.D., R&D Manager, Dionex Corporation
- Dafydd Milton, Ph.D., LC Column Product Manager, Thermo Scientific
- Moderator: Tanuja Koppal, Ph.D.
Q: What determines the use of a newer type of LC column, such as the superficially porous or the sub-2 micron on a traditional HPLC versus running a sample on a UHPLC?
Koerner: Anyone looking to reduce analysis times, reduce solvent usage, and improve chromatographic performance should be looking at using core-shell columns on their HPLC and ultra high-performance liquid chromatography (UHPLC) systems. The back pressures generated by sub-2 micron columns will limit their use on traditional HPLC systems with operating pressure limits ≤400 bar. However, 2.6 micron core-shell particles deliver ultra high efficiencies without generating as much pressure.
Link: In general, one needs to consider what the separation goals are. For example, is a wide array of selectivity options necessary, or is scalability to larger particle sizes important? Superficially, porous particles can offer nearly the same performance as sub 2-μm columns under some conditions, but a limited selection of stationary phases is available, and scalability to larger particle sizes might not be possible in all cases. However, the larger frit in superficially porous columns makes them a better choice for dirtier sample analyses. The benefit of upgrading to a UHPLC instrument is that it offers the flexibility of being able to run any type of column, so a user is not limited to one column technology or the other and can use whichever technology the particular separation requires.
Liu: The need for higher throughput and high column efficiency are the two main considerations when choosing an LC column.
Milton: The main consideration would be the operating pressure. Longer columns packed with sub-2 micron particles will generate back pressures that are above the operating range of conventional HPLC systems. Columns packed with superficially porous particles generate lower back pressures more amenable to conventional systems. Outside of pressure considerations, one of the other important benefits of UHPLC instrumentation is the reduction of system volumes. This goes a long way to limiting dispersion that would have a greater impact when using the shorter, narrower, newer generation of columns.
Q: What are some of the key factors that affect column performance when working with the new column chemistries?
Koerner: When using core-shell columns on conventional HPLC, system dead volume contributions from connecting tubing and detector flow cell volume, as well as detector scan rates, need to be addressed in order to attain the full performance benefits that core-shell columns can provide.
Link: Most users will look toward new column chemistries and selectivities when the standard C18 phases do not provide enough resolution for their separation. Typically, one will need to look at the pH of their mobilephase buffers to make sure they are compatible with the new column being tried, as well as the temperature (if performing separations at elevated temperature). In addition, the choice of the organic modifier can have a big impact. For example, with phenyl columns, the pi-pi interactions can be overwhelming when acetonitrile is used.
Liu: Column performance is affected by several factors, including the desired selectivity, the ruggedness and durability of the column packing, and batch-to-batch reproducibility. When working with the newer chemistries, it is most important to read the column manuals and follow the instructions given to achieve optimum results.
Q: What are some of the improvements taking place in the preparative columns market?
Link: The prep columns market doesn’t show nearly as much in advancement as we see in analytical columns. Mainly, users are interested in scalability and loadability as well as column mechanical stability, so in those areas there tend to be focused improvements. With the recent resurgence of supercritical fluid chromatography (SFC), we are seeing more prep columns targeted for chiral and SFC separations.
Q: What are some of the innovations taking place in guard columns?
Koerner: Disposable guard cartridges have been available for some time and have largely supplanted traditional guard columns and help to extend analytical column lifetimes. However, the impact of added dead volume can be detrimental to the performance of the ultra highefficiency columns. Newer guard cartridge designs must be engineered so that they add zero or very little dead volume, and we can expect to see these being made available in the future.
Link: Many times, the use of guard columns will compromise the efficiency gains of high-performance columns, so often it is better to prepare cleaner samples (by using pre-filtration, for example). That said, I think some of the key innovations in guard columns are in minimizing the effect the guard has on the separation and in maintaining stability. Guard columns aren’t necessarily needed in all cases, but when they are, the goal is to have a minimal impact on the separation while extending the life of the column being used.
Q: How can we increase column life and prevent column contamination, especially when using the new LC columns?
Koerner: Additional attention needs to be paid to mobile phase and sample preparation to ensure that both are particulate-free in order to minimize impact on column lifetimes for the ultra high-efficiency columns based on sub-2 micron and core-shell particles. Simple measures, such as using syringe filters to filter your sample prior to injection, can go a long way toward preventing column contamination.
Link: Simple procedures to increase column life include ensuring the use of high-quality solvents and sample filtration and overall HPLC system maintenance. Often, the reason a column fails or plugs may be particulates resulting from the system or from solvents. So the use of in-line filters in the system sample filtration can often prevent this from happening. To avoid cross contamination, proper flushing of the system (and ensuring the system is flushed prior to removing one column) is key. Many instrument manufacturers offer guidance in the form of injector programs, where steps are made to minimize carryover and cross contamination. This tends to be more important when using high-sensitivity techniques such as liquid chromatography-mass spectrometry (LC-MS).
Liu: Good sample preparation practice is essential to increased column life. In addition, minimizing system dead volume and using dedicated columns will help to extend the life of your columns.
Milton: To extend the lifetime and performance of columns, they can be protected from contamination by sample and solvent debris and interferences from the sample matrix. One of the most cost-effective and efficient ways of trapping these unwanted system components is the use of filters or packed guard columns.
Q: What are some of the key issues around sample prep, carryover, and cleanup, and how can you minimize those problems?
Link: Some key tips around sample prep include proper wetting of the surface when using solid-phase extraction (SPE), as well as using the optimum flow rate with an SPE cartridge. And any time a user is filtering a sample, it’s important to make sure the filter material is compatible with the sample solvent. And, of course, it’s important to make sure the sample solvent is compatible with the HPLC. Being aware of the sample characteristics is important, as compounds that are strongly retained in reversed-phase (RP) HPLC can cause carryover problems. These problems can be mitigated with the use of injector programming features, such as those found on Agilent LC autosamplers.
Milton: For a busy laboratory, sample preparation can be time consuming. Additionally, for high-sensitivity LC-MS applications, effective removal of sample components and matrix interferences that may cause ion suppression is crucial. On-line sample preparation using turbulent flow can be used to quickly eliminate matrix interferences while capturing analytes of interest.
Q: What types of analytes and applications are now being better served or made possible by the new column chemistries?
Koerner: Every application can benefit from the use of core-shell particle columns, since the same chromatographic performance can be achieved with the use of shorter columns, resulting in faster separations (e.g., shorter analysis times) and reduced solvent usage. For chromatographers analyzing very complex sample mixtures, the ultra high efficiencies delivered by these columns can enable increased resolution for analytes that have previously been difficult to separate and identify and accurately quantitate.
Link: Many manufacturers are developing applications focused in the area of clinical types of analyses. These are areas where total workflow solutions are important, coupling not only sample prep with the column but also the LC and MS instruments to show a total solution for clinical analyses.
Liu: Reversed-phase and ion-exchange mixed-mode columns serve well for applications that are challenging to RP or hydrophilic interaction liquid chromatography (HILIC) columns. Their main benefits include adjustable selectivity for greater flexibility in method development, selectivity complementary to RP columns (as well as complementary to themselves under different conditions), and elimination of ion-pairing agents for simplified mobile phase and better mass spectrometry compatibility.
Milton: Retention of polar and hydrophilic compounds with higher sensitivity is now made possible, as these can be retained using techniques such as HILIC or columns packed with porous graphitic carbon without the need for derivitization or the addition of ion-pair reagents to the mobile phase.
Q: What types of changes in LC columns can the users expect to see in the near future?
Koerner: Expect to see additional bonded phase chemistries introduced on core-shell particles that will offer additional stationary phase selectivities to chromatographers.
Link: I think, in general, customers will start to see more and more manufacturers offer superficially porous columns. Just as with the vast number of columns available in sub 2-μm, the same trend will occur with superficially porous particles. Along the same lines, now that the underlying technology has been around for a few years, we’ll start to see more chemistries on these particles giving users more choices in their method development.
Liu: Superficially porous LC columns will find more usage, and hence such columns with more diverse column chemistries will be available from an increased number of manufacturers. Mixed-mode columns will find uses in the applications that are challenging or impossible for conventional reversed-phase HPLC columns.
Milton: Additional novel phases bonded to superficially porous and sub 2-μm particles are becoming more widely available.