Your GC–MS Future Today

Modern Trends in Mixed Mode Chromatography

Ultra-Sensitive and Rapid Analysis of Per- and Ployfluoroakyl Substances in Water

The Lifetime Achievement and Emerging Leader in Chromatography Awards

GOS Determination in Foods Using HPAEC–PAD

Improving on AOAC 2001.02

Identifying Markers of Quality in Curry Powder Using GC×GC–TOF-MS

Michael J. Hennessy Jr. is a man on a mission. The president and CEO of MJH Life Sciences™ was recently included in MM&M’s second annual 40 Under 40 list. The award celebrates inspiring young talent in various areas of pharmaceutical and healthcare marketing who are poised to lead the industry.

As a third-generation member of the family-run MJH Life Sciences, healthcare has always been part of Hennessy Jr.’s life, but he hasn’t used his position to coast on the tailwinds provided to him. Instead, he has fundamentally reinvented the largest privately held, independent, and full-service medical media company in North America by innovating and investing in key growth areas.

Hennessy Jr. received his BBA in marketing from Loyola University Maryland and began his career as a national account manager at MJH Associates in 2010. From there, he took on several sales leadership roles, including the day-to-day management of OncLive®, Specialty Pharmacy Times®, and the Healthcare Specialty Group. In September 2015, he was elevated to president of the company. As president, Hennessy Jr. was responsible for identifying new business opportunities and driving the overall growth of the organization. Shortly after MJH Associates was rebranded as MJH Life Sciences in 2019, he was promoted to president and chief executive officer.

Since the beginning of his career, Hennessy Jr. has demonstrated his vision for creating new and engaging ways to serve healthcare professionals. As senior vice president of the Oncology Specialty Group, he relaunched OncLive.com, propelling the brand to new heights with a digital and video-first approach. He led the launch of a range of video offerings, such as the 

 video programs, that provided the Oncology audience more engaging content options. He then applied this successful model to other brands in the portfolio.

As president, Hennessy Jr. took the initiative to innovate the company’s events offerings, focusing on case-based learning, roundtables, regional seminar series, and award programs. These events built large audiences and gained credibility with industry leaders, further enhancing the MJH content creation model.

After overseeing sustained, organic growth, Hennessy Jr. further strengthened MJH by acquiring UBM Life Sciences Group in January 2019. The acquisition included the addition of seven new offices and more than 220 associates. Hennessy Jr. led the integration of the UBM assets, streamlining the company and focusing on reinvigorating the new brands to deliver on MJH’s vision of helping healthcare professionals improve patient care.

Most recently, Hennessy Jr. employed his visionary leadership to guide MJH through the COVID-19 pandemic. He leveraged the company’s state-of-the-art studios to quickly pivot live events to virtual ones without missing a beat. As a result, the company produced a record number of events that included many new, innovative offerings. In June, MJH launched 

, a first-of-its-kind news channel for healthcare professionals. He then introduced the MJH Life Sciences 

, a partnership with healthcare thought leaders devoted to providing ongoing insight around the pandemic and patient care. MJH has steadily grown under Hennessy Jr.’s leadership to 60 brands, producing 975 events, and reaching 7.3 million unique visitors per month online and 2 million via print.

Most of Hennessy Jr.’s investment has been focused on growth areas in the healthcare industry. He’s created a platform to deliver unparalleled content to audiences in oncology, infectious disease, managed care, and pharmaceutical manufacturing, to name a few, and he continues to add new content where he envisions further growth. For example, the company is launching 

Hennessy Jr. has been agile in focusing on the modern ways audiences consume content, especially since the pandemic began. While continuing to publish MJH category-leading print magazines, his leadership has embraced a digital-first approach focused on well-designed, user-centric websites; video content and video series; podcasts; on-demand content and education; and even digital broadcast. He also led the creation of a state-of-the-art in-house digital studio.

The company’s use of leading-edge marketing approaches has also been a priority. Hennessy Jr. led the implementation of enterprise data and technology platforms that enable MJH’s businesses to drive even greater user-centric media practices. He’s embraced and enabled data-driven programming for MJH and its partners­­—from dynamic web and digital content delivery to automated, omnichannel journey creation to dashboards and analytics.

Beyond MJH, Hennessy Jr. has contributed to the industry at large by fostering and creating personal and content-based relationships with world-renowned key opinion leaders in the medical field and at leading universities, medical centers, hospitals, medical associations, and community cancer centers through MJH’s Strategic Alliance Partnership (SAP) program. The program provides its partners and doctors with a national reach and visibility, utilizing the breadth of MJH’s medical communications platform to showcase cutting-edge initiatives, content, research, and thought leadership that ultimately benefit patients and their families.

This past year, Hennessy Jr. capitalized on an opportunity to grow the business by elevating the SAP program by collaborating with health insurance companies such as Highmark Health and Oscar Health to expand its footprint in the managed care marketplace. Additionally, when forming the MJH Life Sciences COVID-19 Coalition advisory board, he leveraged his existing relationships with several top thought leaders across a variety of key specialties, including Dr. Angela Rasmussen, Dr. Saskia Popescu, and Dr. Paul E. Sax, to help generate the most accurate, up-to-the-minute information on the pandemic’s ever-evolving impact on healthcare professionals and the patients they treat.

Throughout the pandemic, Hennessy Jr. has donated and supported several organizations, including the American Heart Association, NJ Pandemic Relief Fund, Princeton Medical Healthcare Heroes, Make-A-Wish NJ, National Medical Fellowships, Susan G. Komen Foundation, Feeding America, among many others. He also has volunteered his time at the RISE: Greater Goods Thrift Store in Hightstown, New Jersey; HomeFront in Ewing, New Jersey; and the Trenton Area Soup Kitchen (TASK).

Working alongside his father, brother, and cousins in the organization certainly qualifies Hennessy Jr. as a family man. Outside of the office, he loves spending quality time with his wife, Rachel, and two children, MJ and Maggie. He is a fitness aficionado who enjoys running and CrossFit. He also enjoys watching sports—specifically New York Yankees baseball. Relaxing to county music and enjoying the beauty of the Jersey Shore with friends and family is also time he cherishes.

Hennessy Jr. may have been born into the MJH Life Sciences business, but he’s reinvented it in his own image. And while he’s positioned the company for longstanding future growth, don’t expect him to rest on his laurels. He’s no doubt already thinking of ways to innovate the healthcare media landscape even further.

Articles

Michael J. Hennessy Jr., president and CEO of MJH Life Sciences™, parent company to LCGC, was named to MM&M’s second annual 40 Under 40 list.

MJH Life Sciences President and CEO Michael J. Hennessy Jr. Recognized as MM&M 40 Under 40 Honoree

This blog is a collaboration between LCGC and the American Chemical Society Analytical Division Subdivision on Chromatography and Separations Chemistry.

For argument sake, I will forward two 80/20 observations that apply to chromatography:

80% of published applications are for research and development (R&D) studies, and 20% are for routine applications

80% of all chromatographs sold are for routine work, and 20% go to the R&D labs.

That is, of course, not surprising, but it means that we, as chromatographers, often ignore issues that are critical in routine, industrial settings that should see more of our light. So, think about it: Most chromatographs are tasked on day one with a specific analysis and, 20 years later, their final analysis will be the same as the first.

Deploying a chromatograph in a quality control (QC) or process setting is critical to many complex streams in that it provides detailed information on the composition of a huge array of mixtures and provides the means of integrating compositional control strategies in the plant. Day-to-day and instrument-to-instrument variability will frustrate data analysis. Computer-enabled technology can be applied to mitigate the impact of aging instruments and instrument differences which, in turn, lead to a more robust, lower-maintenance world for industrial chromatographs. The key issue is to control retention time drift.

Because peak retention on the column has some variability, this shifting increases the likelihood of misidentifying a peak and, ultimately, risks misreporting a component’s concentration. Chromatographic retention time drift from run to run is always present, but usually is not a significant source of variability. Over time, however, any retention drift amplifies the risk of an error in peak identification, which can lead, in turn, to mistakes in peak tables or in evaluating the overall distribution. Software control of retention time drift is a generic solution to this problem and addressing retention time variation simplifies ownership of a chromatograph by making the data consistent from one month to the next, even one instrument to the next. So, let’s look at software’s role in automating the handling of peak retention times as drawn from the world of chemometrics.

The literature is filled with references, but I restrict my list to two oldies, one drawn from over 50 years ago, and one from more than 20 years ago. In 1965, Kováts described how you can use peaks that are easily identified as markers to calculate relative retention times of neighboring peaks (1). In the late 1990s, groups worked to address the problem of retention time variability by borrowing a multivariate technology that had been applied to voice recognition; one example is Correlation Optimized Warping (COW) described by Nielsen (2). Software, both commercial and freeware, has been available for these approaches since the early 2000s. My team has employed both techniques, separately and in combination, for two decades or so, and has used alignment technology to manage a variety of routine and complex applications, including clinical analysis for the Centers for Disease Control (CDC), systems for economic fraud investigations for the U.S. Food and Drug Administration (FDA), and process hydrocarbon evaluations in refineries and chemical plants.

Figure 1 shows an overlay of the same sample run many times in a three-year period. Because the retention times of peaks move, the traditional way of handling this situation is to rerun the standard and accept new time positions for the peaks. This approach gives a very short-term context and, by forcing frequent recalibrations, lowers the number of samples that can be processed.Hands-on recalibration makes the system more unreliable on-line, where it is harder to attend to these changes. So, we often relegate this type of analysis to a supervised laboratory rather than placing it in a less-supervised position near the sampling point, where the feedback would be timely.

Crude oil chromatograms run between 2014 and 2016.

But what if we could rely on retention times not changing? This is an example of where chemometric alignment technology should be used. For software alignment to work, one chromatogram must be previously chosen to be the alignment standard; the algorithm then adjusts the peak positions in all the other traces to match that standard as closely as possible. Results of this process are seen in Figure 2. After alignment (here using COW) and comparing to Figure 1, we can easily see that consistent peak retention time is achieved.

The result of chemometric alignment on the 2014 chromatograms.

COW-based alignment does not require the alignment standard to be run close to the analysis date, nor does it even need to be run on the same chromatograph. In practice, alignment keeps the peaks in place for a significantly longer time and reduces the work necessary to keep the system calibrated.

The idea is very powerful—that a small amount of software can be added to a chromatographic data system and have that addition open a true plug-and-play capability for the instrument and application. Ostensibly, a company could keep a cold spare chromatograph in inventory. When a process or laboratory unit goes down for any reason, the storage unit would be placed on-line and have its data be completely comparable to all previous runs on its very first injection, possibly without ever performing a traditional calibration. Alignment can also apply this procedure to the historical chromatographic data assembly, resulting in a consistent database ready to be mined.

Of course, this conclusion holds for situations where the column and the method conditions are similar. But if this is the case, the ability to perform more complex chromatographic evaluations in a fully automated manner is achieved.

Nielsen, N-P. V., J. M. Carstensen, and J. Smedsgaard, 

Brian Rohrback is the President and CEO of Infometrix, the dominant independent supplier of chemometrics technology to analytical instrument companies, process analyzer suppliers, and their customers. In industry, he has held positions as a research scientist managing the chromatography group, as an exploration geologist, and as an exploration manager for Europe, Africa, and the Middle East. He has served on the board of directors of several instrument companies and consults for pharmaceutical companies, oil companies, and service organizations. Rohrback holds a B.S. in chemistry (Harvey Mudd College), a PhD in analytical chemistry/geochemistry (UCLA), and an MBA (University of Washington). His publications cover topics in petroleum exploration, chemical plant optimization, clinical and pharmaceutical diagnostics, informatics, pattern recognition, and multivariate analysis. He can be reached at 

 and the American Chemical Society Analytical Division Subdivision on Chromatography and Separations Chemistry (ACS AD SCSC). The goals of the subdivision include

promoting chromatography and separations chemistry

organizing and sponsoring symposia on topics of interest to separations chemists

developing activities to promote the growth of separations science

increasing the professional status and the contacts between separations scientists.

For more information about the subdivision, or to get involved, please visit 

https://acsanalytical.org/subdivisions/separations/

Everyone who is reading this column is interested in chromatography, and I want to shine a light on the industrial side of the application space. 

The LCGC Blog: Controlling Retention Time Drift in Industrial Chromatography

The ALIGN high-flow multi gas generator from Parker-Hannifin is designed as a compact unit providing curtain, source, and exhaust gas to all SCIEX mass spectrometers. According to the company, the generator is a plug-and-play partner for high-end liquid chromatography–mass spectrometry instruments. Parker-Hannifin Corporation, Haverhill, MA 

The ALIGN high-flow multi gas generator from Parker-Hannifin is designed as a compact unit providing curtain, source, and exhaust gas to all SCIEX mass spectrometers. 

Gas generator

LECO’s ChromaTOF Tile data analysis software is designed for GCxGC data analysis. According to the company, the software provides an industry-first date comparison tool that identifies statistically significant differences between classes of samples, and uses the Synovec tile-based Fisher ratio analysis to partition the data into sets of regions (tiles), and compare regionalized date. LECO Corporation, St. Joseph, MI 

LECO’s ChromaTOF Tile data analysis software is designed for GCxGC data analysis. 

Data analysis software

Microlute CP from Porvair Sciences is designed for reproducibility of analyte extraction and recovery from biological, environmental, and chemical samples. According to the company, a hybrid structure enhances the flow-through of samples to maximize interactions between analytes and the solid phase. Porvair Sciences, Wrexham, UK 

https://www.microplates.com/microlute-cp/

Microlute CP from Porvair Sciences is designed for reproducibility of analyte extraction and recovery from biological, environmental, and chemical samples. 

Solid-phase extraction microplates

Tony Taylor is Group Technical Director of Crawford Scientific Group and CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LCâMS and GCâMS for materials characterization, especially in the field of extractables and leachables analysis.

Before we get on to answering this question, for which I will tell you there is no single correct answer, we should take a look at the anatomy of a column and its manufacture.­­­­

As most readers will probably know, the column “walls” are made from silica tubing, typically coated in a strengthening outer coat (outside wall only) of polyimide, which ensures the silica doesn’t break upon flexing. The inner wall of the silica tubing is coated with the stationary phase, which in most cases will be a cross-linked polymer, and sometimes this will be chemically attached (bonded) to the inner wall of the silica tubing. A typical gas chromatography (GC) column cross section is shown in Figure 1.

Typical cross section of a wall coated open tubular (WCOT) capillary GC column.

The silica tubing surface is populated with siloxane bridges and silanol species, and the preparation of this surface prior to stationary-phase coating is critical. The siloxane species (Si-O-Si) are strained and can easily adsorb water from the atmosphere or from reagents used in column preparation. This needs to be very carefully controlled. Controlling the amount, and crucially the “type,” of surface hydroxyl features (-OH) from either silanol (Si-OH) or adsorbed water is critical in determining the level of chemical activity, bleed, and stability of the resulting bonded GC column. Therefore, decades of development have gone into optimizing the pre-treatment and deactivation steps that manufacturers employ prior to the application of the stationary phase. As mentioned above, the quality of reagents and the exclusion of water and other impurities such as residual metal ions, are vitally important when pre-treating and deactivating the bare silica inner surface. Surface treatments such as leaching, acid washing, and the application of the deactivation reagent to block surface silanol species are typically different between manufacturers and will ultimately dictate the quality of the column, especially with respect to the peak shape obtained from polar and ionogenic analytes. The nature of the deactivation agent will also differ depending upon the stationary phase chemistry (primarily the polarity of the stationary phase to be applied) and is designed to be more “wettable” to the stationary-phase chemistry.

Most modern stationary phases are manufactured via a static coating method whereby a mixture of oligomers and polymeric initiators of the desired stationary-phase polymer are filled into the column in solution under an oxygen-free environment. The type and concentration of the oligomers in solution dictate the ultimate stationary-phase chemistry and film thickness. The inlet end of the column is then sealed, and a vacuum is applied to the outlet end, while the column is held at a constant temperature. This causes the solvent to evaporate from outlet to inlet at a controlled rate which deposits the oligomeric species in a homogenous film along the whole column length. The column can then be heated, or catalysts introduced, which causes the deposited oligomeric species to polymerize in the presence of the initiators forming a polymeric film of uniform thickness on the inner wall of the column. Using this method, the polymer crosslinks not only to itself but with surface species on the silica inner wall. These columns are said to be “immobilized,” and are more stable, showing inherently lower levels of stationary-phase bleed. It should be noted that the polymerization process can also be initiated in solution prior to the solvent evaporation step, however, these stationary phases are non-immobilized (or “coated”) and therefore show lower inherent stability.

Any residual oligomers can then be solvent rinsed from the column and the residual rinsing solvent is removed from the column by slowly heating the column in an inert atmosphere.

The specific reagents, conditions, and methods used are closely guarded secrets of manufacturers, and very little is published in the literature or in patents about the specific reagents and methods used.

So now that we know a little more about the column manufacturing process, let’s look at what happens at the end of a column’s lifetime, specifically by defining what might constitute column “death.” From an analytical perspective, the results obtained from analyses or system-suitability tests will probably show loss of efficiency (peak broadening), loss of resolution, or a subtle change in selectivity, a degradation in peak shape (fronting, tailing, or peak splitting), or an inability to distinguish low analyte concentrations from noise or bleed (loss of analytical sensitivity). Of course, these symptoms may be combined, and at some point, we will need to consider replacing the column. No matter how much we trim the front of the column, we just can’t achieve acceptable chromatography or quantitative specification requirements.

So why do we get to this position, how do we decide to place a “do not resuscitate” order on the column, and what can be done to extend column lifetime under our typical operating environment? Well, as heralded earlier, there is really no single correct response, and the column lifetime will ultimately depend on a number of factors including the type of GC column used, the application, the GC method, and your treatment of the column during its lifetime. When I’m teaching a GC class, it’s usually at this point that I say, while a lot of what I’m about to write can be seen as “someone else’s issue to deal with,” and the cost of new column won’t be coming from your salary, we should also bear in mind that not being able to get on with work because the column you just installed is a “dud” can be infuriating.

Perhaps the biggest issue facing even the best columns on the market is loss of stationary phase, which happens on an on-going basis. Our job is to make sure that the rate of loss is a low as we can make it and for the bleed to be consistent. In the case of the polysiloxane-based phases, the presence of oxygen catalyzes the cleavage of backbone siloxane units that were formed during the stationary-phase polymerization process described above, via a “back biting” reaction as shown in Figure 2. While this reaction is catalyzed at higher temperature, it also occurs at room temperature.

Reaction scheme for formation of cyclic polysiloxane bleed products from a dimethyl polysiloxane immobilized GC stationary phase.

The cyclic siloxanes formed are volatile, and elute from the column as bleed, which manifests itself as a rising baseline—and never as discrete peaks on the baseline.

A classic stationary-phase profile with increasing temperature is shown in Figure 3.

 Typical capillary GC stationary phase bleed profile for two different polysiloxane-based stationary phases (Figure reproduced with permission, Agilent Technologies, Santa Clara, CA, USA).

On standing, the stationary phase will become oxidized and will bleed. Obviously one way to protect columns in storage is to cap them—column caps are recommended as opposed to septa! One should also note that the reaction in Figure 1 is also catalyzed by ultraviolet (UV) light, which is why it’s a good idea to keep your capillary GC columns boxed between outings.

On column installation, it’s important to get rid of the bleed products formed during storage to ensure the lowest levels of baseline noise and baseline displacement during temperature programming. This is typically achieved using column conditioning, where the column temperature is elevated slightly above the required upper working temperature and left there for a period of time to drive off the volatile bleed products. It’s very important not to just install the GC column, whack the oven up to the isothermal or gradient temperature maximum stated by the manufacturer, and leave it overnight. You are literally throwing precious stationary-phase out into the detector!! And yes, you should be aware of the upper temperature limits for your columns and stick to them. The gradient temperature maximum (usually the higher of the two figures quoted on the column box or hanger tag) should not be used for more than a few minutes at a time. For more specific information on column conditioning and how to do it, see this article link: 

https://www.chromatographyonline.com/view/gc-column-conditioning-stop-wasting-time-and-money

You will have noticed that the words oxygen and oxidation were used several times in the preceding discussion, generally because oxygen is the biggest GC stationary-phase killer of all, and we need to ensure that we expose the column to the lowest amounts of oxygen possible on an ongoing basis. Yes folks, that means we need to pay attention to our friends the gas filters that are fitted (hopefully!!) to the gas line between the carrier source and our instrument. One of those traps, typically containing a metal catalyst, is there to remove oxygen, and the best of them typically achieve O

 levels below 1 ppb within the flowing carrier gas stream. You need to ensure, on an-ongoing basis, that the trap is not exhausted, and that’s why I recommend the use of an indicating trap typically containing a copper oxide indicator which turns from green to grey as the oxygen levels exiting the trap become too high. This is YOUR responsibility. Don’t leave changing gas traps to someone else, otherwise the answer to the titular question will be, “longer than they do now, because you don’t look after them properly!” Figure 4 shows a “combination” gas filter that reduces levels of oxygen, moisture, and hydrocarbon, but the important point to note is that a quick glance at the gas filters will tell you whether the oxygen filter tell-tale is green (good) or grey (bad). Come on folks, check your traps. It’s really not that difficult, and of course get a replacement trap into that gas line as quickly as possible if you discover that it’s exhausted. As a minimum, gas filter checking should be part of your routine maintenance schedule, but it can also be included in method or instrument SOPs as part of the acquisition set-up sections. 

A typical “combination” gas filter, with the green “oxygen” indicator clearly marked.

Combining these last two points together, extended exposure of the stationary phase to high levels of oxygen at high temperature is the GC column equivalent of a rock and roll lifestyle: they will die young!

I often get asked about the influence of water on GC stationary phases, both as an impurity in the carrier gas, and as a solvent for injection. There is so much information out in the ether about this subject, I’ll just make a quick summary of the main points here. Most bonded phases have nothing to fear with regards to water exposure. The bonding process renders the phase less susceptible to water damage. Coated phases are a different matter, and water should be avoided with these phases as it will significantly shorten the column lifetime. It should be noted, however, that any active sites within the phase can lead to water absorption, which can lead to some degree of irreproducibility in retention. Wax phases are more susceptible to this phenomenon (they are polyethyele glycol (PEG) based), and the more water that is adsorbed, the more irreproducible they will become in terms of both retention and selectivity. It should be said that most manufacturers have developed wax phases that are more water compatible. 

Moisture traps are also typically fitted to the GC carrier gas line, and typically contain molecular sieve or other adsorbent materials. Just like the oxygen trap, these should be changed once exhausted, and modern traps will have an indicator which changes color (check the colors with the trap manufacturer literature).

To ensure gas traps (and columns) last as long as possible, you should regularly check the leak-tight nature of the carrier gas supply lines (using a pressure drop test) and ensure the GC column fittings at the injector and detector are leak free every time the column is changed, and from time to time when columns are left in the instrument over extended periods.

The nature of our sample, analytes, and sample solvents is another major consideration when evaluating expected column lifetime. The sample pH should, ideally, fall within the range 5–9. This can be estimated with pH papers and corrected with sample dilution (if sensitivity requirements allow), or with adjustment using volatile buffers (solid buffers should be avoided at all costs), again with the caveat that one needs to consider any chemical interactions with the analytes of interest. The more the sample pH deviates from the required operating range, the quicker the column bleed will become intolerable, and the column will be unusable.

There are many applications which require the analyte to be derivatized to impart volatility or reduce analyte polarity, and any excess derivatising reagent will potentially damage the stationary phase within the column. The degree of damage will depend upon the type of derivatizing reagent used, which are in order of most to least damaging: perfluoro acid anhydride acylation reagents > silylating reagents (especially for PEG based columns) > acylation reagents > perfluoroacylimidazoles. Wherever possible, the derivatized sample should be evaporated to dryness to remove as much of the volatile derivatizing reagent as possible prior to reconstitution in a suitable solvent.

Of course, many of us associate the “cleanliness” of our samples with the expected column lifetime, and the nature of the sample matrix as one of primary factors in causing column damage. While this is undoubtedly an important factor is assessing expected column longevity, aside from what has been discussed above, there is much that can be done to avoid column damage, including the usual steps such as more thorough or more highly selective sample preparation that will help to isolate the analytes of interest from matrix components. As most column damage due to fouling by involatile matrix components occurs in the first few meters of the column, it is always worthwhile considering the use of a guard column, which can be changed once chromatography deteriorates, as an alternative to continuous trimming of the analytical column which will result in slightly lowered efficiency and the possible movement of analyte retention times outside the identification windows set for them within the data system. Interestingly, as the stationary-phase film is disrupted through chemical degradation, it will migrate down the column and pool into a “bubble” of very thick phase a little way down the column. This will cause severe peak broadening and an apparent loss of efficiency, and it is also the reason that such seemingly miraculous recoveries can be made by column trimming. It is also the reason why iterative trimming of short lengths of column (10–20 cm) from the inlet side can suddenly result in a huge performance recovery. Many variations of guard columns are available that can be coated in the desired stationary phase, uncoated but deactivated, or built into the analytical column, in which case no column connection is necessary, but the guard has a finite lifetime as it is an uncoated region of the column prior to the coated and immobilized phase. If sensitivity requirements allow, we could consider applying a higher split ratio when introducing the sample. And if splitless injection is used, the judicious use of a glass wool plug will also help to protect the column from less volatile matrix components. All of these measures will help to prolong column lifetime.

It is very important for us to note that GC column dimensions and the chemical nature of the stationary phase are primary factors in dictating expected column lifetime. The more polar the stationary phase, the more the column will bleed and perhaps the shorter we can expect the column lifetime to be. Remember that PEG and Wax phases are the most polar and therefore likely to last for the least amount of time. Thicker stationary-phase films will inherently bleed more, and one might suspect that they will last a shorter time, however this isn’t necessarily the case. Unless we are dealing with extremely volatile analytes, the effects of stationary-phase loss will be less pronounced simply because there is more phase present. Thicker films also “mask” the effects of active groups on the silica surface, and therefore a lot of phase loss will need to occur prior to seeing effects such as peak broadening, tailing, and a loss of sensitivity at lower analyte concentrations. The trick with thicker film columns is to condition them properly and use the guidelines in the link that I provided above.

It’s important to have a reference point for making the decision on whether a column has reached the end of its useful lifetime. This may take the form of system-suitability specification for a particular method, reference chromatogram from when the column the new column was installed and working well, and so on. Once the column begins to show signs of ageing, remember that good column cutting, leak free column installation, and trimming of the column are ways to temporarily stave off the need for column replacement.

So, as I said right at the start, the expected column lifetime will depend on how careful you are about storing, installing, conditioning, and protecting it by paying attention to the sample preparation, use of guard columns, and the use of oxygen and moisture-free carrier gases. There are no tables of expected column lifetimes, because lifetime depends on how we treat the column, and not what our columns can do for us, but for what we can do for our columns!

Tony Taylor is the Chief Scientific Officer of Arch Sciences Group and the Technical Director of CHROMacademy. His background is in pharmaceutical R&D and polymer chemistry, but he has spent the past 20 years in training and consulting, working with Crawford Scientific Group clients to ensure they attain the very best analytical science possible. He has trained and consulted with thousands of analytical chemists globally and is passionate about professional development in separation science, developing CHROMacademy as a means to provide high-quality online education to analytical chemists. His current research interests include HPLC column selectivity codification, advanced automated sample preparation, and LC–MS and GC–MS for materials characterization, especially in the field of extractables and leachables analysis.

The question, which is often asked of our technical support and applications chemists, is one to which I often reply, in the words of John F Kennedy, "Ask not what your column can for you, ask what you can do for your column.” OK, JFK substituted “column” for “country” in his version of the quotation, but as you will see, it’s a very relevant premise!

The LCGC Blog: How long should my GC column last?

Many consider you to be a leader in the study of separation mechanisms and methods of detection, with a view to developing mathematical models to improve fundamental understanding of these aspects of chromatography. What would you say are your most important contributions to the development of new chromatographic and electrophoretic methods of analysis?

Trying to identify my most important contributions is exceedingly difficult because of the wide range of chromatographic fields in which I have worked and the very large number of students and collaborators who have contributed. My publications list in excess of 150 co-authors and for this reason I feel that our contributions should be viewed as a whole rather than trying to single out specific achievements. However, I will mention below just a couple of broad areas where I feel that our contributions have been significant.

I have always been interested in chromatographic retention mechanisms and electrophoretic migration mechanisms, especially in the derivation of mathematical models which describe these mechanisms. Once a suitable mathematical model is available, method optimization can be accelerated greatly because retention time can be predicted for a wide variety of conditions and then used to identify the optimal conditions. My group has published widely on such retention models for IC and electrophoresis and we have tried to focus on models which can be applied with minimal input data. We have derived IC retention models for isocratic elution, gradient elution, and complex eluent profiles involving multiple sequential isocratic and gradient steps and these models have underpinned commercial IC simulation and optimization software sold by Thermo Fisher Scientific which has had wide uptake by users.

My group has also maintained a strong focus on the separation of metal complexes. We have explored a wide variety of ligand types, but our greatest successes have been with metallocyanide complexes. These complexes are of great importance in a wide range of industrial process, but perhaps the best example is in the extraction of gold from its ores. This is usually undertaken by complexing the gold with cyanide, adsorbing the gold cyanide complex onto activated carbon, and then desorbing with hydroxide. Operators of this gold extraction process need to know how much gold cyanide is in solution during the leaching process, how much free cyanide is available, and how much cyanide has been consumed by complexes of other metals, such as iron. We developed a “cyanide analyzer” which could supply all of the required information in a single run.

You and your research group have recently developed a miniaturized deep-ultraviolet light emitting diode (LED)-based detector for use in a portable capillary-scale liquid chromatography (LC) system (1). This detector was incorporated into a briefcase-sized portable capillary HPLC system. In your estimation, how will this device improve field analysis using HPLC methods?

The universality of UV detection increases at lower wavelengths, and the absorptivity of most organic molecules also increases at lower wavelengths. Thus, deep UV detection increases sensitivity and also widens the range of compounds which can be detected. In the case of a miniaturized, portable instrument there is a strong necessity to minimize weight and also heat production. The deep-UV LED is a perfect solution to these challenges and the particular LED that we evaluated is a prototype, pre-production version that was supplied to us for evaluation.

You have reviewed the literature from 2015–2020 on the topic of predictive models for retention times in LC (2)., What can you tell us about the state of the art in retention-time modeling? What additional of future work would be helpful to the field of retention modeling?

Method development in liquid chromatography is generally a slow process because of the need to equilibrate the system with each new mobile phase that is to be evaluated. For this reason, accurate prediction of retention time is a very desirable goal. Retention time can be calculated from mathematical models which are derived from a sound understanding of the chromatographic retention mechanism, but such models usually require some experimental input and these models are applicable only when the actual retention mechanism is well understood. For these reasons, the focus in the field of retention prediction has been to use statistical approaches to model large databases of retention data, based on the chemical structure of the analytes for which retention times are sought. This process, known as 

quantitative structure-retention relationships

 (QSRR), enables retention times to be predicted with sufficient accuracy (1-5%) to choose the best chromatographic system for a desired separation, based only on the structures of the analytes to be separation. However, once the optimal system has been selected there is usually a subsequent step in which the fine details of the mobile phase composition are optimized, and this generally involves some experimental input.

We have recently completed a major QSRR project in which we worked with industrial partners (Pfizer, Thermo Fisher, and LC Resources) to devise some new approaches to the QSRR process. We have found that the most significant step in the development of a statistical QSRR model is the choice of the retention database compounds which will be used to train the model. It is tempting to think that using a large number of training compounds might give the best results, but in fact the reverse is true. In particular, if one selects only those training compounds which have chemical similarity to the target compounds, good prediction accuracy can be achieved using fewer than 10 training compounds. It also turns out that best results are achieved when a new QSRR model is made for each individual compound (that is local models are derived rather than global models). In my view, the future directions of QSRR research will involve applications in nontargeted metabolomics, use of more comprehensive retention databases involving a wider variety of analytes and stationary phases (most probably accessed by crowd sourcing), application to the separation of enantiomers, and the use of the QSRR methodology to gain better insight into chromatographic retention mechanisms. Better access to very fast computers will also speed up the QSRR process.

You have continued research and development of a miniature UV absorbance detector for capillary LC based on LED technology (3). This work is important for miniaturized analysis systems. How is your specific research approach different from your contemporaries for LC detection?

There are several groups working currently and in the recent past on the development of a miniaturized, portable LC instrument and some of these groups have produced very nice instruments. Our focus differs somewhat in two aspects. First, our research is strongly directed toward the needs of industry, and in particular the pharmaceutical industry. We are actively collaborating with a local instrument manufacturer (Trajan Scientific and Medical) and a major pharmaceutical company (Pfizer) to design our portable instrument as a flexible platform which can be used right across the drug development portfolio, from drug synthesis and design through to manufacturing. This flexibility in application requires a platform where detectors can be changed easily and sample preparation steps, such as dilution, can be incorporated. The second point of difference is cost. Most importantly, we are not trying to make a miniature UHPLC instrument capable of high-speed runs using high pressures. Rather, we are using low-cost syringe pumps capable of medium pressure, coupled with low-cost switching valves and LED-based detectors to come up with a purpose-built, inexpensive instrument that can be used in a wide variety of applications. We are not trying to replace benchtop LC systems in the pharmaceutical industry but rather to complement them with a new instrument that is inexpensive and can therefore be applied in numerous at-line and on-line situations.

What do you consider are the most important research publications you have produced science over your career in this field?

As stated above, I am reluctant to choose particular publications because to do so would require me to highlight some collaborators rather than others. However, the one publication that I am happy to mention is a book on IC which I published in 1990 (Paul R. Haddad and Peter E. Jackson, 

Ion Chromatography: Principles and Applications

, Elsevier, Journal of Chromatography Library, Vol. 46). Peter and I are very proud of this book—it is nearly 800 pages long, it required a monumental effort to write, and when published it was the most comprehensive IC text available. We feel that we achieved a nice balance between a very comprehensive theoretical treatment of all relevant topics and an equally comprehensive tabulation of applications. In our view the book offered something to people working across the entire IC spectrum, from fundamental researchers to users trying to address specific problems. The book has been cited over 700 times and has won several awards, including inclusion in the Laboratory Equipment Top 10 Products for 1990.

regarding what you would consider to be the most important new areas of research in chromatography?

I will confine my comments here to liquid chromatography. LC is now a very well-established analytical technique and one might expect its development to now be slow. However, progress continues to be rapid, with ground-breaking new developments appearing continually and even entirely new LC technologies emerging. In my view important areas of research will be continued development of new stationary phases to improve peak resolution and selectivity for problem analytes (such as enantiomers), improved peak capacity, increased use of 3D printing, and new developments in predictive tools (with QSRR being but one example). Improved peak capacity is perhaps the most urgent target because current LC systems cannot cope effectively with the huge number of analytes present in biological systems. For this reason, the use of LC in “omics” systems will not reach its full potential until we can produce LC systems with perhaps 10 times the peak capacity of current approaches. For this, 2D and 3D chromatographic systems will provide the key, but to achieve the necessary peak capacity they will need to be coupled with high-resolution stationary phases and high-resolution mass spectrometers. A further key technology that will contribute to higher peak capacity is 3D printing, with the ultimate goal being to print a multidimensional separation system.

What words of advice would you share with young researchers just getting started, or even undergraduates considering a future career in science? Do you have any specific advice for those interested in separation science as a career?

Separation science is a great career, with endless possibilities and directions. The advice that I always give to my students is to see their skills as general rather than specific and to recognize that all forms of chromatography are connected. After doing an undergraduate degree and then a PhD, many students see their skills as being confined to the specific problem on which they have worked during their PhD. They can then be unwilling to look for a job in other areas of chromatography. I try to emphasize that what they have learned in one area of chromatography can be easily translated into another. I often relate my own experience as an early career researcher. I undertook a PhD in the field of molecular fluorescence spectroscopy applied to inorganic ternary complexes. When I returned to the same university as a junior staff member some five years later, my department head (who had also been my PhD advisor) instructed me to develop an interest in HPLC. At that time, I did not even know what the acronym HPLC meant, but I recognized that my PhD had provided me with generic skills in problem solving, experimental hypothesis and design, literature searching, and report writing and that these skills would be sufficient to provide a strong base for moving into a new field.

(1) S.C. Lam, L.J. Coates, V. Gupta, H.J. Wirth, A.A. Gooley, P.R. Haddad, and B. Paull, Ultraviolet absorbance detector based on a high output power 235 nm surface mounted device-type light-emitting diode, 

(2) P.R. Haddad, M. Taraji, and R. Szücs, Prediction of Analyte Retention Time in Liquid Chromatography, 

(3) M. Hemida, L.J. Coates, S. Lam, V. Gupta, M. Macka, H.-J. Wirth, A.A. Gooley, P.R. Haddad, and B. Paull, Miniature Multiwavelength Deep UV-LED-Based Absorption Detection System for Capillary LC, 

 Lifetime Achievement in Chromatography Award winner. He received his PhD in analytical chemistry from the University of New South Wales, Australia in 1975, and his BS degree in 1971 from the same institution. He also holds a Diploma of Military Studies (Science) awarded in 1969 by the Royal Military College, Duntroon, Australia. In 1996, he was awarded a Doctor of Science by the University of New South Wales, Australia.

He is an Emeritus Distinguished Professor at the University of Tasmania, Australia and was the foundation Director of the Australian Centre for Research on Separation Science (ACROSS). He is best known for his research on the theory, mechanism and applications analytical separation science in liquid phases, with particular emphasis on the separation and quantification of ionic species.

He has performed research in such separation techniques as high performance liquid chromatography (HPLC), ion chromatography (IC), capillary electrophoresis (CE) and capillary electrochromatography (CEC). The general aim of this research has been to study separation mechanisms and methods of detection, with a view to developing mathematical models improving fundamental understanding of these aspects and to apply this understanding to the development of new chromatographic and electrophoretic methods of analysis. Other research work involves the study of detection methods, with an emphasis on potentiometric detection using reactive metallic electrodes and the theory and application of indirect methods of spectrophotometric detection. The separation of complexed metal ions and sample handling methods have comprised further major research themes.

Paul Haddad of the University of Tasmania in Australia is distinguished as the foundation director of the Australian Centre for Research on Separation Science (ACROSS). He is well known for his research in many analytical techniques, including high performance liquid chromatography (HPLC), ion chromatography (IC), capillary electrophoresis (CE) and capillary electrochromatography (CEC). He is the winner of the 2021 LCGC Lifetime Achievement in Chromatography Award, which honors an outstanding and seasoned professional for a lifetime of contributions to the advancement of chromatographic techniques and applications. He recently spoke to us about his research work and career.

A Lifetime of Contributions for Advancing Research in Separation Science: Paul Haddad, the Winner of the 2021 Lifetime Achievement in Chromatography Award

 (1), Merck reported a miniaturized automation platform for nanomole-scale synthesis of pharmaceuticals combining the use of robotics with emerging liquid chromatography (LC) and mass spectrometry (MS)–based high-throughput analysis (HTA) techniques. This publication has drawn the attention of many laboratories across both academic and industrial sectors. Why was this research important to Merck and the pharmaceutical industry? What were the major analytical challenges you encountered in enabling this platform?

At the forefront of new synthetic endeavors, such as drug discovery or natural product synthesis, large quantities of material are rarely available, and timelines are tight. With this platform, more than 1500 chemistry experiments can be carried out in less than a day, using as little as 0.02 milligrams of material per reaction. At that point, analytical chemistry quickly became the bottleneck in this endeavor. Our team came up with an HTA methodology based on mixed-(multiple injections in a single experimental run) MISER LC–MS that enabled analysis of 1536 reactions in about 2.4 h. It was a very collaborative project among a multidisciplinary team of organic and analytical chemists, a phenomenal experience.

In other work you have carried out fundamental studies in ultrafast chiral separations, and reported new applications for high-throughput analysis (2) and 2D-LC (3), overcoming limitations of traditional approaches. Would you explain the significance and meaning of this work for the readers of 

Ultrafast chiral chromatography offers tremendous potential for high-throughput enantiopurity assays, with analysis time that is competitive with sensor-based analytical approaches, while providing great selectivity. At the time of this publication (2), investigations of high-speed chiral separations in the academia were quickly growing. However, most of those reports were fundamental in nature involving simple mixtures to prove a concept. We joined forces with academia to develop ultrafast chromatographic enantioseparations for a variety of complex pharmaceutically related drugs and intermediates, showing that sub-minute resolutions were possible in the vast majority of cases by both supercritical fluid chromatography (SFC) and reversed-phase LC (RPLC). Half of these chiral mixtures were separated in an impressive time window ranging from 5 to 20 s using readily available instrumentation without any modification. New applications were also introduced, illustrating how such methods can be routinely developed and used for ultrafast high-throughput analysis to enable enantioselective synthesis investigations and enzymatic processes.

In addition, our work laid the foundation for the use of ultrafast chiral chromatography as a second dimension of 2D-LC analysis (3). This paper introduced a variety of multiple achiral x chiral and chiral x chiral 2D-LC examples (single and multiple heart-cutting, high-resolution sampling, and comprehensive) using highly efficient chiral selectors (sub-2-μm fully porous and 2.7 μm fused-core particles) in the second dimension. This concept enabled the separation and analysis of complex mixtures of closely related pharmaceuticals and synthetic intermediates, including chiral and achiral drugs and metabolites, constitutional isomers, stereoisomers, and organohalogenated species.

You have also published a recent work that introduces multifactorial peak crossover (MPC) via computer-assisted chromatographic modeling to address challenging coelutions of critical pairs and poor chromatographic productivity of purification methods (4). How is this work important for industrial pharmaceutical laboratories? How do you envision MPC assisting a broader pharmaceutical analytical community?

MPC is a software-based technique that focuses on mapping the separation landscape of pharmaceutical mixtures to quickly identify spaces of peak coelution crossover, which enables one to conveniently switch the elution order of target analytes (4). We can gain massive productivity increases (shorter cycle time and higher sample loading) by simply moving a target peak from the tail to the front of an undesired component. MPC chromatography dramatically reduces the time spent developing productive analytical- and preparative-scale separations. This concept can also be used to improve signal-to-noise ratio substantially, enabling straightforward ppb detection of low-level target components with direct impact in the quantitation of metabolites and potential genotoxic impurities (PGIs).

What issues and problems would you define as previously ignored or neglected specifically in the field of new and important pharmaceutical analysis methods? What major developments do you see as important in automating screening methods, and in multidimensional chromatography? What is your vision for improvements that could be made in separation instrumentation, columns, data processing algorithms, or high-speed computing power?

Every day brings something new and different to these labs. Because Merck’s pipeline is very rich and diverse, it is important to upgrade our analytical toolkit constantly. Analytical chemistry is quickly becoming the bottleneck in the development of more complex targets. Certainly, pushing the boundaries of analytical innovation today will help us overcoming many of the pharmaceutical challenges of tomorrow. One of my current research projects focuses on the integration of modern automated screenings with computer-assisted modeling approaches to accelerate method development. Despite recent advances in this area, current multidimensional chromatography technology requires a series of arduous method development activities poorly suited for a fast-paced industrial environment. I believe that moving away from “guru dependency” is very important to our future success. Recent contributions are laying the foundation for systematic ways to facilitate method development through automated multicolumn 2D-LC screening (5) and computer-assisted optimization of 2D-LC separations (6), which are key in securing the viability of 2D-LC as a mainstay for industrial applications.

What were some of the key challenges you encountered during your work recently? What would you consider to be the most useful contributions of your work?

The development of novel and increasingly challenging process chemistries requires a commensurate level of analytical innovation to develop meaningful and reliable assays of reaction outcomes, with rapid data turnaround, to enable decision making toward the next round of experiments. The time constraints of developing robust quantitative methods prior to each processing step can easily lead to sample analysis becoming a bottleneck in synthetic route development. I found this extremely challenging, and in my view, it pushes you out of your comfort zone. Simply put, process chemistry and target molecules dictate the kind of analytical tool you will end up using, no matter your background or preferences. In this regard, our recent research on introducing new “generic or more universal” chromatographic workflows helped us tremendously to minimize the time spent developing new analytical assays, while also facilitating method transfer to manufacturing facilities and application in regulatory settings. Our recent review paper in 

What are some key aspects that motivate you? Would you share some of your work and organizational habits that have helped you be productive and successful professionally?

Everything gets easier when you find a meaningful purpose in life. I’ve accepted the fact that any innovation or invention comes with a risk of failure. Although I always try to make the right choices, sometimes I don’t succeed, but every day I get back up and move forward. Over the years, I became more disciplined and incorporated very good habits in my routine that help me to stay focused and eliminate distractions.

What can you share with our readers regarding your next area of interest for your research?

Pioneering measurement science to accelerate the development of new medicines and vaccines is my focus at MRL. I’m very interested in incorporating more automation and predictive algorithms to my routine, as well as feedback-controlled instrumentation. Artificial intelligence, machine learning, and deep learning advances are also capturing my attention.

(1) A. Buitrago Santanilla, E. L. Regalado, T. Pereira, K. Bateman, L-C. Campeau, S. Berritt, Y. Liu, M. Shevlin, Z-C. Shi, J. Voigt, J. Schneeweis, C. J. Welch, R. Helmy, P. Vachal, I. Davies, T. Cernak, and S. Dreher, Nanomole-scale high-throughput chemistry for the synthesis of complex molecules, 

(2) C.L. Barhate, L.A. Joyce, A.A. Makarov, K. Zawatzky, F. Bernardoni, W.A. Schafer, D.W. Armstrong, C.J. Welch,and E.L. Regalado, Ultrafast chiral separations for high throughput enantiopurity analysis, 

(3) C.L. Barhate, E.L. Regalado, N.D. Contrella, J. Lee, J. Jo, A.A. Makarov, D.W. Armstrong, and C.J. Welch, Ultrafast chiral chromatography as the second dimension in two-dimensional liquid chromatography experiments, 

(4) I.A. Haidar Ahmad, V. Shchurik, T. Nowak, B.F. Mann, and E.L. Regalado, Introducing multifactorial peak crossover in analytical and preparative chromatography via computer-assisted modeling, 

(5) H. Wang, H.R. Lhotka, R. Bennett, M. Potapenko, C.J. Pickens, B.F. Mann, I.A. Haidar Ahmad, and E.L. Regalado, Introducing Online Multicolumn Two-dimensional Liquid Chromatography Screening for Facile Selection of Stationary and Mobile Phase Conditions in both Dimensions, 

(6) D.M. Makey, V. Schurik, H. Wang, H.R. Lhotka, D.R. Stoll, I. Mangion, E.L. Regalado, and I. Haidar Ahmad, Mapping the separation landscape in two-dimensional liquid chromatography: blueprints for efficient analysis and purification of pharmaceuticals enabled by computer-assisted modeling, 

(2021). doi.org/10.1021/acs.analchem.0c03680 

(7) E.L. Regalado, I.A. Hadar Ahmad, R. Bennett, V. D’Atri, A.A. Makarov, G.R. Humphrey, I. Mangion, and D. Guillarme, The emergence of universal chromatographic methods in the research and development of new drug substances, 

Erik Regalado, the 2021 winner of the Emerging Leader in Chromatography Award, is a Principal Scientist in the AR&D department at Merck Research Laboratories (MRL) where he leads the Method Screening and Purifications group. His industrial research focuses on analytical and preparative enabling technologies that accelerate the development of new pharmaceuticals, including automated method screenings, multidimensional chromatography, HTA, ultrafast and computer-assisted separations. Regalado completed a Postdoctoral Fellowship at MRL (2013-2015), received a PhD in Chemistry from the University of Havana (UH), Cuba in collaboration with the University of Nice Sophia Antipolis, Nice, France (2011), a Master of Science in Organic Chemistry (2007), and a Bachelor of Science in Chemistry (2003) from UH.

Erik Regalado is a Principal Scientist in the Analytical Research and Development (AR&D) department at Merck Research Laboratories where he leads the Method Screening and Purifications group. His industrial research focuses on analytical and preparative enabling technologies for the development of new pharmaceuticals. He is studying automated methods, ultrafast and computer-assisted analysis, multidimensional chromatography, and many other methods. He is the 2021 winner of the LCGC Emerging Leader in Chromatography Award, which recognizes the achievements and aspirations of a talented young separation scientist who has made strides early in his or her career toward the advancement of chromatographic techniques and applications. He recently spoke to us about his current research and career. 

Accelerating the development of new pharmaceuticals: Erik Regalado, the 2021 winner of the Emerging Leader in Chromatography Award

The most commonly used designs for modern liquid chromatography (LC) pumps produce mobile-phase streams with small short-term variations in mobile phase composition. Understanding the origin of these variations and their effects on chromatographic performance can help us develop high-performing methods. In this installment, we focus on the effect of these mobile-phase composition “waves” on detector baselines.

This month’s installment of “LC Troubleshooting” is motivated by communication I had with readers in recent months about mobile-phase composition, pumps, and mixing. Although these topics have been discussed in this column frequently in the past, they are moving targets to some extent because pump technology for liquid chromatography (LC) continues to evolve. Therefore, user expectations for pump performance in demanding applications also change. As an example of the ongoing importance of these issues, below is an excerpt of an e-mail I received recently from an LC user:

“We have two [brand name LC instruments] with quaternary pumps that are acting up. If we try to run the system using 50% A and 50% B, we get retention time shifts. If we pre-mix the solvent and just run channel A, all is fine. Gradients make the problem worse...so obviously there is a problem with the mixing.”

In this installment, I review the operating principles of LC pumps that rely on low- or high-pressure mixing approaches, describe how waves of solvent composition can develop in the mobile phase, and evaluate the potential impacts of these waves on detector baselines. In next month’s installment, I will discuss the results of simulations that show how these waves can impact variability in retention time along with some solutions to these problems. One could devote an entire book chapter to these topics, so I discuss the highlights here, providing a concise overview of LC pump technology. For readers that are interested in learning more, I encourage considering the following resources: the books by Kromidas (1) and Snyder and Dolan (2) have chapters dedicated to modern LC pump technology, details about performance specifications, and descriptions of tests that can be used to evaluate pump performance; two “LC Troubleshooting” articles by John Dolan in 2006 (3) and 2014 (4) describe case studies that illustrate what can happen when things go wrong in the pump; and Choikhet and co-workers (5) and Gritti (6) discuss in great detail the impact of imperfect pump performance on LC applications involving trifluoroacetic acid (TFA) in the mobile phase. These are all excellent resources for those looking to add to their LC troubleshooting knowledge.

Review of LC Pumping Principles—Low- and High-Pressure Mixing Designs

Figure 1 illustrates the basic principles of the two of the most common designs for pumping systems used in LC. In the case of the high-pressure mixing approach, two independent pump heads, each capable of producing a high-pressure stream of a mobile-phase component, draw in and discharge solvent at a consistent flow rate (for isocratic operation). For example, if the total flow rate through the column is 1.0 mL/min, and the desired mobile-phase composition is 40:60 acetonitrile:water, then one pump head discharges acetonitrile continuously at 0.4 mL/ min, and the other pump head discharges water continuously at 0.6 mL/min. The two streams discharged from the pump heads then converge and pass through a mixer, and the mixed mobile phase proceeds to the sampler, and eventually the column. In the case of the low-pressure mixing approach, there is only one pump head that pressurizes the mobile phase to drive it through the column. The mobile-phase composition is determined by assembling small “packets” of individual solvents in a serial fashion into a mobile-phase stream that is drawn into the high-pressure pump. In most modern pumps of this design, the volume of each solvent packet is determined by the length of time a solenoid-type valve is open between the solvent bottle and the proportioning valve unit. Furthermore, these times are also related to the volume of each stroke of the high-pressure pump and the mobile-phase flow rate. For example, suppose the stroke volume is 100 μL, the desired mobile-phase composition is 40:60 acetonitrile:water, and the flow rate is 1 mL/min. The period of each pump stroke will be 6 s; the solenoid for the acetonitrile line will be open for 2.4 s, drawing pure acetonitrile into the tubing leading from the proportioning valve to the pump head. Then, this valve closes, and the solenoid for the water line opens for 3.6 s and pure water is drawn into the tubing. This completes one cycle of mobile-phase composition proportioning. The solvent composed in this way is mixed extensively as it travels through the high-pressure pump head itself, and an additional mixer is positioned between the pump and the sampler.

FIGURE 1: Block diagrams for the two most commonly used designs of high performance liquid chromatography (HPLC) pumps in use today: (a) binary pump with high-pressure mixing; and (b) quaternary pump with low-pressure mixing.

Origins of Solvent Waves and Their Impacts on Detector Baselines

Each of the pump designs discussed above has several strengths and weaknesses. In both cases, however, the mobile-phase composition at the pump outlet will not be perfectly smooth (that is, no variation in composition during isocratic operation; in gradient elution, the change in composition over time would ideally be smooth without any short-term noise). The primary causes of the deviations of the actual mobile-phase composition from what is programmed are different for the two designs. In the case of the low-pressure mixing approach, it is intuitive that there would be short-term variations in composition on the time scale of one pump stroke. During the pump stroke, there are times when the fluid entering the high-pressure pump is literally all A or all B. This is illustrated in Figure 2b. The resulting variation in mobile-phase composition can be smoothed to a large degree with effective mixing downstream from the high-pressure pump, but completely eliminating the variation would require a large volume mixer. If the detector is capable of detecting small variations in composition (for example, refractive index detection, or ultraviolet [UV] detection in the case where mobile-phase additives absorb UV light, such as TFA), then they will be observable in the detector signal as “waves,” or short-term noise. Adding a large mixer introduces other problems, such as a large delay between the time of a programmed change in composition and when that change arrives at the column (in gradient elution this appears as the “gradient delay” or “dwell” time). Thus, the configurations of these pumps as received from manufacturers reflect a compromise between doing enough mixing to smooth out these waves to a large extent and not adding a mixer that is so large that it causes other problems. Indeed, pump manufacturers offer mixers of different volumes that allows the user to choose a larger volume mixer for applications that are expected to be especially sensitive to short-term variations in mobile-phase composition (for example, applications involving TFA).

FIGURE 2: Conceptual illustration of the origin of mobile phase composition waves in the case of (a) high-pressure mixing and (b) low-pressure mixing designs used in modern pumps.

The primary origin of solvent waves in the case of high-pressure mixing is fundamentally different. In this case, if the flow rate from each pump head were perfectly consistent over time, the composition of the mixed mobile phase would be perfectly consistent over time. But, in a reciprocating piston design (which is the dominant design in use today), there are small changes in the flow from each pump head at the end of a piston stroke due to the imperfect operation of check valves. These small changes in flow are illustrated in Figure 2a. If these flow rate changes are different for channels A and B, and they are not perfectly synchronized in time, then there will be a small, short-term variation in the composition of the mixed mobile phase. The lengths of the resulting waves in this case tend to be shorter in comparison to the low-pressure mixing case, as illustrated in Figure 2. These waves can also be greatly minimized by introducing a mixer between the solvent convergence point in the pump and the LC column. However, the same challenge exists here as with the low-pressure mixing design: Completely eliminating the waves requires a large mixer, so what we use in practice represents a compromise between smoothing the mobile-phase composition and having a low gradient delay volume (which is essential for fast gradient elution separations, for example).

As mentioned above, the mobile-phase composition waves that flow from the pump can impact the quality of detector baselines (as measured by noise and drift) if the detector signal is dependent on the composition of the mobile phase itself. Most of the time, this can be avoided or minimized through judiciously choosing the conditions and instrument parameters. For example, acetonitrile is attractive as a mobile-phase organic solvent modifier for reversed-phase liquid chromatography (RPLC) when using UV detection at low wavelengths (< 230 nm). If the mobile- phase components are effectively transparent to the detector, then small variations like waves in the composition will not impact the quality of the detector baseline signal. Sometimes, though, this is impossible, or at least very difficult to avoid. One well known and studied example is the case where TFA is used as a mobile-phase modifier for RPLC separations of peptides involving UV detection. TFA is attractive because it tends to improve peak shapes for peptides and increase retention for hydrophilic peptides. However, a disadvantage of TFA in this context is that it absorbs a significant amount of UV light at 214 nm, which is the wavelength typically used for peptide mapping applications. Furthermore, the TFA itself is somewhat retained by RPLC stationary phases. When a mobile-phase composition wave travels through the column, the acetonitrile-rich part of the wave will decrease the local retention of TFA, dumping more of it into the mobile phase where it will absorb more UV light. This is a very complex situation that cannot be thoroughly discussed here, but there are at least two excellent papers (5,6) that describe all of the factors involved and demonstrate the impact of different chromatographic variables on baseline quality when using TFA in the mobile phase with UV detection. I strongly encourage readers interested in learning about this situation in more detail to consult these papers.

In a previous installment of “LC Troubleshooting”, I discussed why mobile-phase mixers are needed following LC pumps, and the types of situations when a change in the type of mixer might be needed (7). Figure 3 shows the effect of increased mixer volume on the noise level in UV detector baselines when using TFA in the mobile phase delivered by a binary pump. Although the 35 μL mixer might be adequate for other applications, a larger volume mixer is helpful for reducing baseline noise in this case because of the increased sensitivity of the baseline noise to mobile-phase composition waves when using TFA as an additive.

FIGURE 3: Comparison of ultraviolet (UV) absorbance signals (214 nm) obtained with different mixers in use. The pump was a high-pressure binary mixing system (Agilent 1290, Infinity II). The column was a 30 mm x 2.1 mm i.d. Agilent SB-C18, and gradient elution was used. Solvent A was 0.1% trifluoroacetic acid (TFA) in water, solvent B was 0.1% TFA in acetonitrile (ACN), and the gradient ran from 2–40% B in 4 min.

In this column, I have reviewed the operating principles of modern LC pumps based on low- and high-pressure mixing designs and explained how these pumps produce mobile-phase streams with small short-term variations in mobile-phase composition. These composition “waves” can negatively affect detector baseline quality and also retention time variability. In next month’s installment of “LC Troubleshooting”, I will continue exploring this topic by discussing the results of simulations that illustrate the effect of method parameters including the amplitude of the composition waves, flow rate, and pump stroke volume on retention time precision.

The HPLC Expert II: Find and Optimize the Benefits of your HPLC/UHPLC

High-Performance Gradient Elution: The Practical Application of the Linear-Solvent-Strength Model

 (John Wiley & Sons, Hoboken, New Jersey, 2007).

(5) K. Choikhet, B. Glatz, and G. Rozing, 

, 461605 (2020). https://doi.org/10.1016/j. chroma.2020.461605.

 is the editor of “LC Troubleshooting.” Stoll is a professor and the co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota. His primary research focus is on the development of 2D-LC for both targeted and untargeted analyses. He has authored or coauthored more than 60 peer-reviewed publications and four book chapters in separation science and more than 100 conference presentations. He is also a member of LCGC’s editorial advisory board. Direct correspondence to: 

A review of the operating principles of modern liquid chromatography (LC) pumps based on low- and high-pressure mixing designs, and a look at how these pumps produce mobile phase streams with small short-term variations in mobile phase composition, with a focus on the effect of these mobile-phase composition “waves” on detector baselines.

LCGC Europe: Surfing on Mobile Phase, Part 1: Origins of Mobile-Phase Composition Waves and Their Effects on Detector Baselines

Commercialization of columns that provide multiple modes of chromatographic separations have recently been on the rise. For example, combinations of retention modes, such as ion-exchange and reversed-phase, often enable the separation of complex mixtures of analytes not possible using single-mode columns. In this work, recent trends in what is often referred to as “mixed-mode” phase are investigated. Particular attention is paid to recent fundamental research, stationary phase development and design, and areas of application.

 reviews of newly launched high-performance liquid chromatography (HPLC) columns, it was observed that several stationary phase developments fell into a category that can be characterized as “mixed-mode” (1,2). The idea of mixed-mode chromatography is not new. However, the observation prompted the question, “What is the current status of mixed-mode chromatography?”

An initial search through the literature and various websites revealed that there have been a multitude of publications, including reviews, that have written about mixed-mode chromatography in recent years. For example, West and others published a review of mixed-mode chromatography that covered many important developments, including combinations of reversed-phase liquid chromatography (RPLC) and ion-exchange chromatography (IEC), as well as hydrophilic interaction liquid chromatography (HILIC) and ion-exchange (3). Zhang and Liu published another notable review that focused on mixed-mode chromatography applications in pharmaceutical and biopharmaceutical analyses (4). The reader is referred to these reviews and references within for a more in-depth discussion of the technology.

Mixed-mode chromatography remains somewhat ill-defined. West and co-workers defined the term as the combined use of two (or more) retention mechanisms in a single chromatographic system. Adding to this, Gilar and co-workers further define the term as the use of multiple dominant retention mechanisms (5). The dominant designation is an important one as all chromatographic systems involve at least some minor contributions from retention mechanisms other than the primary ones. These minor, but usually important, contributions are often referred to as 

. For the purposes of this column, we will refer to mixed-mode chromatography where multiple retention mechanisms play a dominant role in the overall chromatographic performance. Mixed-mode systems may be achieved via a number of different routes. However, the combination of partition (RPLC or HILIC) chromatography and ion-exchange seems to be associated most closely with the term.

In their description of new phases that combined partitioning and anion-exchange chromatography, Pohl and Liu noted that mixed-mode columns address a number of key challenges in chromatographic method development (6). Using reversed-phase columns only can result in poor polar analyte retention, basic analyte peak tailing, and column “dewetting” under high aqueous mobile phase content. They go on to note that ion-exchange can be used to tackle some of these issues, but ion-exchange alone is not suitable to separate molecules based on their differences in hydrophobicity. The combination of the two modes of chromatography often provides the potential to address a number of analytical challenges. Mixed-mode chromatography has played a significant role in modern chromatographic practices and has been applied to areas such as oligonucleotides, peptides, proteins, metabolomics, pharmaceutical analysis, and natural product studies, among many others (7).

In an attempt to take an even more recent pulse on the level of interest and utilization of mixed-mode chromatography, a non-exhaustive search of publications in the past year along with a perusal of well-known column vendor websites was conducted. The limited search revealed a significant number of papers and technical notes in this realm, indicating continued interest. What follows is a brief synopsis of what was found, broken down into fundamental studies, new stationary phase designs, and applications of the technology.

The application of mixed-mode chromatography is often limited, because of the complexity of the resulting chromatography. With more mechanisms playing a significant role, additional parameters that control these mechanisms need to be taken into account during method development and practice of the resulting procedures. In addition, it is often difficult to find a full description of surface chemistry from vendors or detailed characterization results (8). Fundamental studies that provide the user with information to help guide method development and understand both the potential and limitations of stationary phases is of utmost importance.

Lämmerhofer and associates used chromatographic, molecular modeling and electrochemical techniques to characterize chiral zwitterionic materials (7). Although the target phases were commercialized for use in chiral separations, the group notes significant achiral utility. The columns studied were described as cinchona carbamate stationary phases decorated with cyclohexylsulfonic acid and carbamate residues and are thus likely to provide multiple dominant retention mechanisms. Indeed, the group found the phases to exhibit moderate hydrophobicity, the potential for HILIC operation, and ion-exchange character. A unique aspect of this research was that investigators used a sequential building and analysis of intermediary versions of the stationary phases to better understand the individual structural contributors to retention mechanisms.

Gilar and co-workers investigated the chromatographic attributes of several commercially available columns, including single-mode as well as a recently launched mixed-mode column combining C18 and anion-exchange chemistry (5). The group focused their efforts on understanding the ion-exchange interaction potential of the columns across a wide pH range. Fundamental information regarding the interactions available across a number of different conditions is important to method developers for both selecting and utilizing various stationary phases. For instance, the researchers were surprised to find that the ion-exchange interactions for the mixed-mode phase dropped off at a pH value much lower than the ligand p

 value. The observation was attributed to the impact of ionized surface silanols on the overall surface charge and demonstrates both the complexity and the need to carefully study mixed-mode systems.

The majority of the published research over the past year has focused on the development of new stationary phases and their characterization. What follows is not intended to be an exhaustive list, but it should provide an overview of the types and breadth of stationary phases being investigated across the globe in this field.

Shields and Webber reported on the development of a mixed-mode, reversed-phase cation-exchange system based on a thiolyne reaction. The intent was to develop a mixed-mode column with low pH stability. The authors used the separation of monoamine neurotransmitters to demonstrate both the ion-exchange and partition properties of the phase (9). Guo and others published a paper describing the interesting combination of MOF-235, polyethylene glycol, and silica, in a core–shell-based format. The authors indicate a combination of HILIC and ion-exchange is possible with the phase composition (10).

Li and associates described the use of modified dialdehyde cellulose as a substrate for developing stationary phases that exhibit both HILIC and ion-exchange properties. The authors note facile functionalization and suggest this approach as the basis for further stationary phase development (11).

Several stationary phase developments have been recently published by Wang and co-workers. The authors describe a poly(ethyleneimine) embedded N-acetyl-L-phenylalanine stationary phase that is shown to exhibit RPLC, HILIC, and ion-exchange characteristics (12). In a second paper from the group, a stationary phase co-modified with N-isopropylacrylamide and aminophenylboronic acid is described.

The resulting phase was also characterized as providing both hydrophobic and hydrophilic retention capabilities along with anion-exchange properties (13). Wang and associates also reported on the incorporation of ionic liquids with C18 and cyclodextrin moieties bonded to silica supports as potential stationary phases for liquid chromatography (LC) (14). The group characterized the phases as exhibiting hydrophobic and ion-exchange character as well as the potential for use in the HILIC domain. A positive comparison with commercially available, single-mode columns was provided.

Heydar and Hosseini published a paper describing the preparation of a novel, silica-based stationary phase using 9-methylacridine and 9-undecylacridine (15). The phases were shown to exhibit hydrophobic and hydrophilic partitioning as well as anion-exchange characteristics. Finally, Wolrab and associates investigated a series of zwitterionic and strong cation-exchange based mixed-mode phases under RPLC, HILIC, and supercritical fluid chromatography (SFC) conditions (16). The authors noted that ion-exchange interactions appear to prevail over others for the phases studied. However, other interactions, such as partitioning, can be enhanced or attenuated using various mobile phase conditions.

It is clear from the number of reports over a short period of time, as well as from the diversity of approaches, that interest in developing mixed-mode chromatography stationary phases continues.

Mixed-mode chromatography is powerful, but complex. As noted above, when multiple dominant retention mechanisms are invoked, many variables must be controlled to produce a robust and repeatable method. Mixed-mode chromatography is therefore most often applied only to complex systems. The following are examples of mixed-mode applications found in recent literature.

The combination of anion-exchange and RPLC was used to analyze artificial sweeteners in surface waters. Anionic sulfamates (acesulfame, cyclamate, saccharin), zwitterionic dipeptides (aspartame, neotame), and polar derivatives of natural products (sucralose, neohesperidin dihydrochalcone [NHDC]) were all efficiently separated on an octadecylsilane (C18)-strong anion exchanger (SAX) combination phase, as shown in Figure 1. One of the major advantages of the mixed-mode approach noted by the authors was the ability to inject large volumes of sample, presumably because of the accumulation effect of the ion-exchange character (17).

FIGURE 1: Optimized separation of seven artificial sweeteners on a mixed-mode stationary phase. Reproduced with permission from reference (17). ACN is acetonitrile.

Zhang and associates recently reported on the use of mixed-mode chromatography for the analysis of oligonucleotides. The use of ion-pair reversed-phase chromatography (IP-RPLC) appears to be the most heavily employed mode of separation for oligonucleotides to date. However, anion-exchange, HILIC, and mixed-mode methods have also been successful. According to the authors, the use of mixed-mode separations for oligonucleotides dates back to the early 1980s. The authors also note that RPLC and ion-exchange mixed-mode approaches have displayed improved separations over either mode alone. In addition, it is speculated that multidimensional liquid chromatography (mLC) is poised to deliver much of what mixed-mode chromatography can do and is expected to continue to grow in this space (18). Lämmerhofer and co-workers also proposed the use of a chiral zwitterionic phase, which exhibits both partition and anion-exchange properties, for the first dimension separation of oligonucleotides using RPLC as the second dimension (19).

The application of mixed-mode chromatography for the separation of underivatized amino acids was demonstrated by Moussa and associates (20). The traditional amino acid analysis routine consists of an ion-exchange based separation followed by reaction with ninhydrin or similar reagent. The traditional approach is noted as being time-consuming, nonspecific, and requires a dedicated analyzer. The authors used a “trimodal” column consisting of anion-exchange, cation-exchange, and hydrophobic properties to separate 52 amino acid-related compounds in 18 min with minimal sample preparation. The authors noted that the bimodal systems investigated did not provide the necessary separation of critical pairs.

Sze and co-workers reported on the utility of employing ion-exchange in combination with reversed-phase and HILIC separations for the improved analysis of deamidation in proteins (21). The authors note that deamidation of proteins results in several species of very similar hydrophobicities that are thus difficult to separate using partition chromatography alone. The authors suggest that the use of electrostatic interactions—coupled with HILIC chromatography, along with improved sample preparation and advanced mass spectrometric techniques—will help serve to fill deficiencies in this important area of research.

Another application of mixed-mode chromatography published in 2020 tackled the separation of pentacyclic triterpenoids using a mixed-mode, weak-anion-exchange stationary phase (22). The critical pairs of erythrodiol and uvaol, as well as oleanolic acid and ursolic acid, were only resolved with a combination of reversed-phase or HILIC with ion-exchange mechanisms, whereas both RPLC and HILIC alone were found to be insufficient. As shown in Figure 2, the mixed-mode column utilized provided the separation of 10 analytes in approximately a 7-min run time.

Mixed-mode chromatography columns are on the rise. This article reviews recent fundamental research, stationary phase development and design, and areas of application.