When high performance liquid chromatography (HPLC) users are confronted with questions about how to improve upon the performance of an existing or recently developed method, there are many technological options to consider. This series offers guidance for a systematic approach to method improvement that can save time and resources and likely produce more robust methods. Part 2 explains options adjusting selectivity (peak spacing or elution pattern) when dealing with relatively simple mixtures—which are not always easy to separate—with a focus on reversed-phase LC separations.

As explained in a recent article (1), this article series was prompted by a question I was asked during a visit to a local laboratory here in Minnesota, USA. The question was essentially, “What are my options for improving an old high performance liquid chromatography (HPLC) method that works, but probably is not ideal?” There are many options, but it can be difficult to know where to start. This series of articles is intended to make it more manageable to think about how to proceed. Not all the options we will discuss will be available to all users, but it is valuable to understand what the full array of options looks like, so that you can feel confident and make an informed decision about how to proceed.

In last month’s instalment of “LC Troubleshooting”, I kicked off this series of articles by reviewing and discussing some foundational concepts that form the basis of a systematic approach to thinking about how to improve the resolution (and, indirectly, speed) of a separation. I discussed how different separation challenges (for example, simple vs. complex mixtures) can require different approaches to improving resolution, and talked through a menu of options to improve performance, including both kinetic (that is, factors affecting peak width) and thermodynamic (that is, factors that affect peak speak spacing) factors. I also discussed the importance of resolution as a metric of separation performance because it is a measure of peak separation that quantitatively takes into account both the widths of peaks and the distance between them (that is, the distance between the apices of the peaks). Finally, I discussed the influence of different factors on resolution, including retention (as measured by retention factor, 

). The relationship between resolution and these factors is often examined using the Purnell equation, and one of the key takeaways is that the influence of the selectivity of resolution is greater over a large range than the influences of retention and efficiency. Thus, it is always wise to think about how one might change selectivity as a means of improving resolution.

In the second instalment of this series, I focus on options to improve the performance of separations of relatively simple mixtures. In this case, because it is usually possible to fully resolve the components of the mixture, we can also think about how to improve the speed (throughput) of the separation; however, our discussion of speed will be addressed in a future instalment. Many of the details of this discussion are applicable to the specific case of reversed-phase LC separations.

For the purposes of discussion in this series of articles, I think of a “simple mixture” as one containing a small number of compounds. What constitutes a number that is small enough for the mixture to be considered simple is discussed in the next section. In a very rough sense, I always consider a mixture with fewer than five components to be simple, and a mixture with more than 100 components to be complex. What happens between five and 100 really depends on the chemistry of the analytes and the goals of the separation. Readers interested in this aspect are referred to the discussion of “regular” and “irregular” samples by Snyder and Dolan (2). Although this is also a useful framework for thinking about how to approach method development, we will not discuss this framework here. It is very important to note here that just because I refer to a mixture as “simple” does not mean it will be easy to separate. Indeed, a mixture of just two analytes can be very difficult to separate, as in the case of a pair of enantiomers. In most cases, it will be impossible to resolve such a pair of compounds, unless a chiral stationary phase is involved.

What Are My Chances of Separating the Mixture I Have?

One way to answer this question about the probability of separating a particular mixture is to use the statistical overlap theory (SOT), which was developed mainly by Davis and Giddings in 1983, with their seminal paper introducing the idea (3). The SOT is based on a number of assumptions that may not be strictly consistent with the characteristics of a real method development problem; however, I think it is still a very useful means of thinking through the chances of actually separating a particular mixture at hand. If SOT predicts that the probability of separating the mixture is less than 1%, then it would probably be a major waste of time and resources to try to fully resolve the mixture, and we would be better off thinking about alternatives, such as two-dimensional (2D) separations or multiple methods (see below for more on this). One of the major assumptions of SOT is that peaks are randomly distributed throughout the resulting chromatogram when a mixture is separated. For a diverse mixture of analytes, the distribution of peaks is usually close enough to random for SOT to be applicable, but there certainly are exceptions to this. For example, separations of homologous series of analytes (for example, alkanes and alkylbenzenes) produce chromatograms with highly ordered patterns of peaks. SOT cannot reasonably be applied to separations of samples that are dominated by compounds like this. In our discussion here, we only scratch the surface of SOT; readers interested in learning more are referred to the original paper by Davis and Giddings (3), and the subsequent literature that refers to this early work (4).

When considering simple mixtures, we are usually most interested in fully resolving all the components of the mixture. In SOT, this outcome is given the symbol P

 is the number of components in the sample, and 

Conceptually, peak capacity quantifies the number of peaks that can fit perfectly side-by-side in a given separation space without overlapping. Under isocratic conditions, this can be calculated easily from the desired resolution between adjacent peaks (

), and the retention factor of the most retained component in the separation (

Most calculations of peak capacity in the literature involve a resolution of 1, but if we want to baseline resolve them for quantitative purposes, then we need at least 1.5, and I have used that number here. Table 1 shows the peak capacities calculated using equation 2 for some combinations of 

 that are relevant in everyday practice of isocratic reversed-phase LC. The 

 values span a range from short (5) to long (20) separations, and the plate numbers span a range from what can be achieved easily on a short column with almost any instrument (5000), to what can only be achieved with modern columns and high-performing instruments (40000). We see that the range of peak capacities that results is approximately 20 to 100. Realizing a peak capacity beyond 100 requires gradient elution conditions, 2D separations, or both; these are topics we will address in future instalments in this series.

Now that we have a sense for the range in peak capacities that can be expected under a typical array of conditions, we can turn back to the original question: What is the probability of separating a given mixture with these peak capacities? Figure 1 shows this probability for mixtures in the range of 2–50 components, and peak capacities of 20, 100, 300, or 1000.

There are a number of important takeaways we can glean from this result. First, with a peak capacity of 20, the probability of resolving even a relatively simple five-component mixture is just 8%. This low probability is a harsh reality of statistics applied to chromatography. It is absolutely true that we can significantly improve these odds by adjusting the mobile phase chemistry, stationary phase chemistry, or both, to reorganize the peak pattern, but in the absence of this effort, the likelihood of fully resolving the mixture on the first try using a short separation and modest column are disappointingly low. In the process of method development, we should never be surprised if we cannot resolve our mixture on the first go. Second, when using a higher peak capacity separation of 100, we see that the probability of resolving the five-component mixture increases dramatically to 60%. This is still far from a “sure thing”, but at least now the odds of success on the first try are better than 50%. Now, if we follow the blue line to the right, we see that the probability drops rapidly, reaching 1% for a 16-component mixture. Beyond 16 components, the likelihood of fully resolving the mixture is vanishingly small, and it will take a great deal of effort in method development to find conditions that provide complete resolution of the mixture. Finally, we see that improving peak capacity to 300 (achievable with a single reversed-phase LC column, under gradient conditions) improves things significantly, moving the 1% mark out to a 27-component sample. Giving ourselves a reasonable chance of resolving mixtures more complex than this requires even higher peak capacities, which can only be provided by 2D separations in most cases, at least under practically useful conditions.

An alternative to 2D separation that is sometimes used for samples of intermediate complexity is to use multiple conventional LC methods to target and resolve different subsets of the components of a mixture. For example, a C18 column might be used to resolve components 1–12, and a phenyl column may be used to separate components 13–20. The information provided by the two methods run separately for each sample can then be combined to provide the information needed for all 20 analytes in the mixture, whereas developing a single method to yield the same information may be very difficult and require a great deal of method development time and resources.

Improving Resolution Through Thermodynamic Adjustments: Changing the Selectivity

As discussed in the preceding section, the likelihood of separating even relatively simple mixtures using a single column under isocratic conditions is dismally low. Fortunately, we have at our disposal a number of levers we can use in method development to overcome the harsh reality of the statistics of random distributions and reorganize peaks to make better use of the space in our chromatograms. In LC, the chemistries of both the mobile and stationary phases can have significant impacts on selectivity, and thus the distribution of peaks in a chromatogram. In general, changing the mobile phase is easier experimentally, but has less of an impact on selectivity; changing the stationary phase is more expensive and harder (but not impossible) to automate, but has more potential for providing large changes in selectivity.

 When it comes to adjusting the mobile phase, in reversed-phase LC, both the solvent type and the aqueous or organic solvent composition influence selectivity. Currently, we do not know how to predict which organic solvent will provide the most useful selectivity for a given mixture of molecules, thus it is most common in practice to simply do a solvent screen early in method development to see which solvent is most likely to provide the needed selectivity. Figure 2 shows that the impact of the solvent type on the peak pattern can be significant. The dashed lines connecting the peaks for specific compounds are drawn in to indicate which compounds are impacted the most, and that elution order reversals can occur.

 the amount of organic solvent (that is, the organic:aqueous ratio) in the mobile phase that will yield the selectivity needed for a successful separation. However, the good news is that once a solvent is chosen, the retention behaviour for a given molecule is consistent enough that a retention model can be built from a few initial retention measurements. With this model in hand, optimal conditions can be found by simulation and then verified by experiment. The development of this general approach began in the 1980s with the work of Snyder and Dolan and coworkers (5), and the approach has enjoyed great success through the years, with development of several commercially available software packages along the way (such as DryLab and LC Simulator). Figure 3(a) shows an example of how a retention model can identify mobile phase compositions that yield selectivities that looks promising (see dashed lines C and D) to fully resolve the mixture. Figure 3(b–d) show chromatograms simulated using mobile phase compositions highlighted by the dashed lines in the plot. In Figure 3(b) we see coelution of mefenamic acid and 4-n-hexylaniline, which is predicted by the crossing of the green and purple lines in Figure 3(a). In Figures 3(c) and (d), we see that the mixture is fully resolved, but the conditions in (d) are more attractive because they yield resolution of the mixture in a shorter time.

Using this approach to improve isocratic separations is straightforward in practice and requires a few initial “training experiments” where the mobile phase composition is deliberately varied so that the dependence of retention on mobile phase composition can be established. These data can then be fed into the software that allows identification of candidate conditions that are likely to provide resolution of the sample mixture in the shortest analysis time.

In addition to changing the organic solvent type and amount (as shown in Figures 2 and 3), changing the mobile phase pH can also be a powerful way to adjust selectivity, particularly when the sample mixture contains ionogenic analytes. This is a complex topic that requires too much discussion to address here; however, it has been covered extensively in previous instalments of “LC Troubleshooting”; interested readers are referred to those prior instalments to learn more (6).

Changing the stationary phase can also be an effective means of adjusting selectivity. As I often tell my students, the good news is that we have approximately 1000 different reversed-phase LC columns to choose from in the marketplace. The bad news is that in practical method development, we don’t have the time or resources to try them all, so we have to choose a few to try. Figure 4 shows an example of the dramatic impact that changing the stationary phase can have on the elution pattern for a simple mixture. The extent to which the changes are more or less different than the change observed when changing the organic solvent type (as in Figure 2) will depend on the chemistry of the analytes and stationary phases involved. Our ability to predict which stationary phases are the right ones to try is pretty limited, but there are some tools to help with this.

The hydrophobic subtraction model (HSM) of reversed-phase selectivity, developed by Snyder and Dolan and coworkers (7), provides a means of identifying column pairs that are either similar or different. In the context of method development where we want to adjust selectivity by changing the stationary phase chemistry, the HSM can be used to suggest candidate columns to try. Currently, data for approximately 750 columns are available and free web-based tools can be used to interrogate the results. Readers interested in learning more about the model and use of the tools are referred to previous articles in 

(8), and the websites hosting the tools (

https://apps.usp.org/app/USPNF/columnsDB.html

Many HPLC users are confronted with questions about how to improve upon the performance of an existing method. Fortunately, modern HPLC technologies (that is, instrumentation, columns, and software) provide many possible avenues to make improvements. However, the number of possibilities can be intimidating, making it difficult to decide upon a particular path to improvement. In this instalment of “LC Troubleshooting”, I have discussed options for adjusting selectivity (that is, peak spacing or elution pattern) when dealing with relatively simple mixtures, with a focus on reversed-phase LC separations. In LC, both the chemistry of the mobile phase and the stationary phase strongly influence selectivity, thus changing one or both of them can be a viable means of bringing about changes in selectivity. In the case of the mobile phase, changing the type and amount of organic solvent, as well as the pH, are all important factors to consider. In many cases, changing the stationary phase chemistry is the most impactful, albeit more expensive, means of changing selectivity. These are all important options to keep in mind when thinking about how to improve a separation.

(1) Stoll, D. R. What Are My Options to Improve My Separation? Part 1: Foundational Concepts. 

(2) Snyder, L. R.; Dolan J. W. Introduction. In 

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

(3) Davis, J. M.; Giddings, J. C. Statistical Theory of Component Overlap in Multicomponent Chromatograms. 

(4) Schure, M. R.; Davis, J. M. The Simple Use of Statistical Overlap Theory in Chromatography. 

LCGC Europe – Recent Developments in UHPLC and HPLC

(5) Snyder, L. R.; Dolan, J. W.; Lommen, D. C. Drylab Computer Simulation for High-Performance Liquid Cromatographic Method Development. 

(6) Dolan, J. W. Back to Basics: The Role of pH in Retention and Selectivity. 

(7) Dolan, J. W.; Snyder, L. R. The Hydrophobic-Subtraction Model for Reversed-Phase Liquid Chromatography: A Reprise. 

(8) Dolan, J. W.; Stoll, D. R.; Snyder, L. R. How to Take Maximum Advantage of Column-Type Selectivity. 

Articles

What are the options available when adjusting selectivity (peak spacing or elution pattern) when dealing with relatively simple mixtures?

What Are My Options to Improve My Separation? Part 2: Likelihood of Separation, Adjusting Selectivity for Simple Mixtures

Since the publication of the first tenets of green chemistry about 25 years ago, the conversation about sustainability and chemistry has slowly grown, and today seems at a fever pitch, as companies are adding sustainability officers, universities are adding curriculum, and chemistry-related processes being updated regularly. In this instalment, we will being exploring the impact of green chemistry on analytical chemistry and gas chromatography (GC). We will define green chemistry, and discuss what it is and what it is not. For example, it is not “chemistry lite”. We will then briefly review relevant literature on green analytical chemistry, and begin a conversation on green gas chromatography and the many areas in which GC-based methods can be made “greener”.

The 12 Principles of Green Chemistry were introduced in the late 1990s and have seen rapidly increasing interest over the past decade (1–3). Table 1 lists these principles of green chemistry along with principles of green analytical chemistry. More recently, the United Nations has promulgated their 17 Sustainable Development Goals (UN SDGs), which are guiding policymaking in governmental and corporate venues (4). The 12 Principles of Green Chemistry and the UN SDGs guide sustainable practices in the chemical sciences and in analytical chemistry research and development. Thinking about green chemistry and sustainability in chemical science can trace their roots to the early work of Lovelock, the inventor of the electron capture detector, and the classic book that is credited with starting the environmental movement, 

Not surprisingly, there has been much politics and public discourse around the broad topics of sustainability and the UN SDGs. The American Chemical Society (ACS) clearly states that green chemistry is “not politics”, “not a public relations ploy”, and “not a pipe dream” (3). For educators like myself, it is also not “chemistry lite”. Using the 12 Principles of Green Chemistry in teaching chemistry encourages the use of more modern techniques and ideas at earlier points in the curriculum, and green chemistry is clearly the future of chemical education.

The 12 Principles of Green Chemistry have been adapted to analytical chemistry, with a useful recent review provided by Nowak, Wietecha-Pozluszny, and Pawliszyn (8). In 2013, Galuszka and co-authors outlined 12 Principles of Green Analytical Chemistry (also listed in Table 1) (9). Taken together, the principles of green and green analytical chemistry provide an interesting roadmap for analytical method development and optimization. While the 12 Principles of Green Chemistry are clearly geared towards synthesis and manufacturing, some clear parallels are seen in waste reduction, atom economy, reduced energy usage, miniaturization, and improved safety. Interestingly, and specific to gas chromatography (GC), both sets of principles call for elimination of derivatization. The analytical principles also call for multi-analyte methods, another principle that directly encourages chromatography.

 in which a process is considered holistically and can be broken down into individual steps, which are then evaluated for their overall impact on the process, related external processes, and sustainability, is another aspect of green chemistry and sustainability. The classic work by Meadows describes systems thinking in detail, with examples where the whole process can be negatively impacted by a seemingly positive change in one area (10). Analytical chemists already apply a form of systems thinking when developing methods, especially those with goals such as high speed, low cost, and easy to use. In developing a full method, we are taught to consider the many steps in the process, using flow charts like Figure 1, which is a block diagram of the steps in a typical GC method, with system thinking-related additions to reflect some of the external forces impacting the method.

When optimizing the method, we think towards the overall goal of the analysis, and then optimize each step towards that goal. In addition to the usual analytical chemistry steps of sample preparation, instrumentation, and data collection, systems thinking teaches us to consider all related aspects of the method, including where the samples come from, how they are transported to the laboratory, the number of samples, reagents, and consumables, time and effort in data analysis, and where the samples, reagents, and materials go after the analysis is finished. Energy and personnel inputs are included in Figure 1 but not attached to a particular step in the method, as they impact all steps.

An example of an unintended consequence that systems thinking might expose could be seen in a switch from liquid–liquid extraction (LLE) to solid-phase microextraction (SPME) for the sample preparation, an obvious choice for making a GC method greener. LLE generally has the advantages of simplicity and low cost, with disadvantages of high solvent utilization. SPME, while clearly greener, may have higher costs, depending on fibre lifetime. More interestingly, equilibrium in LLE is generally rapidly achieved due to intimate and complete mixing of the two liquid phases, while SPME may require longer equilibration or heating of the extraction vessel, both of which might negate some of the green benefit. Longer equilibration time might then slow down the chromatographic analysis, causing the instrument to idle for longer time between runs, further reducing the benefit. Systems thinking and thinking holistically are therefore a critical part of truly making any analytical method greener.

A further aspect of systems thinking places the analytical method into the context of the whole life cycle of the product or process being measured. Systems thinking assists with the identification of bottlenecks or delays in processes that can be remedied, much in the way that chemists define the rate determining step in a reaction mechanism. Systems thinking underlies the current attempts to evaluate analytical and chromatographic methods for greenness. As we will see below, evaluating a method involves examining the sustainability aspects of each step in the process and all materials used. Two methods for evaluating the greenness of analytical methods are discussed below.

The Analytical Method Greenness Score (AMGS) was proposed by Hicks and associates in 2019 (11). This metric provides a tool especially suited to chromatographic methods, and was demonstrated for high performance liquid chromatography (HPLC), ultrahigh-pressure liquid chromatography (UHPLC), and supercritical fluid chromatography (SFC). Its main focuses are on the mass of solvents used for sample preparation, instrument operation, health and environmental measures for the solvents, energy utilization for both the solvents and instrument, and the number of sample injections expected, including standards. In short, AMGS is related to instrument energy, solvent energy, and solvent safety. The authors note that energy usage is a major contributor to the greenness of any analytical method, and that this can vary widely, depending on the application and technique. They note that chromatographic systems integrated with additional instruments, such as a mass spectrometer, have significantly higher energy usage than the chromatograph alone. I further note that hyphenated techniques also may require a more specialized and expensive operator.

The RGB Additive Colour Model was developed by Nowak and Koscielniak for evaluating analytical methods according to analytical performance (red), productivity and effectiveness (blue), and safety and eco-friendliness (green), as seen in Figure 2 (12). Methods are given a numerical score from 1–100, based on how effectively they address each of these method goals. An interesting question about using this idea for method development or optimization lies in whether the three colours are competitive or complementary. Do productivity and performance have to be sacrificed to gain safety and eco-friendliness? Do practical methods sacrifice performance and greenness? Do high performance methods, such as those with extremely low detection limits, have to sacrifice practicality and greenness? These questions still require answers.

There are several approaches that can make GC more sustainable, some of which we have discussed in recent instalments, including miniaturization and carrier gas choice. We discussed a sample preparation example above.

In an earlier instalment, we discussed several configurations and principles for smaller gas chromatographs that can perform the routine tasks generally done on larger systems (13). Today’s miniature gas chromatographs can perform all of the tasks necessary for most routine analysis applications. The sustainability advantages include smaller laboratory footprint, less heat generation and lower power consumption, and potentially simpler operation. One sustainability challenge in implementing miniature instruments is that replacing the existing instrument itself has up-front capital and sustainability costs that must be considered.

For several reasons, including high inertness, excellent column performance, slow peak broadening and, until recently, ready availability, helium is the most widely used carrier gas for capillary GC. Today, ongoing helium shortages are wellknown, and result from helium being obtained as a by-product of fossil fuel production (14). From a sustainability perspective, helium is probably the worst choice for carrier gas. Mainly on cost and availability, but now with an eye towards long-term sustainability, hydrogen and nitrogen are now more widely considered as carrier gases.

As a replacement for helium, hydrogen is an obvious choice, and has been discussed in the literature almost since the inception of capillary GC. From a chromatographic perspective, hydrogen offers faster separations and greater separation efficiency at high flow rates than either helium or nitrogen. From our sustainability perspective, hydrogen requires either the purchase of high pressure cylinders or a hydrogen generator, which is an additional capital expense items that may cost nearly as much as the gas chromatograph itself.

Nitrogen is classically considered the poorest choice of the three gases, from a chromatographic point of view. According to classical van Deemter plots, nitrogen rapidly loses efficiency as the flow rate is increased to practical values. However, in temperature programming, the effects noted in van Deemter plots do not apply. In a previous instalment, we discussed how nitrogen can provide very similar chromatographic performance to helium in many temperature programmed situations (15). Nitrogen is also more difficult to use with GC–mass spectrometry (MS), as it is slightly ionizable at the usual 70 eV used in electron ionization ion sources, so its signal can overload a mass selective detector. For the best combination of greenness and chromatographic performance in capillary GC, I recommend nitrogen as the carrier gas, followed by hydrogen (if nitrogen use is not possible), and then helium.

Green and sustainable chemistry are here to stay. As we move forwards in analytical science and chromatographic method development, principles of green chemistry and sustainability will guide our work. Applying principles of green chemistry to analytical methods will have the added benefit of making them more rugged, robust, and reproducible, while reducing waste, consumption, and cost. I encourage gas chromatographers to begin embracing green chemistry by examining their own methods, and considering sustainable changes, such as using nitrogen carrier gas, smaller, less power consuming instruments, and solvent-limited or solvent-free sample preparation.

The author’s work on green gas chromatography is supported by a grant from the American Chemical Society Green Chemistry Institute’s Pharmaceutical Roundtable and collaborations with Seton Hall University’s Academy for Green Chemistry, Sustainability, and Stewardship.

 3rd Ed. Royal Society of Chemistry, 2016.

(3) American Chemical Society Green Chemistry Institute, 

(4) United Nations Sustainable Development Goals, 

(5) Snow, N. H. Selectivity and Sensitivity: The Electron Capture Detector (ECD), Its Unique Inventor James Lovelock (1919– 2022), and GAIA, 

(8) Nowak, P. M.; Wietecha-Posłuszny, R.; Pawliszyn, J. White Analytical Chemistry: An Approach to Reconcile the Principles of Green Analytical Chemistry and Functionality. 

(9) Gałuszka, A.; Migaszewski, Z.; Namiesnik, J. The 12 Principles of Green Analytical Chemistry and the SIGNIFICANCE Mnemonic of Green Analytical Practices. 

(11) Hicks, M. B. et.al., Making the Move Towards Modernized Greener Separations: Introduction of the Analytical Method Greenness Score (AMGS) Calculator. 

(12) Nowak, P. M.; Kościelniak, P. What Color Is Your Method? Adaptation of the RGB Additive Color Model to Analytical Method Evaluation. 

(13) Snow, N. H. Let’s Get Small: Powerful Gas Chromatography in Small Packages, 

(14) Pflum, M. NBC News, “The Fate of America’s Largest Supply of Helium is Up In The Air.” 

https://www.nbcnews.com/science/science-news/fate-americas-largest-supply-helium-air-rcna69309

(15) Snow, N. H.; McCann, S. P; Handzo, B.; Rana, H. Go With the Flow: Thinking About Carrier Gas Flow in GC. 

Exploring the impact of green chemistry on analytical chemistry and GC is the focus of this instalment of "GC Connections".

Green Chemistry: What Is It (and What Is It Not)? And How Does It Apply to Gas Chromatography (GC)?

This article covers liquid chromatography (LC) columns and accessories commercially released after Pittcon 2022 through the 2023 conference held in Philadelphia, USA. As in the past, 

sent out a survey in late 2022 and early 2023 asking vendors to supply information on products launched over the past year. Note that new products for gas chromatography (GC), LC instrumentation and software, and sample preparation are covered elsewhere. Information for this article was obtained over the course of many months; thus, it is possible that some information has been missed or misinterpreted. The reader is encouraged to check with specific vendor sites for additional product releases as well as more detailed information on product usage and attributes. Links to vendor sites are provided where applicable.

The vendors that responded to the survey and their new liquid chromatography (LC) products are listed in Table 1. Initial categorization of the products into small and large molecule analyses as done in recent reviews (1–3) was difficult this year because of the number of product line launches that included columns designed for both. The most reasonable segregation this year seemed to be oriented more towards the column technology the product lines were based on rather than their intended use. Many of the products and product lines released this year were based on recent, established technologies, such as superficially porous particle (SPP) substrates. Many others were extensions of existing product lines with new surface chemistry or bonding processes. Products that fit into these descriptions as well as new chiral and size-exclusion chromatography (SEC) columns were placed in the first category entitled, “New Product Lines and Product Line Extensions”. A second category based on column hardware format was also deemed appropriate. Products in this category that were designated “Nontraditional Column Formats” include the recently introduced 1.5-mm internal diameter (i.d.), capillary, and pillar array columns. The final column-based category includes a number of product lines that have been introduced based on inert column hardware, continuing a major trend from last year (4). Accessories are also an important component to any LC system. That is why the last section is devoted to “non-column” new products that also contribute to successful liquid separations.

New Product Lines and Product Line Extensions

Chromanik Technologies Inc. released two new stationary phases for small molecule analysis. The SunShell C18 is built on a 90 Å, 3.5-μm SPP surface. A “unique endcapping” process using high temperature and hexamethyldichlorotrisiloxane as the endcapping reagent is reported to result in an extended pH range of 1.5–10. The company also launched Sunniest PFP & C18, a mixed pentafluorophenylpropyl and octadecyl phase on a 120 Å, 5-μm fully porous particle (FPP). The mixed phase is more hydrophobic and more stable than the PFP stationary phase alone. More information can be found on the 

Fortis Technologies Ltd. introduced a new line of columns (Evosphere) based on monodisperse, FPP architecture. Monodispersity is reported to “add a new layer of efficiency” while retaining high capacity for increased loading (as in preparative chromatography). According to the company, this is an alternative approach to gaining efficiency compared to using SPP-based phases. The new line is based on a 100 Å pore size that is available in 1.7-, 3-, and 5-μm particle sizes. Numerous surface chemistries are available, including C18 AR (C18 and aromatic mixed bonding), C18/PFP, diphenyl, phenyl-hexyl, AQUA (proprietary polar alkyl blend), C12, RP18‑amide, and PFP. For more information see Evosphere on the 

GL Sciences introduced a new SPP-based C18 column branded as InertCore Plus C18. According to the company, the InertCore Plus C18 columns are subjected to rigorous internal quality control (QC), resulting in superior reproducibility because of shell layer homogeneity. More information can be found at the 

 website. The company also launched InertSustain PFP, a column based on 1.9-μm, 100 Å, and FPP architecture. The column is recommended for highly polar molecules.

Nacalai Tesque, Inc. introduced the Cosmosil RNA-RP1 column for the separation of large ribonucleic acid (RNA), messenger RNA (mRNA), and deoxyribonucleic acid (DNA) analytes, a major area of focus for the analytical community over the past few years. The column is based on a 5-μm, “super wide” fully porous silica particle modified with C18. More information can be found at the 

A new entrant this year, Scion Instruments, released Scion Hysil columns to the market. With both a C8 and C18 surface chemistry, the Hysil phases use a “hybrid” substrate that has a particle size of 5 μm and a 120 Å pore. The company reports a pH range of 1–12 and notes that the columns are versatile for reversed-phase applications. It is also conveyed that the C18 is compatible with fully aqueous mobile phases. More information can be found on the 

United Chemical Technologies released a line of their own SPP‑based columns over the past year. The columns are based on a 2.7-μm, 90 Å particle that includes C18, pentafluorophenylpropyl (PFPP; note that the PFP designation may or not include the propyl spacer), and DA surface chemistries. The C18 is noted as being suitable for most acidic, neutral, and basic compounds. In addition to reversed‑phase applications, the PFPP can be used in both normal‑phase and HILIC separations, and provides excellent retention and selectivity of amine-containing compounds as well as charged bases. The DA phase is described as a proprietary polyaromatic phase suitable for aromatic, conjugated, and unsaturated analytes. It is also noted that the DA phase has the ability to analyze compounds that are difficult to resolve on C18. For more information, please refer to the 

ColumnTek, LLC continued to build their portfolio by introducing Enantiocel AA (L). The column is intended for the enantioseparation of amino acids, hydroxy acids, and amino alcohols via a ligand-exchange mechanism using copper ion interaction. This is a silica-based column that has been modified with (L) penicillamine and is available in 3- and 5-μm particle sizes.

Daicel launched the Chiralpak IM column, an immobilized cellulose tris (3-chloro-4-methylphenylcarbamate) based on fully porous silica. The company notes that this is an immobilized version of their Chiralcel OZ phase and it provides expanded solvent compatibility. The columns, available in both 3- and 5-μm particle sizes, are suitable for supercritical fluid chromatography (SFC), normal‑phase, reversed-phase, and polar organic high performance LC (HPLC) and ultrahigh-pressure LC (UHPLC) modes. For more information, please visit the 

Regis Technologies, Inc. also introduced a new set of columns over the past year. The Whelk-O1 Core columns are built on a 2.7‑μm SPP silica with their Whelk-O1 chiral stationary phase (based on 1-[3,5-dinitrobenzamido]-1,2,3,4,- tetrahydrophenanthrene), which results in faster and more efficient separations compared to similar conventional fully porous silica columns. It is also notable that the phase is available in both R,R and S,S forms should specific elution order be required. More details may be found at the 

To go with their newly released reversed-phase columns for oligonucleotide analyses, Nacalai Tesque Inc. also launched Cosmosil RNA-SEC-1000 and Cosmosil RNA‑SEC-2000 for the separation of RNA molecules based on size. The company notes that the 5-μm FPP columns are modified with a “hydrophilic functional group”. For more information on these and related products, please visit the 

Shodex launched a new SEC column called Protein LW-403 4D. The column is described as a 1.9-μm SPP phase with a hydrophilic surface that is suitable for analyzing proteins with high molecular weight. For additional details, please visit the 

Tosoh Bioscience recently commercialized the TSKgel UP‑SW3000‑LS SEC column. The 2-μm, 250 Å, diol-modified FPP silica-based column is intended for the analysis of biomolecules with SEC–multiangle light scattering (MALS) and SEC–mass spectrometry (SEC–MS). The column features fast equilibration times, high robustness, and lot‑to‑lot reproducibility. Low “shedding” reduces noise in MALS applications. For more information, please visit the 

Advanced Materials Technology, Inc. introduced the Halo 1.5 column line of columns for both small molecules, peptides, and proteins based on their 2.0‑and 2.7-μm SPP technology. The 1.5‑mm i.d. columns are reported to provide increased sensitivity from conventional UHPLC systems, higher ionization efficiencies from LC–MS systems, and higher efficiencies compared to 1-mm i.d. columns on UHPLC systems, while reducing solvent consumption (50% compared to 2.1-mm i.d.). According to the company, it is an “easy to implement” microflow solution. The columns are available in multiple surface chemistries for small molecules including C18, LPH (a sterically-protected C18), and AQ‑C18 (aqueous-compatible C18) in their 90 Å pore size material. For peptides, an ES‑C18 chemistry is available in the 160 Å format. A C4 and a diphenyl phase are also available for protein analyses utilizing a 1000 Å pore size. Additional information may be found at the 

Focusing more on peptide and protein analyses, MilliporeSigma also introduced a line of 1.5-mm i.d. columns under the name BIOshell 1.5-mm i.d. UHPLC columns. The company notes that the 1.5-mm i.d. geometry enables the chromatographer to operate at lower flow rates (saving solvent) without compromising efficiency or sensitivity. The SPP structure, combined with wide pore diameters, enables rapid and high efficiency separations of peptides, monoclonal antibodies (mAbs), bispecific antibodies, and antibody–drug conjugates (ADCs). This line of columns includes C18 (160 Å), C4, and diphenyl (both 1000 Å) surfaces on a 2.7-μm SPP substrate. For additional information, please visit the 

Bruker Scientific LLC has developed three new capillary columns geared towards peptide analyses. The columns are based on 1.5-μm, 100 Å fully porous C18 and endcapped particles that differ in dimensions. PepSep Max dimensions are 25 cm (length) × 75 μm (i.d.), whereas the PepSep Ultra and the PepSep Xtreme are 15 cm × 150 μm and 25 cm × 150 μm, respectively. The columns also feature “interchangeable tips”, which reportedly offer robust “twist on and off” connections. More information can be found at the 

Thermo Fisher Scientific introduced the μPAC Neo HPLC columns, described as micromachined 2.5-μm diameter, C18-modified pillars in a silicon wafer. Columns are available in 50 cm and 110 cm lengths and are intended for nanoLC–MS proteomics. Please visit the 

Shimadzu launched a large number of columns under the name Shim-pack Scepter Claris over the past year. The commonalities are a bioinert coating applied to the column body and frits, as well as a wide pH range feature. A variety of pore sizes (80, 120, and 300 Å) as well as a number of particle sizes (1.9, 3, and 5 μm) are available. Surface chemistry options include C18, HD-C18 (high density), C4, C8, Diol-HILIC, PFPP, and phenyl. For which phases are available on given supports, please visit the 

 website. The company recommends the columns for analyzing metal-coordinating analytes and hydrophobic compounds such as nucleic acids, proteins, and lipids that adsorb to stainless steel surfaces.

Waters Corporation launched a number of new lines of columns with their metal-passivated column hardware termed MaxPeak High Performance Surface technology. Common to each of these newly branded columns is the Premier designation. Under the Acquity and XBridge Premier BEH banners (hybrid particles 1.7 and 2.5 μm, 130 Å), C8, phenyl, and hydrophilic interaction liquid chromatography (HILIC) (bare BEH particles) phases were developed. The C8 is noted as being less retentive than standard C18 phases and exhibiting high pH stability. The phenyl phase provides complementary selectivity to C18 phases, especially for polyaromatic compounds, and exhibits the same high pH stability as its C8 kin. The HILIC phase also shows pH stability and is used for highly polar compounds. Under the name Acquity and XSelect Premier HSS columns, both a C18 and a PFP phase were launched. The columns are built on 1.8- and 2.5-μm, 100 Å FPPs, with high coverage noted. The C18 exhibits high retention and exceptional peak shapes for bases, whereas the PFP provides alternative selectivity to C18 and separation of closely related compounds with sharp peaks for bases. On the Cortecs Premier line (1.6- and 2.7-μm, 90 Å or 120 Å SPP supports), three chemistries were released, including C18, C18+, and T3. The C18 is described as a “full coverage C18 phase with endcapping”, and it is intended for general use. The C18+ adds a weak anion-exchange character to the C18 that produces excellent peak shapes for bases when using formic acid or other low ionic strength acidic mobile phases. The T3 is noted as a “mid‑coverage C18 phase, with endcapping”. The column is recommended for both nonpolar and moderately polar molecules, and it is 100% water-wettable. More details can be found on the 

 website. For large molecules, Waters released the Acquity Premier and XBridge Premier Protein BEH C4 columns. The pH-stable hybrid particle columns are available in 1.7‑and 2.5-μm particle sizes with a 300 Å pore size. The C4 surface chemistry, combined with the particle and column technology, results in a column line suitable for intact mass and sub-unit analysis of proteins, including those that are phosphorylated. See the 

YMC Co., Ltd. introduced their own line of bioinert columns labelled YMC‑Accura Triart. The columns are built on inert hybrid silica particles (1.9-, 3-, and 5-μm and 120 or 300 Å pore sizes) and packed in hardware (columns and frits) deactivated with a bioinert coating. The columns are noted for their high pH and temperature stability and are recommended for oligonucleotides, (phospho)peptides, (phospho)lipids, and other coordinating substances. Stationary phases include C18, C18 ExRS (extended pH and stability), Bio C18, Bio C4, phenyl, PFP, and a diol phase for HILIC separations. For additional information, please visit the 

Knauer released a new line of column heating devices with varying capacities and features. Two channel systems for heating of the eluent are noted. For further details, please visit the 

MicroSolv Technology Corporation introduced a line of polyetheretherketone (PEEK) column hardware. According to the 

 website, their all-PEEK HPLC column hardware is extruded from Victrex PEEK for maximum reproducibility and the finest chromatography. A small amount of graphite (20%) is added to the formulation in order to achieve a black surface sheen. There are no other chemical or physical treatments added to the process, making these columns more biocompatible. Columns, frits, and endfittings are available in 2-, 4-, and 7.5-mm i.d. formats.

Optimize Technologies released EXP Ti-REX fittings, a new hand-tight tip‑sealing solution. The reusable fittings are used for connecting PEEKsil and fused silica tubing and are rated to 20,000 psi.

Restek Corporation launched the Sidewinder LC column heater. According to the company, the unique design completely encloses any LC analytical column up to 25 cm in length. Two lengths of heater jackets are available where the short column holder accommodates columns up to 10 cm in length, whereas the long column holder holds columns up to 25 cm in length. The control module provides optimum heating performance (accuracy to within 1 °C) and stability to within 0.1 °C. The Sidewinder controller has fast 10 Hz sampling for improved responsiveness. The RS232 control allows external programming. For additional information, visit the 

Novilytic developed a new proteomics kit that works with your existing HPLC system. The Proteometer-L kit is designed for rapid analyses of intact IgG and IgG fragments containing the Fc region in a harvested cell-free culture filtrate (CFF). The kit measures (mAb) titer and relative aggregate content without the need for sample purification. According to the company, the kit offers the following advantages over existing methods for determining mAb titer and aggregate content: two critical quality attributes in one assay as critical quality attributes can be directly analyzed in crude culture filtrate (no additional purification needed), linear response from 0.5–16 mg/mL mAb, very small sample volume required (minimum 0.5 μL), and fast (10 min) runtime. For more information, please visit the 

Many of the new products and product lines introduced for LC over the past year are either new product line extensions (new chemical surfaces for existing brands) or whole new series based on advanced particle technologies, column hardware developments, or both. Some of the released lines are new to the companies, but they are based on established particle designs (SPP, for example). In another case, a new product line has been launched based on a new monodisperse, fully porous particle. Other new product lines are the result of new or newer technology combinations. For example, there are a few cases where SPP technology and hybrid particle designs are being combined to form improved or alternative column lines. In other products, new column formats are being combined with advanced particle technologies. It is also noted that the trend towards more inert column hardware continues to be significant based on the several collections of columns that have emerged. It is clear that LC vendors continue to put emphasis into new particle and column hardware technologies, as well as continue efforts to best combine these advances to tackle an assortment of separation challenges.

(1) Bell, D.S. New Chromatography Columns and Accessories for 2020. 

(2) Bell, D.S. New Liquid Chromatography Columns and Accessories for 2021. 

(3) Bell, D.S. New Liquid Chromatography Columns and Accessories for 2022. 

(4) Muraco, C.E.; Bell, D.S. Highlights from the 50th International Symposium on High Performance Liquid Phase Separations and Related Techniques (HPLC 2022). 

is a research fellow in Research and Development at Restek. He also serves on the EAB for 

and is the editor for “Column Watch”. His main objectives have been to create and promote novel separation technologies and to conduct research on molecular interactions that contribute to retention and selectivity in an array of chromatographic processes. His research results have been presented in symposia worldwide. Direct correspondence to: 

Events

This article covers LC columns and accessories commercially released after Pittcon 2022 through the 2023 conference held in Philadelphia, USA.

New LC Columns and Accessories: What to Know for 2023

This yearly report on new products introduced in the preceding year, since March 2022, covers sample preparation instrumentation, supplies, and accessories.

Our annual review of sample preparation products covers the previous year, since March 2022. However, we’ve offered a little latitude for products introduced since the pandemic. It is becoming increasingly more difficult to identify new products. Several vendors are avoiding exhibiting at general analytical conferences in favour of more focused meetings. Others, due to the pandemic or following other trends, are increasing their marketing via social media or other digital means. In late 2022, the LCGC staff submitted a survey to vendors of sample preparation products. Responses to this survey are compiled in this review, as are new product introductions during the past 12 months noted via direct mailings, email, and other means. Additionally, vendor exhibits at the 2022 Eastern Analytical Symposium and Pittcon 2023 were visited. During the pandemic, many vendors slowed research and development (R&D) activities. This year demonstrated a rebound in products resulting from more intensive R&D. Sorbents and accessories for solid-phase extraction (SPE) led the way, as in previous years.

This review is presented in four sections. First, a journal devoted to sample preparation is discussed. Next, SPE sorbents and products are discussed. Third, instrument-based sample preparation technologies are presented. Finally, attention is turned to other sample preparation accessories and supporting technologies. To assist the reader with some of the details behind these new products, each section presents a tabular summary of the associated products. In all cases (except the new journal), the new products we uncovered are presented in the annotated table, while the text highlights particularly worthwhile products. 

Researchers developing sample preparation technologies and methods have published their progress in an assortment of general analytical, chromatography, and separation science journals in the absence of a dedicated sample preparation journal. Elsevier addressed this need with the introduction of an open access journal, 

. The journal website (1) describes the journal as:

“(P)ublishing high-quality original research, reviews, tutorials and opinion articles covering with all aspects of sample preparation. The journal welcomes the submission of original manuscripts on all fundamental and applied aspects of sample preparation such as new extraction principles, new materials, new extraction techniques or technologies, fundamentals related to operational understanding and optimization, miniaturization, automation, hyphenation, chemometrics, green sample preparation, and impactful applications in e.g. environmental, food and beverages, pharmaceutical, bioanalysis and biomedical. Contributions aiming to evaluate the effect of the various sample treatments using state-of-the-art approaches for the multivariate data analysis of designed experiments are welcomed... (A)ll articles must be consistent with green analytical chemistry principles and propose sustainable practices that will benefit the society and the environment.”

The international editorial board comprises several academic researchers predominantly associated with the Sample Preparation Study Group and Network of the European Chemical Society-Division of Analytical Chemistry, which we described in March 2020 (2).

For several years, the pipette tip approach to SPE has seen increased interest. This year, Millipore Sigma introduced two Dispersive Pipette Extraction (DPX) products. First is the Swift HLB-DPX tips. The pipette tips use a proprietary hydrophilic-modified styrene polymer available in a box of 96 and with a variety of bed weights for greater capacity with lower elution volumes. The product design allows for direct sample loading without the traditional conditioning step. The robust methodology used is resistant to overdrying. Next is DPX tips with HybridSPE technology. The sorbent material is loosely contained within the tip and is designed for the selective isolation of phospholipids from biological matrices. Following protein precipitation with acetonitrile, biological samples are extracted with the DPX tip for targeted removal of endogenous phospholipids.

Based on the established Empore technology, CDS Analytical developed micropipette Stage Tips for the rapid, small-volume desalting and fractionation of peptides and proteins. The products are housed in chemically resistant polypropylene material for use with organic solvents or acidic or basic conditions. The NanoPak-C line of SPE micropipette tips from Millennial Scientific is expanded with two new offerings. Nanopak-C Monolith SPE Micro-Pipette Tips incorporates monolith SPE principles to extract and purify femtomole to picomole amounts of analytes for chromatographic or mass spectrometric or polymerase-chain reaction of small molecules, proteins, RNA, or DNA. The dispersive product, NanoPak-C Dispersive SPE Micro-Pipette Tips, features similar attributes with a porous-carbon network. The sterile and autoclavable product permits use in aseptic environments and may be applied to COVID-19 testing and research. Moving to traditional SPE, GL Sciences built on their InsertSep product line of SPE cartridges. InertSep MA-2 is a methacrylate-based weak anion exchanger in a six-millilitre cartridge with a 150-mg bed. The InertSep MA-2 has only ion-exchange mode capability—for example, for the selective extraction of ionic species such as acidic polyfluoroalkyl substances (PFAS). A combined weak ion exchange and reversed-phase material is available in the InertSep WAX FF. The base material is polymeric SDB for application to PFAS and acidic compounds.

 (NBE), is a development from Tecan and features a unique dual-chamber design that combines a reaction vessel with an SPE column (see Figure 1). The upper portion of the column is the reaction reservoir, which is separated from the microparticulate SPE sorbents with a novel pocket seal air lock and filter. When positive pressure is applied, sample can flow from the reaction reservoir. The reservoir can be used for digestion or incubation prior to SPE.

In the high-throughput well-plate format, Orochem introduced the Panthera Delux MicroElute Plate featuring both normaland reversed-phase chemistries. The plate is in the 96-well format and provides a high sorbent bed and minimal void volume. It is compatible with most liquid handling systems. Another alternative format of SPE is the in- or on-needle approach. The μSPEed Cartridge introduced by ePrep features a one-way valve that allows sample to aspirate through the needle-mounted cartridge into the syringe and focus of analytes onto the sorbent bed. A wide variety of phases are available. The μSPEed takes a step towards automation with the ePrep digiVOL. The digiVOL is a handheld digital syringe capable of dispensing 0.1 μL to 5 mL of aqueous or organic solvents. The syringe is controlled with an app that also features a calibration standards mode. The final area of SPE products are automated workstations. Three new products emerged in this area. First is the Thermo Scientific AccelerOme sample preparation platform automating sample preparation for liquid chromatography– mass spectrometry (LC–MS) proteomics. Pre-built validated methods and kit-format reagents reduce error, along with UV measurement of peptide concentration prior to LC–MS analysis. The Gerstel FreeStyle Automated SPE workstation is a modular design for ease of addition of a variety of capabilities with over 40 sample vessel and SPE column racks and up to 10-litre samples. The workstation can accommodate up to 15 solvents. Finally, UCT developed the HydraFlow four-channel cartridge manifold for water extractions. The liquid channel-switching design allows transition from sample collection to waste discharge.

Table 1 provides a summary of each of these SPE products.

Automated Sample Preparation Workstations and Instrumentation

A number of new instrument-based sample preparation systems were developed in the review period. Based on the number of new products, the most popular dealt with thermal analysis methods. CDS Analytical introduced its 6000 series of pyroprobes. The 6150 pyroprobe interfaces with any standard gas chromatograph (GC), and supports multi-ramp and multistep capability up to 30 temperature profiles per sample. Meanwhile the 6200 pyroprobe possesses a built-in focus trap using reactant gas, such as oxygen or air, to collect analytes. A thermal desorber for dynamic headspace sampling is available with the 6200. The Gerstel Thermal Extractor separates pyrolysis and related thermal extraction methods from thermal desorption so the thermal desorption and pyrolysis may be performed sequentially on the same sample. Optimized sample holders for liquids and solids are available. EST Analytical presented three new systems for volatiles analysis. The Evolution2 purge and trap concentrator provides higher resolution than previous models. By using an eight-port valve, the desorbed sample goes directly to the gas chromatograph. Also for purge and trap analysis, the Centurion has separate processing stations for soils and water. Multiple samples can be extracted from the same sample vial. The Nexis autosampler platform allows combustion analysis, sample handling up to 250 μL, and total sulfur analysis. An Italian company, HTA, presented the 2000H series of headspace autosamplers powered with artificial intelligence (AI). The AI support optimizes instrument parameters, tracks use of consumables, and predicts maintenance needs. Markes International’s Centri-90 is an automated sample concentrator for headspace trapping, solid-phase microextraction (SPME), and SPME-Arrow enrichment. The system is applicable to food safety testing, volatile organic compound analysis, hazardous air pollutant monitoring, and other applications.

Three digestions systems are featured during the past year. CEM presented the Blade and the MARS 6 microwave digestion systems. The Blade offers advanced vortexing and a single-model cavity for high watt density. The MARS 6 uses contactless in situ light-emitting technology for superior temperature control. Twelve samples may be digested simultaneously. Waters developed the RapiZyme protein digestion for clean protein digestions based on chemically modified recombinant trypsin.

Two systems for dried-blood spot (DBS) analysis are recently brought to market. The Thermo Transend DSX-1 DBS system features a flow-through desorption to streamline sample workflow. No need for disc punching of samples. Captainer AB introduced the Captainer B 50 microsampling card for DBS analysis. DBS can be placed into a 96-well plate for extraction.

Thermo Fisher developed Extreva Accelerated Solvent Extraction (ASE) as an improvement to previous systems by combining sample extraction, in-cell cleanup, and solvent evaporation. The ePrep Sample Preparation Workstation includes filtering, SPE, microSPE, liquid–liquid extraction, and robotic syringe cleanup in an autosampler rack.

Table 2 provides a summary of these automated sample preparation instruments.

Sample Preparation Accessories and Related Products

The field of sample preparation is so broad, and there are any number of established and emerging technologies identified as sample preparation, that commercial developments and new product introductions are often in seemingly scattered areas. An attempt is made here to unify these varied product offerings.

Advanced Material Technology designed the DandyVice sample preparation filtering aid for ergonomic considerations in routine laboratory tasks. Membrane Solutions introduced a series of filtration products including 96-well plates, efficient endotoxin removal liquid filters, 15-mL centrifuge filters, MedCap UCT PES capsule filters, and PES Black Gridded membranes. Orochem Technology Cell Lysate Filters are a 96-well plate for clarification of cell debris, colloids, precipitates, and aggregates from target proteins.

The Organomation Nitro-Gen converts a compressed air stream to 95–99% pure nitrogen. The KineTek Analytical FlexMixer is a multiple gas blending and diluting system for calibration of gas analyzers, exposure control studies, or gas storage.

Sampling Systems introduced SteriWare Ultra HDPE sampling scoops, which are sterilized and double bagged. Table 3 provides a summary of these sample preparation accessories and related products.

The post-pandemic timeframe is seeing an uptick in sample preparation technologies from users, vendors, and suppliers. New developments in the past year are reviewed and expected to be the harbinger of continued developments in the next few years. With further development, definitions of future trends will emerge.

https://www.sciencedirect.com/journal/advances-in-sample-preparation/

(2) Raynie, D. E. Sample Preparation in the Rearview Mirror: Looking Back on 2019. 

This yearly report on new products introduced at Pittcon and in the preceding year covers sample preparation instruments, supplies, and accessories.

New Sample Preparation Products and Accessories for 2023

 spoke to Leon Barron from Imperial College London, UK, about his work developing a fully miniaturized workflow incorporating a three-dimensional-printed passive sampler device (3D-PSD) to monitor contaminants of emerging concern (CECs) in river water.

Q. You have recently developed a fully miniaturized workflow incorporating a three-dimensional-printed passive sampler device (3D-PSD) to monitor contaminants of emerging concern (CECs) in river water (1). What chemicals of emerging concern are you investigating and why?

 Our research interests lie in improving the understanding of the occurrence, fate, and potential impact of chemicals in the environment. As approximately 350,000 chemicals are currently licensed for manufacture and sale globally (2), this sets a very large challenge to us as analytical chemists. Indeed, it has been estimated that we have exceeded our safe threshold for “novel entities” such as chemicals to ensure healthy and sustainable life on Earth, and this issue arguably represents the third greatest planetary crisis behind climate change and biodiversity loss (3). Of all of these substances, chemicals of emerging concern (CECs) are those that have been detected in the environment but that do not fall under any regulatory framework, with concerns being held about their potential environmental impacts. This work was the focus of Alexandra Richardson’s PhD (1). We focused on a more “modest” set of (just) 164 substances, including drugs (both medicinal and illicit), personal care products, and pesticides, as well as some of their metabolites and transformation products.

We have traditionally focused on CEC residues mostly in water, including wastewater, surface and ground water, and drinking water, and we generally see that chemical residues pervade all of these aquatic compartments. We then assess the environmental risks, including through biomonitoring, and effect assessment in selected species. Recently there has been an increased concern in the UK about untreated wastewater, including “combined sewer overflows” (CSOs), where the wastewater in sewers and the capacity to treat it is exceeded and it is discharged directly into river catchments. We have been trying to better understand the scale of this issue from a chemical residue perspective (4). Still one of our most difficult questions to answer is, “Where should we take a sample to reliably give us the information we need to identify and mitigate CEC sources?”. Many sources of chemicals in the environment are not as obvious and may be diffuse in nature. Understanding what, where, when, and why these chemicals occur in the environment is therefore critical to understanding their potential risks.

Q. Why did you decide a 3D-PSD was necessary for this type of analysis? What benefits does this device offer compared to previous methods?

 Traditionally, most disk-type passive samplers have used a standard 47 mm format, but nothing prevents these being bigger or indeed smaller. In our case, we wanted to make something smaller, cheaper, multifunctional, and more suited to larger-scale monitoring programmes. At the time, we thought that the fastest way to make prototypes was to use 3D-printing because we did not have access to a workshop or any advanced manufacturing equipment. As a result, concept designs were made into working prototypes to trial on the same day. It was not initially our intention to just focus on 3D-printed materials in the final design, but in the end, the “print-on-demand” flexibility it offered, along with its performance, meant that we have kept using it. In our open access paper, we provided the computer-aided design (CAD) files for anyone to 3D-print and trial the final design themselves, as well as a step-by-step guide on how to use it. The materials cost for each device including a compatible transport cap was <£3 GBP and the print time was 1.93 h. Of course, these designs can be made from other materials if needed, and also using different manufacturing techniques. We have since made a Teflon-based design that works well too, but this was milled, not 3D-printed. The most important benefit of this new miniaturized design was the ability to configure up to five different sorbents in the same disk. We have performed extensive testing on hydrophilic-lipophilic balanced (HLB) sorbents, as well as anion and cation exchangers in river water. Over ten 9-mm disks could be punched out from just one commercially available 47-mm sorbent disk—enough for two fully loaded 3D-printed passive samplers. When different selectivities were combined, this expanded the chemical space for these samplers, and also provided added confidence for detection of new substances with increasing orthogonality of the sorbents used. There were several other advantages including, but not limited to: (a) reduced plastic usage—we only print what we need, when we need it and with no supplier delivery lead time; (b) shipment to and from sites is very cost-effective if needed; (c) they can be reused several times (cleaning in a small volume of methanol is enough to re-deploy and we have used some over 10 times); (d) manufacturing of devices was performed on a standard, relatively low cost and readily available benchtop 3D-printer; and (e) the design can be easily modified if needed to include more sorbent chambers, shape, or functionality for different applications, for example, for microbiological characterization.

Q. What were the main challenges involved in developing the 3D device? Could this device be adapted for other types of environmental analysis?

) is correlated with sampler surface area, one of the biggest challenges was how to manage a reduction in sensitivity in comparison to standard 47 mm samplers. Rapid advances in mass spectrometry (MS) mean that direct injection analytical methods are now emerging that can enable detection in water at the ng/L concentration level, even using standard injection volumes of 10 µL. We developed analytical methods using highly sensitive MS instruments to recover much of the losses due to reduced surface area (5,6). In addition, as multiple sorbents can be housed in a single device, pooling extracts from multiple disks increases sampling surface area and therefore sensitivity, if needed to. As the smaller 9 mm disk sorbents are “punched” out from a larger commercially available disk, we have found that the sensitivity can actually be tailored to fit the analytical system performance, as R

 seemed scalable from those generated on traditional 47 mm passive samplers of similar design.This is also useful where the number of compounds for which usable R

 data from the literature can potentially be used in a semiquantitative way to extrapolate mass on sampler to concentration in water.

Aside from sensitivity challenges, we wanted to use resin materials in the housing that would be compatible with trace analysis and with typical reagents, such as acids, bases, and organic solvents. Before designing passive samplers, we were working on another project focusing on 3D-printing of fritless solid-phase extraction (SPE) cartridges for sample clean-up in forensic explosives analysis, which laid the foundations for this work (7). Ten different resins were trialled using both stereolithography and fused filament deposition 3D-printing techniques. We found that stereolithography printing in a specific methacrylate-based resin offered the best results in terms of low chemical-leaching and chemical reagent stability, and resulted in a non-porous, robust, and reusable device with high degrees of reproducibility. One perceived drawback is that the methacrylate housing also later showed surface accumulation of CECs—especially those with log

 > 3. This sorption to the housing did not markedly affect uptake onto the embedded sorbent disks. However, given that the material was so stable in most organic solvents, as well as mineral acids and bases, 9-mm sorbent disks could actually be printed in this same resin material for passive sampling of these less polar substances. In other preliminary work, we were also able to further functionalize its surface to potentially tailor selectivity further in the future.

More recently, we have found these to be adaptable to related and even other environmental applications and compartments. For example, we found that, when fitted with multiple sorbent chemistries, these show promise as surrogates for biomonitoring, particularly for benthic invertebrates under exposure to CECs. This addresses the “3Rs” principle to Reduce, Replace, and Refine the methods we use for animal testing. Interestingly, we’ve already trialled these in “Citizen Science” type studies working with local communities to deploy samplers in their catchments and these seem to work pretty well at scale. We’ve already assessed several urban catchments with the help of local environmental conservancy groups to help them understand where pollution sources occur, and the feedback has been largely very positive. Furthermore, these samplers are currently being fitted with different sorbent materials more suited to air sampling—both as proxy and wearable devices. For example, we are currently working on the NERC-funded WellHome project (8) where we have adapted these samplers for indoor measurements of (semi)-volatile organic chemicals in homes in the White City area in London using comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–TOF-MS) and liquid chromatography quadrupole time-of-flight mass spectrometry (LC–QTOF-MS). In addition, we are starting to explore their potential application in the terrestrial environment to understand chemical transport and impacts on microbiology. For example, we are working on another NERC-funded project focusing on the role that compost plays in fungal resistance to azoles.

Q. Can you elaborate on the other steps in the analytical workflow that were miniaturized for investigating CECs?

 Interestingly, this relatively simple decision to scale-down the passive sampler resulted in many other opportunities for miniaturization—some of which really made a major impact. First, reducing the scale of passive samplers massively reduced the calibration and extraction requirements, especially the volume of solvents, energy consumption, and need for larger bespoke rigging. Traditional 47-mm passive sampler disks use 30–50 mL of organic solvents such as methanol for extraction of CECs. In this case, we required only 1.6 mL per disk. An added advantage was that disks fitted neatly into commercially available single-use SPE cartridge barrels for extraction in a standard manifold. The larger passive samplers often require tailored apparatus for this that require rigorous cleaning in between extractions. As already described above, more sensitive LC–MS/MS was required and much of this focused on miniaturization of some component steps (5). For example, the separation of all CECs was performed using a 3 × 5 mm biphenyl guard cartridge. This meant that rapid separations for quantitation could be performed for 164 CECs and SIL-IS in ~5 min using only 2.75 mL of mobile phase per run, enabling 260 injections per day. The rapid organic solvent gradient boosted sensitivity and the small injection volume (10 µL) and standard flow rate (0.5 mL/min) gave an optimized dilution factor on-column to maximize the sensitivity even further down to the single digit ng/L concentration level in river water matrix. What was also critical here for robust quantification was fast MS. We were able to define 20–30 datapoints per chromatographic peak for each of two transitions per compound, as well as one transition for 36 stable isotope internal standards. Measurement was also very stable across large sequences, even in very dirty samples like influent wastewater (yes, we do routinely inject untreated wastewater into our LC–MS/MS systems and have done so for about five years now). The relatively higher organic proportion in the LC eluate across the day, coupled with a low injection volume, keeps everything surprisingly clean.

Although this analytical method was applied to river water passive sampler extracts in this work, it was also required to measure the CEC concentrations in exposure water (here, <50 ng/L) during calibration and to derive R

. Normally, at this concentration level, SPE is often required for robust measurements, which, in turn, requires a much larger calibration tank to yield enough water for CEC preconcentration. This direct injection approach meant that we were able to calibrate 60 passive sampler disks in just a 5 L beaker. The knock-on effect and beyond of this passive sampling work is that our requirement for freezer storage space for water samples has been significantly reduced, and we can perform a full standard addition quantification on most water samples, if required, on as little as 1 mL of water with N>5 calibrants using the same sample as a matrix. A single upright freezer can therefore house thousands of samples! We also use the same LC–QTOF-MS approach to perform direct injection suspect screening at the same level, albeit with a slightly higher injection volume (40 µL) and a longer 10 cm column for added selectivity for larger numbers of CECs.

Q. What were your main findings using this method in practice?

 We conducted a one-week deployment in summer 2021 in the River Wandle (South London, UK) and at two sites down- and up-stream of a major wastewater treatment facility and on a river with multiple CSO vents discharging multiple times a year. Surprisingly, for such a small, low-cost, and relatively simple device, we managed to detect quite a few CECs. In total, we detected 80 CECs using our targeted LC–MS/MS methods and a further 113 using suspect screening with LC–QTOF-MS on the same extracts. Of the first 80, six were European Union Water Framework Directive priority substances*/watch-list compounds (atrazine*, simazine*, sulfamethoxazole, terbutryn*, trimethoprim, and venlafaxine). In comparison, only 41 CECs were detected in the water samples using direct injection LC–MS/MS, showing the added sensitivity gains offered by the passive sampler. However, this direct injection method for water analysis was still very useful to rapidly identify where exactly the wastewater treatment plant discharge point was and to know where passive samplers should be deployed. In this preliminary work, we managed to calibrate the samplers for 39 CECs and were able to generate time-weighted average concentrations for 10 CECs detected in extracts from the river using R

data and were able to compare this with concentrations measured in the water directly. Concentrations were generally very similar to those measured in water, and errors of 23 ±17 ng/L were recorded for eight compounds, which was very encouraging. However, three- to fivefold higher concentrations of two compounds (venlafaxine and lidocaine, respectively) were found in passive sampler extracts by comparison. However, it was also unclear whether the grab samples of water taken before and after deployment were actually representative of the full weeks’ CEC exposure in the water, especially with respect to any unknown CSOs that may have occurred during that time. In addition, laboratory R

 data can only go so far in terms of representing CEC uptake on passive samplers in the environment and is dependent on temperature, salinity, and biofouling, among additional variables.

Even more interesting was that this was one of the first studies not only to deploy a 3D-printed passive sampler for so many CECs in the environment but also that successfully performed suspect screening on the extracts to potentially understand a wider complement of CECs in the catchment. Of the 113 compounds tentatively identified in the extracts (almost 1/10th of our library complement), 69 were pharmaceuticals, 20 were pesticides, 12 were pharmaceutical metabolites, three were controlled drugs, and two were controlled drug metabolites. Importantly, a large difference between the CECs detected above and below the wastewater treatment plant was observed (38 upstream and 98 downstream). Taking into account that other monitoring programmes have focused on the London area, this was a larger number of compounds detected by comparison and prompts a concern about the risks of contamination, particularly in the smaller tributaries of the River Thames where dilution factors may be low in receiving water for wastewater and CSO discharges. Two interesting examples are shown in the paper relating to the azoles, fluconazole and fipronil, both now included on the European watch list (9). In particular, the Norman network currently reports that fipronil’s lowest predicted no-effect concentration is 0.77 ng/L. In the UK, Environment Agency monitoring data has estimated concentrations of fipronil to be 10 ±20 ng/L (from 2013–2021), and so is of particular concern. It was detected in all extracts and at higher signal intensity in the downstream site. Fipronil is used generally as a veterinary parasiticide, and recent concerns around the practice of prophylactic treatment of companion animals have been raised, as it occurs frequently at wastewater discharge sites (10).

 As one of the main challenges with CECs in the environment is scale, manufacture of these miniaturized passive samplers devices and the high throughput and sensitive analytical methodologies are vital, but they also create another scale-related challenge: data. In all, we now monitor 300–400 CECs quantitatively at high frequency using a small number of targeted LC–MS/MS methods, and >1200 qualitatively with LC–QTOF-MS using fully curated libraries of retention time, MS, and MS/MS. However, this is really only the tip of the iceberg. To extend our suspect screening capability further both in water and with our passive samplers, we have recently used open access LC–HRMS libraries of tens of thousands of compounds, together with machine learning-based retention time prediction tools that we have developed in-house, to help short-list new compounds for confirmation (11). This approach has been effective at reducing the number of viable suspects by roughly two-thirds in comparison to using full-scan HRMS data alone. We are now also using nontargeted analysis to potentially understand chemical contaminants in drinking water, and this brings with it several more challenges of scale that may benefit from use of these passive samplers (12).

Like many environmental chemists, our analyte list is expanding rapidly to include many more classes of chemicals, and now includes “forever chemicals” such as perfluoroalkyl substances (PFAS), disinfectant by-products, endocrine disruptors, plastic-related, and industrial chemicals among many others. Reliable data handling workflows for untargeted and suspect screening are now our biggest challenge and there is tremendous opportunity to work in an interdisciplinary way to better understand the meaning of the data we generate using these chemical analysis tools for global environmental health.

(1) Richardson, A. K.; Irlam, R. C.; Wright, H. R.; et al. A Miniaturized Passive Sampling-Based Workflow for Monitoring Chemicals of Emerging Concern in Water. 

, 156260. DOI: 10.1016/j.scitotenv.2022.156260

(2) Wang, Z.; Walker, G. W.; Muir, D. C. G.; Nagatani-Yoshida, K. Toward a Global Understanding of Chemical Pollution: A First Comprehensive Analysis of National and Regional Chemical Inventories. 

, 2575–2584. DOI: 10.1021/acs.est.9b06379

(3) Lawton, G. Why Chemical Pollution is Turning into a Third Great Planetary Crisis. 

(4) Munro, K.; Martins, C. P. B.; Loewenthal, M.; et al. Evaluation of Combined Sewer Overflow Impacts on Short-Term Pharmaceutical and Illicit Drug Occurrence in a Heavily Urbanised Tidal River Catchment (London, UK). 

, 1099–1111. DOI: 10.1016/j.scitotenv.2018.12.108

(5) Ng, K. T.; Rapp-Wright, H.; Egli, M.; et al. High-Throughput Multi-Residue Quantification of Contaminants of Emerging Concern in Wastewaters Enabled Using Direct Injection Liquid Chromatography-Tandem Mass Spectrometry. 

, 122933. DOI: 10.1016/j.jhazmat.2020.122933

(6) Egli, M.; Hartmann, A.; Wright, H. R.; et al. Quantitative Determination and Environmental Risk Assessment of 102 Chemicals of Emerging Concern in Wastewater-Impacted Rivers Using Rapid Direct-Injection Liquid Chromatography—Tandem Mass Spectrometry. 

(7) Irlam, R. C.; Hughes, C.; Parkin, M. C.; et al. Trace Multi-Class Organic Explosives Analysis in Complex Matrices Enabled Using LEGO-Inspired Clickable 3D-Printed Solid Phase Extraction Block Arrays.

, 461506. DOI: 10.1016/j.chroma.2020.461506

. https://www.imperial.ac.uk/school-public-health/environmental-research-group/the-wellhome-study/ (accessed 2023-04-24).

(9) Commission Implementing Decision (EU) 2022/1307 of 22 July 2022 establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council (notified under document C(2022) 5098) (Text with EEA relevance) C/2022/5098, 

, 117–120. http://data.europa.eu/eli/dec_impl/2022/1307/oj

(10) Preston-Allen, R. G. G.; Albini, D.; Barron, L.; et al. Are Urban Areas Hotspots for Pollution from Pet Parasiticides? 

(11) Richardson, A. K.; Chadha, M.; Rapp-Wright, H.; et al. Rapid Direct Analysis of River Water and Machine Learning Assisted Suspect Screening of Emerging Contaminants in Passive Sampler Extracts. 

(12) Ciccarelli, D.; Christopher Braddock, D.; Surman, A. J.; et al. Enhanced Selectivity for Acidic Contaminants in Drinking Water: From Suspect Screening to Toxicity Prediction. 

, 130906. DOI: 10.1016/j.jhazmat.2023.130906

Featured Interview

LCGC Europe spoke to Leon Barron from Imperial College London, UK, about his work developing a fully miniaturized workflow incorporating a three-dimensional-printed passive sampler device (3D-PSD) to monitor contaminants of emerging concern (CECs) in river water.

Monitoring Contaminants of Emerging Concern in the Environment

The Chromatographic Society (ChromSoc) and the Separations Science Group of the Analytical Division of the Royal Society of Chemistry are pleased to announce 

The meeting has been separated into six separate sessions spread across two days. Fabrice Gritti (Waters, USA), a previous ChromSoc medal recipient, will begin the day with a presentation on the development of vacuum-jacketed columns and future improvements in LC–MS. This will be followed by complementary talks by Simone Dimartino (University of Edinburgh, UK) on the development of 3D-printed devices, and Bert Vankeerberghen (Vrije Universiteit Brussels, Belgium) on novel Si-based formats to obtain perfect order in chromatographic columns—both interesting and novel approaches to improving column performance.

The second session will start with a presentation by Dave Bell (Restek/

’s editor of Column Watch, USA) on the latest advances and trends in column and separation technologies. Jennifer Field (The Open University/Shimadzu, UK) will then discuss column characterization and method development strategies for 1D and 2D analysis of peptides.

The third and fourth sessions will address the challenges of targeted and nontargeted analysis covering a range of hot topics. This will include Giorgio Blom (AstraZeneca, Sweden) and Brunhilde Guessregen (Merck Group, Germany) on the challenges and latest developments in nitrosamine analysis and an overview of methods for their quantification, respectively.

Stefan Bieber (AFIN-TS, Germany) will kick off the second day and will present on the possibilities, advances, and current challenges in untargeted analysis. Martin Jones (University of Birmingham, UK) will follow on nontargeted metabolomics as a new approach methodology for toxicology and chemical risk assessment research.

The analysis of new modalities and biological therapeutics will be the theme of the fifth session. Valentina D’Atri (University of Geneva, Switzerland) will cover advances in the analysis of new modalities, including AAVs and bispecific antibodies, with Raphael Ewonde (Vrije Universiteit Brussels, Belgium) presenting on advances in mAb analysis and optimization of separations using HIC.

In the final session, Lewis Couchman (ASI Ltd/RSC SSG, UK) will provide an update on the progress in ultra-rapid bioanalysis by LC–MS. The final talk will be from Stef Molenaar (University of Amsterdam, Netherlands) on advances in the field of in silico retention modelling and method development (LC and SFC).

The important exhibition and vendor presentation programme will ensure this event will be both thought-provoking and well attended.

For further information on the meeting, submission of abstracts, or sponsorship, please visit: 

https://www.rsc.org/events/detail/76489/emerging-separations-technologies

https://chromsoc.com/category/events-news/

The Chromatographic Society and the Separations Science
Group of the Analytical Division of the Royal Society of Chemistry are
pleased to announce Emerging Separations Technologies 2023 is to
be held 24–25 May 2023, at RSC Burlington House, London, UK.

May 2023 | LCGC Europe

What Are My Options to Improve My Separation? Part 2: Likelihood of Separation, Adjusting Selectivity for Simple Mixtures

Green Chemistry: What Is It (and What Is It Not)? And How Does It Apply to Gas Chromatography (GC)?

New LC Columns and Accessories: What to Know for 2023

New Sample Preparation Products and Accessories for 2023

Monitoring Contaminants of Emerging Concern in the Environment

Event: Emerging Separations Technologies 2023

Suspect Screening of Lupin-Produced Phytotoxins in Environmental Samples