Gas Chromatography Meets Non-Targeted Screening

Enhancing Performance of Analytical Methods

Ion Mobility–Mass Spectrometry (IM–MS)

Bioanalysis of Small-Molecule Drugs and Metabolites in Physiological Samples by LC–MS, Part 1

Where Has My Efficiency Gone? 

Facilitating Gas Chromatography Separation of Complex Mixtures

The Role of Microextraction Techniques 

Roxana Eggleston-Rangel is an application scientist at Phenomenex. She received her bachelors and masters degree in chemistry and biochemistry from California State University (Los Angeles, USA). Roxana is an LC–MS/MS enthusiast currently working on developing LC–MS/MS methods, with a focus on nano- and micro-flow separations.

While mass spectrometry coupled to liquid chromatography (LC–MS) has proven to be a powerful tool in the area of discovery omics (prote-, lipid-, metabol-, food-, and glycomics), omics research is often challenged by the complex nature of its samples. The identification and quantification of molecules of interest can prove particularly difficult when dealing with small amounts of samples and complex sample matrices (biofluids and single-cell analysis). Liquid chromatography column miniaturization can overcome these challenges. Reducing the column inner diameter results in a reduction in chromatographic dilution and increased sensitivity, allowing for more efficient tandem mass spectrometry sampling (MS/MS) and thus a higher number of molecule identifications.

Mass spectrometry coupled to liquid chromatography (LC–MS) has proven to be a powerful tool in discovery omics. MS advances in mass accuracy, sensitivity, resolving power, and scanning speeds over the last 20 years have made it possible to analyze molecules with high confidence and sensitivity (1,2). The coupling of a liquid chromatographic system to a mass spectrometer has allowed the profiling of biological samples by analyzing thousands of metabolites simultaneously. In proteomics, deep proteome coverage and post-translational modification (PTM) identification of phosphorylation, glycosylation, and other sites has been accomplished (3). In the world of foodomics, LC–MS allows for the crucial analysis of foods for nutritional content and contamination, which is of great concern to producers, governmental agencies, and the consumer (4).

Though there is no doubt that LC–MS is a powerful tool in discovery omics, it is often challenged by the complex nature of its samples. Metabolomics biomarkers tend to be present in extremely low amounts in biological matrices containing high concentrations of other compounds and contaminants, making these matrices extremely complex (5). Consequently, the search for metabolomic biomarkers can be hampered when using analytical-flow LC–MS (Table 1). Analytical LC–MS tends to favour the detection of metabolites already present at a high concentration; this is in part due to the difficult ionization that requires the use of high mass spectrometer source temperatures, voltages, and nebulization gases. Similarly, lipidomic analysis often uses nonvolatile salts, which exhibit poor desolvation and ionization efficiency (6,7). Ionization is not the only challenge. Sample amount can also be a dramatic limiting factor, such as in the case of biomedical and discovery proteomics that deal with low amount of samples, for example, single cells, biopsies, and tissues that do not provide the millions of cells or hundreds of micrograms of protein that are required in common proteomics workflows (1).

Liquid chromatography column miniaturization can overcome these challenges. Reducing the column inner diameter results in a reduction in chromatographic dilution. This dilution happens when a sample amount is injected into the column and it gets diluted by the surrounding solvents. In this case, less solvent means less chromatographic dilution, which is the result of using a smaller inner column diameter (Figure 1). A reduction in chromatographic dilution results in an increase in ion sensitivity, allowing for more efficient MS/MS sampling and thus a higher number of molecule identifications (2).

Theoretically, scaling-down the column internal diameter (i.d.) from 2.1 mm to 0.300 mm results in a sample concentration of 49-fold, which should translate into a 49‑fold increase in sensitivity (8,9). Realistically, this is not always the case; sensitivity is measured by ion intensity, which depends on other factors besides sample concentration, such as the ionization nature of the analyte, and the LC–MS equipment (emitter type) and source parameters. Nonetheless, as shown in Figure 2, scaling‑down the column inner diameter from 2.1 to 0.300 mm can result in an increase in sensitivity of almost 10‑fold for 50 ng of oxycodone.

 = column inner diameter and f2>f1 and d2>d1, a decrease in column inner diameter also requires a decrease in flow rate to maintain the same (or similar) linear velocity; with a reduction in flow rate comes an increase in ionization efficiency. In mass spectrometry, electrospray ionization (ESI) is used when analytical-LC flow rates are used (Table 1). Analytical flow produces a large volume of liquid at the MS emitter, which makes ionization efficiency troublesome. Consequently, ESI uses high voltages, high temperatures, and high amounts of nebulization gases to produce gas phase ions from these large charged liquid droplets.

Flow reduction decreases droplet size at the MS emitter and increases the concentration of charged analytes (Figure 3). Typically, a voltage of 1.5–3 kV is applied, allowing for those analytes to transition from liquid to gas phase without the need of nebulizing gases, high temperatures, and high voltages that tend to hamper ionization of analytes present in low amounts. In comparison to ESI used in analytical flow, nano-ESI (NSI) produces a stable spray that can provide better sensitivity of analytes containing undesirable but perhaps necessary amounts of salts, such as in the case of discovery lipidomics (6), but more importantly it provides better sensitivity overall.

Column miniaturization reduces chromatographic dilution and greatly improves ionization efficiency. This combination makes nano-flow a highly desirable technique in the world of omics when only low amounts of sample are available and better ionization is needed because of the addition of nonvolatile solvents or salts. Besides improved sensitivity, higher signal-to-noise (

), and lower limits of quantitation (LLOQ), it also reduces solvent consumption, which reduces costs and benefits the environment. At the same time, with a reduction in flow rate there is sometimes the need to increase gradient length. An increase in gradient length reduces sample throughput. Nonetheless, when sample throughput, sensitivity, and robustness are desired, micro-flow is a great intermediate option (Figures 4 and 5). Figure 5 illustrates the increase in sensitivity achieved by column miniaturization when scaling-down the column inner diameter from 0.300 mm (micro-flow) to 0.075 mm (nano-flow).

Despite the many benefits of low‑flow chromatography, there are also a few considerations that must be taken into account. When moving towards nano- and micro-flow, one must consider the dwell volume. This is the volume it takes from when the gradient is formed to when it reaches the head of the column and is responsible for gradient delays. In the case of analytical flow, a dwell volume of 5 µL does not cause a noticeable gradient delay, but the same dwell volume when operating at 250 nL/min would cause a gradient delay of about 20 min. To minimize this volume, low-flow instrumentation must be plumbed with 10–75 µm i.d. capillary tubing.

Extracolumn volume refers to the volume found between the injector and the detector and is responsible for mixing effects that lower observed efficiency as it adds extracolumn broadening. It can be found in any of the components of the LC system (needle seat, frits, tubing, connections) and can contribute to peak broadening. To minimize extracolumn volume effects, low‑flow chromatography uses miniaturized LC systems in which each of its components transports only a small volume in comparison to conventional LC systems. Furthermore, to minimize or avoid extracolumn volume effects, every single connection must be routinely inspected for leaks, which can be very time-consuming. A leak in the low‑flow world is almost always undetectable by the naked eye and it requires a highly experienced operator to detect such leaks.

In addition, column clogging and overloading also need to be considered as microscopic solids or, in the case of proteomics, undigested proteins can clog the small inner diameter column, rendering it unusable. Thus, the user must be careful to properly clean the sample prior to injection and use a trap (precolumn) column to eliminate undesired contaminants from reaching the miniaturized column. When using a trap column, the trap is in line with the column, the sample is loaded into it, and undesirable particles go to waste. Once the sample is concentrated into the trap and free of contaminants, it is directed towards the column for analysis. Using a trap column can help to avoid nano- and micro-column contamination and to extend the column’s lifetime.

Column miniaturization can bring many challenges, but there is no denying that the benefits outweigh these. The increase in ion intensity brought by low-flow chromatography has made it possible to identify biomarkers for clinical research (10) and to identify thousands of proteins using less than 2 ng of protein, and hundreds in single cells (11) as well as metabolites (5). The biomedical advancements and gains in fundamental scientific knowledge brought by micro- and nano-flow separations make this a powerful analytical technique.

L. Yi, P.D. Piehowski, T. Shi, R.D. Smith, and W.J. Qian, 

E. Shishkova, A.S. Hebert, and J.J. Coon, 

C. Aydoğan, F. Rigano, L.K. Krčmová, D.S. Chung, M. Macka, and L. Mondello, 

N. Danne-Rasche, C. Coman, and R. Ahrends, 

A.E. El-Faramawy, S.K.W. Michael, and B.A. Thomson, 

H.D. Meiring, E. van der Heeft, G.J. ten Hove, and A.P.J.M. de Jong, 

S.R. Wilson, T. Vehus, H.S. Berg, and E. Lundanes, 

, (2015) DOI: https://doi.org/10.4155/bio.15.92

Y. Zhu, G. Clair, W.B. Chrisler, Y. Shen, R. Zhao, A.K. Shukla, R.J. Moore, R.S. Misra, G.S. Pryhuber, R.D. Smith, C. Ansong, and R.T. Kelly, 

is an application scientist at Phenomenex. She received her bachelors and masters degree in chemistry and biochemistry from California State University (Los Angeles, USA). Roxana is an LC–MS/MS enthusiast currently working on developing LC–MS/MS methods, with a focus on nano- and micro-flow separations.

Articles

Reducing the column inner diameter results in a reduction in chromatographic dilution and increased sensitivity, allowing for more efficient tandem mass spectrometry sampling (MS/MS) and a higher number of molecule identifications.

Why Use Miniaturized Columns in Liquid Chromatography? Benefits and Challenges

Solvent effects were described in detail by Konrad Grob in the early 1980s when he observed that solvents retained by the stationary phase influenced retention of early eluting analytes and could lead to peak distortion. The effects of stationary phase polarities with varying solvents examined in split mode and using significantly less solvent were not the main focus of Grob’s work. Split injection eliminates the injection port as a contributor to variation in retention times. This article examines different stationary phases, solvents, and compounds using different split ratios demonstrating impacts to selectivity as a function of analytes interacting with the solvent and the stationary phase.

A colleague of mine, Corby Hilliard, was faced with a chromatographic anomaly while performing split analysis. Two different solvents—1-propanol and toluene—contained the same complex mixture of analytes, but one compound in particular shifted 0.2 min. An overlay of the two chromatograms revealed that decane exhibited a retention time of 5.98 min with 1-propanol as the solvent and 6.18 min with toluene as the solvent (1). Using Konrad Grob’s book on split and splitless injection, a reference was made to solvent soaking (2) and from there we had a hypothesis to test (Figure 1).

In capillary gas chromatography (GC) there are five common techniques for vaporizing a sample and transferring it onto the inlet of the analytical column: split, splitless, direct, PTV, and on-column injections. The most commonly used techniques are split and splitless, where the sample is vaporized using heat and transferred to the head of the column. In splitless injection, the split valve is closed long enough to allow most of the vaporized sample and solvent in the injection port liner to be transferred to the analytical column, which can take anywhere between 30 and 90 s. Since the split vent valve is closed, the sample transfer moves at the speed of the column flow, typically in the 1–2 mL/min range (3). Note that the volumetric flow rate into the inlet is lower as the carrier gas is compressed. In split mode, the split rate can be adjusted with a high degree of precision using modern equipment. Jack Cochran generated a 6-point calibration curve that covered a split range from 400:1 (1 ng on-column) to 10:1 (36.4 ng on-column) with a correlation coefficient of 0.99948 (4). A notable observation from this work highlighted a 3 s delay in elution of analytes when split ratios were set too low and amount of analyte on-column was too high. He found that by increasing the split ratio and adjusting the on-column concentration to 8 ng, using a 0.25 mm inner diameter (i.d.) × 0.25 µm film thickness, produced good peak shapes and stable retention times. Increasing the film thickness would also accommodate more analyte and solvent on-column.

There are two different approaches for refocusing vaporized sample from the inlet to the head of the column: solvent focusing and analyte focusing. The difference between these two techniques is the initial temperature of the column oven. Analyte focusing begins with an oven temperature that allows the solvent to move through the column as a vapour at the time of injection and requires a large difference in boiling point between the solvent and higher molecular weight analytes. Solvent focusing requires an oven temperature 20 °C below the boiling point of the sample solvent and allows the solvent and analyte to focus and wet the stationary phase. This can result in a solvent effect as described by Konrad Grob, where the condensed solvent acts as a temporary stationary phase contributing to changes in retention based on the polarity of the solvent, analyte, and column (1). In this work, six different compounds were made up in five different solvents analyzed on three different stationary phases, examining high and low split ratios to precisely control the amount of solvent on the column. The compounds and solvents represent a range of polarities, vapour pressures, and boiling points.

Three columns were chosen with orthogonal selectivity addressing a wide polarity range: 100% polydimethylsiloxane (PDMS), “1‑type phase”; polyethylene glycol (PEG), “wax phase”; and a 20% diphenyl–80% polydimethylsiloxane phase, “20-type phase”. Columns dimensions were the same for the phases evaluated (30 m × 0.25 mm, 0.5-μm 

f columns [Restek]). Compounds chosen were nonpolar (hexane, benzene, toluene), intermediately polar (chloroform, methyl tert butyl ether), and polar (ethanol). The solvents used were methanol, 2-propanol, acetonitrile, 1-propanol, and 2-ethoxyethanol. The starting temperature for this study was 40 °C, assuring that the solvent was fully retained at the head of the column so we could measure its impact. Standards were made up at 5000 parts‑per‑million, therefore a 200:1 split resulted in 25 ng on-column concentration and a 25:1 split in a 200 ng on‑column compound concentration. Instrument conditions were as follows: column flow: 1.1 mL/min, 8.55 psi, 37.88 cm/s; oven: 40 °C (hold 12 min) 30 °C/min to 225 °C (hold 5) [23 min]; MS scan: 35 to 300 AMU.

Analyses for this study were performed in triplicate using a 1 µL fast autosampler injection. Based on earlier studies, it was determined that 2-ethoxyethanol does not influence retention of the analytes tested regardless of the split ratios or stationary phases. For this reason, the six compounds were spiked into this glycol ether and used as a retention check standard before and after sample sets to verify consistent retention times. Surprisingly, the majority of triplicate runs were within 0.01 min (Figure 2). This work was originally performed in our laboratory to evaluate impacts on selectivity, however, no differences were observed.

From our work with hand sanitizers, we found problems with peak distortion of acetaldehyde in the presence of percent levels of methanol and ethanol using a cyanopropyl-type stationary phase. A similar approach was taken with this work in that we used methanol as the solvent and examined the performance of ethanol relative to a nonpolar compound, in this case benzene. Using a PEG stationary phase and injecting a methanol standard with a 200:1 split ratio, our retention times matched those found in Figure 2. However, when the split ratio was adjusted to 25:1, which introduced eight times more solvent, ethanol was retained on a layer of condensed methanol at the head of the column. Ethanol eluted just before benzene with a 200:1 split and eluted after benzene using a 25:1 split (Figure 3).

The results found in Figure 3 were not completely surprising because we are dealing with two highly polar alcohols; however, when analyzing a mid-polarity compound, such as chloroform, predicting the outcome is more complex. Konrad Grob wrote a paper specifically about solvent effects and chloroform and for this reason we added this compound into our test set. The paper describes chloroform as being “partially trapped”, which results in peak distortion (5). Using a 100% PDMS column and acetonitrile as the solvent, hexane and chloroform retention times were within 0.04 min using a 200:1 split. Using a 25:1 split, hexane was less retained and chloroform was slightly more retained and, as predicted by Grob, the peak shape was broad and distorted, indicating the analyte was “partially trapped” by acetonitrile (Figure 4).

For our final notable result, we examined three different split ratios: 200:1, 50:1, and 25:1. We found an example where there was retention at and in the solvent front, which, in this case, was acetonitrile. The column used was a PEG and the two compounds examined were chloroform and toluene. At 200:1, chloroform eluted at 8.28 min and toluene was 9.10 min, which again matched our control runs and is a clear indication of no solvent effects. At a 25:1 split, chloroform was in the solvent with a retention time of 8.15 min and toluene was more retained at 9.42 min. As the solvent increased, chloroform was less retained in the solvent than it would be in the PEG phase. Toluene was more soluble in an acetonitrile layer than in the column phase (Figure 5).

The four primary examples have demonstrated different solvent effects similar to that described by Konrad Grob four decades ago. Interestingly, even 0.04 μL of solvent retained in the column acts as a temporary stationary phase, influencing retention based on the polarity of the stationary phase, solvent, and analytes. In some cases, predictions can be made about the change in retention characteristics specific to solvent effects. For mid-polarity compounds, predictions are far more challenging.

Special thanks to Jaap de Zeeuw (Restek, Middelburg, The Netherlands) for his technical advice and review.

 has managed a team of chemists in Restek’s innovations laboratory since 2004. Before taking the reins of the laboratory, he spent seven years as an environmental chemist and was critical to the development of Restek’s current line of volatile GC columns. Prior to joining Restek, he operated a variety of gas chromatographic detectors conducting method development and sample analysis. Chris holds a B.S. in environmental science from Saint Michael’s College, USA.

Different stationary phases, solvents, and compounds using different split ratios demonstrating impacts to selectivity as a function of analytes interacting with the solvent and the stationary phase are examined.

Practical GC: Selectivity Differences Resulting from Analytes Interacting with the Solvent and Stationary Phase

Q. Protein-based drugs are becoming increasingly important; however, the inherent complexity of proteins can present some challenges during processing, formulation, and storage. What are some of the issues that protein‑based drugs present and what effect do they have?

 Compared to small-molecule drugs, proteins are often more sensitive, for example, the proteins are often sensitive to pH, salt concentration, and organic solvents. This has implications mainly for the stability of a protein-based drug, it should not degrade nor aggregate and should maintain its activity. Failure to address these issues may lead to a loss in efficacy or, in the worst case, the immunological responses in the patient.

Q. Protein analysis has relied heavily on size-exclusion chromatography (SEC) in combination with a suitable detector. What are the advantages and disadvantages of this technology for this purpose?

 SEC is a well established workhorse technique that offers high resolution and is relatively easy to operate. If a liquid chromatography (LC) instrument is available, the only additional hardware that is needed is the SEC column. The disadvantages come into play when analyzing samples under specific conditions, such as in a pharmaceutical formulation. Successful SEC separations often require a certain level of salts to be used in the mobile phase, which might not be appropriate for the protein or formulation under investigation. In particular for biological matrices, when analyzing for protein complexes or other large components, the packed column may either get clogged or the sample degraded because of the high shear forces in the column.

Q. Understanding how protein therapeutics behave following administration is a difficult but crucial step. Can you speak to the importance of understanding the interactions of protein therapeutics post-administration and the difficulties of studying them?

While the quality of the protein drug product is, of course, important, it matters little if the protein-based drug fails upon administration. For example, if there are undesirable interactions, such as a fast and irreversible association between the protein drug and blood components, it will have a negative effect on efficacy. The challenge is to study the therapeutic drug in the vast amounts of endogenous proteins and other components present in the body. To be able to follow the component of interest, some type of selective detection is needed.

Q. Sample preparation is always an important consideration for complex analyses. What sample preparation is required when analyzing protein-based drugs? Are there any issues in carrying out sample preparation on these complex proteins or the matrices?

The sample preparation is very much governed by the analytical method used. The commonly applied sample preparations used for small-molecule drugs, such as precipitation with organic solvents, salts or changes in pH, or liquid–liquid extraction, are not always possible to use for protein drugs because they may severely alter the protein state, which is what we are analyzing.

Q. In a 2018 study you investigated the suitability of AF4 for high-resolution separation of proteins and other components in serum and plasma and the separation of whole blood without sample pretreatment (1). What are the features of AF4 that make it suitable for these analyses?

The open separation channel of AF4 offers certain benefits, mainly that the sample components will experience relatively lower shear forces and that the surface area is much smaller than in a packed column. The smaller surface area considerably decreases sample interaction with the separation device. The open channel also makes it less susceptible to clogging as a result of large-sized components in the sample.

Q. What were the conclusions of this study?

That it is possible to monitor a fluorescently labelled antibody in serum and plasma samples with very little sample preparation. The only sample preparation was a dilution step, which was mainly performed to ensure accurate functioning of the autosampler.

Q. This paper was followed up with another study published in 2020, which coupled surface plasmon resonance (SPR) to AF4 for the analysis of active fractions of bio-separations in real samples, such as serum or saliva (2). What exactly is SPR and what are the benefits of using it for this purpose?

The analysis of proteins in biological fluids requires a selective detection. While the use of fluorescently labelled analytes works well, it may not always be desirable or possible to fluorescently label the analyte. Labelling may affect analyte behaviour and therefore the option to detect the unlabelled protein is very attractive.

Surface plasmon resonance is a phenomenon in which small changes on a surface, such as adsorption of protein, can be monitored. In essence, the refractive index of the surface changes if there is sample interacting with the surface. The phenomenon is used in a wide range of instruments and can be employed for studying a wide range of materials—proteins, polymers, and nanoparticles to name a few. We used SPR in an immunoassay configuration, with the surface coated with an antibody selective against our target analyte. In this configuration, SPR offered the sensitive and selective label-free detection needed for the complex matrices we were investigating.

Q. What are the future applications of this methodology and could it potentially be used in other areas?

We believe that the AF4–SPR combination could be of interest in pharmaceutical development during discovery phases, as well as in later stages. Both AF4 and SPR have individually been applied to many other types of analytes, so the combination has the potential to be applicable to a wide range of analytes. One particularly interesting avenue would be to use it for selective detection of nanoparticles and viruses in biological fluids.

Q. What other applications is AF4 commonly used for?

AF4 has been used for analyzing macromolecules in solution and colloidal dispersions. These areas are pharmaceutically relevant and AF4 is especially interesting since the complex samples often cover a wide size range that few other techniques have the ability to separate. It may be proteins, biopolymers, polymers, and nanoparticles, or combinations of them. There are continuous improvements in the technique, allowing higher resolution and sensitivity through different channel designs. In particular, I believe that combining AF4 with different detectors, such as SPR or mass spectrometry (MS), can offer synergies and answer questions that none of the techniques could by themselves.

We are currently looking into combining AF4 with another selective detector—MS—for the analysis of proteins in biological fluids. We are heavily involved in the NextBioForm consortium, which addresses the formulation of next‑generation biologics (3). Part of this research focuses on understanding and controlling chemical degradation, such as isomerization and deamidation, in relation to structure and conformation of therapeutic proteins in solution. Another area focuses on understanding interface-induced aggregation of therapeutic proteins.

M. Leeman, J. Choi, S. Hansson, M. Ulmius Storm, and L. Nilsson, 

, 4867–4873 (2018). https://doi.org/10.1007/s00216-018-1127-2

M. Leeman, W.M. Albers, R. Bombera, J. Kuncova-Kallio, J. Tuppurainen, and L. Nilsson, 

, 117–127 (2021). https://doi.org/10.1007/s00216-020-03011-x

Lars Nilsson is Professor of Formulation Technology at Lund University in Sweden, and co-founder of the spinoff CRO SOLVE Research and Consultancy AB, which offers analysis by AF4, MALS, and other techniques as a service. His research focuses on applied colloid chemistry and formulation technology, as well as development and application of AF4 methodology for the separation and characterization of biomacromolecules and nanoparticles within the life sciences. The AF4 work is currently directed towards analysis of therapeutic proteins and peptides in product formulation and biological fluids.

Lars Nilsson from the Department of Food Technology, Engineering and Nutrition, Faculty of Engineering, in Lund, Sweden, discusses the evolution of asymmetrical flow field-flow fractionation (AF4) in bioanalysis for the analysis of therapeutic proteins.

Advances in Asymmetrical Flow Field-Flow Fractionation (AF4)

I was recently researching sample preparation techniques we are developing in the laboratory, and, as is typical these days, I disappeared down somewhat of an Internet rabbit hole. Whilst I thought my knowledge of sample preparation techniques was relatively comprehensive, I quickly found that this wasn’t the case. This exercise also reminded me of how much we love an acronym—and oh boy is this particular area rich with these! When I started my career, I can clearly remember being bamboozled by the language of analytical sciences, and I often wonder how younger scientists feel when faced with such a huge array of acronyms in the literature, let alone how they gather an understanding of which techniques are applicable in which situations.

I wonder how many readers would recognize all of the acronyms shown in Table 1. Have a go and see how many you get (you may need to scroll up a little in order to hide the answers within the text below). I’ll provide the answers and some further details on each technique in the body of the article.

I know that during my research there were certainly techniques that I had not previously encountered and I wanted to share some of this in the hope that it provides a useful overview of sample preparation techniques you might not have previously considered for your applications. The degree of selectivity achieved by a sample preparation technique is often a primary reason for selection and I’ve also included some information on the relative selectivity of each technique. As my own application involved high analytical throughput, I’ve also included some information on whether there is currently an automated solution for the technique available on the market.

Increasingly stringent regulatory requirements across a range of industries as well as the analytical requirements within our own businesses drive our sensitivity to the very limits of instrument capability. Therefore, I’ve also included information on the ability of each technique to enrich the analytes from sample to final prepared extract.

The acceptor phase (typically an immiscible organic) and donor phase liquids are shaken or mixed to transfer analytes to the acceptor phase based on partition co-efficient. The nature of the acceptor solvent and donor phase pH and ionic strength can be adjusted to favour the transfer of analytes. Widely applicable to liquid samples, especially in the aqueous phase, and simple to implement.

SALLE: Support-Assisted Liquid–Liquid Extraction (also Solid-Liquid Extraction or Supported-Liquid Extraction [SLE])

The donor phase is percolated into and supported within a bed of purified, highly polar solid substrate (diatomaceous earth is typical) with high surface area. The acceptor phase (typically an immiscible organic) is slowly passed through the substrate bed and analyte transfer occurs between the thin layer of donor phase coating the substrate particles, containing the enriched analyte, and the slowly moving acceptor phase. The donor or acceptor phase may be buffered or pH adjusted to promote analyte transfer. Widely used for biological, food, and environmental applications.

Non-ionic or zwitterionic surfactants are used above the critical micelle concentration to form micelles that contain the target analytes. When the temperature is raised above the cloud point, the so-called 

 undergoes phase separation from the bulk aqueous phase and can be removed via decantation or aspiration. CPE is different from the two closely related techniques of coacervative extraction (CAE) and aqueous surfactant two-phase (ASTP) extraction in which ionic surfactants may be used and phase separation is induced using pH or ionic strength. Whilst CPE was initially focused on the extraction of inorganics, the technique has recently found applications in the extraction of biologics, as well as priority pollutants from water.

The use of ultrasound to enhance extraction efficiency. Whilst the technique is used widely to ensure extraction from complex and intractable matrices, such as natural products, many liquid-based extraction techniques can benefit from the enhanced extraction efficiency when using ultrasound. Typically, the use of ultrasound will enable the volume of the donor solvent to be reduced and the technique is often promoted as a greener alternative to traditional liquid extraction techniques. Widely used in the extraction of biologics and natural products.

A non-microwave absorbing acceptor solvent is used with a sample containing a high dielectric constant substance such as water. The sample is rapidly heated using microwave energy and analyte transfer occurs into the acceptor solvent. A variation of the technique uses a microwave absorbing substrate, which is added to the acceptor solvent to induce rapid heating. The technique can use open or closed vessels, the latter being a subgroup of the PFE techniques (see below), provided that the material of construction is non-microwave absorbing. MAE is widely used in the analysis of natural products, polymers, and foods.

ASE: Accelerated Solvent Extraction (see PFE)

A trade name used widely for pressurized fluid extraction.

PFE: Pressurized Fluid Extraction (also Pressurized Solvent Extraction [PSE]

A liquid-solid extraction technique in which the sample and solvent are placed in a sealed container and heated well above the normal boiling point of the extraction solvent. The combination of temperature and pressure results in highly efficient extraction, which is usually completed in a few minutes. Correct selection of the extraction solvent is critical in determining the selectivity of the technique. Smaller volumes of extraction solvent may be required due to the use of increased temperature and pressure and therefore the technique is viewed as a greener alternative. The technique has been used widely in the environmental, food, chemical, petrochemical, and pharmaceutical industries.

A version of pressurized fluid extraction in which the extraction solvent is water. The superheating of the aqueous solvent changes the dielectric constant and water is said to adopt “solvent-like” properties. The technique is promoted as a greener alternative to traditional PFE using organic solvents.

Supercritical solvents, such as carbon dioxide, are used to extract analytes—typically from solid samples. Supercritical fluids have gas-like diffusivity but liquid-like solvating properties, resulting in enhanced extraction efficiency from intractable matrices. The technique requires specialist equipment that can precisely maintain the extraction solvent above its supercritical point using a combination of back pressure and temperature. The extractants can be collected in a cold-trap, liquid-trap, or onto an absorbent. Modifiers can be added to the supercritical fluid to alter its polarity and hence enable the extraction of more hydrophilic analytes. The technique is a greener alternative to those that use organic solvents. Supercritical fluid extraction has been used extensively and has published applications across a diverse range of industries.

A solid or liquid sample is first heated in a sealed container to release volatile target analytes. The gaseous headspace is then sampled using a gas-tight syringe or loop autosampler. Phase ratio, temperature, extraction time, and agitation can all be used to optimize the extraction efficiency. Static headspace extraction can be used wherever volatile analytes are targeted in solid or liquid samples.

The solid or liquid sample is heated and the volatile target analytes in the headspace vapour (purging) or from within the liquid sample (sparging) are constantly swept from the sample volume using an inert gas. Volatilized analytes are trapped downstream onto an adsorbent material or via cryogenic means, prior to release into the chromatograph via strong heating. Typically, dynamic headspace extraction is used where analyte preconcentration is required.

An extraction method using a solid substrate, typically coated with a stationary phase of a selected chemistry used to trap, wash, and subsequently elute target analytes. The sorbent is available in tube, cartridge, and well-plate formats. A wide range of substrate chemistries are available and are analogous to the phases used in reversed- or normal-phase high performance liquid chromatography (HPLC), and can also be ionic or mixed-mode for improved analyte specificity. The process typically consists of conditioning the sorbent, applying the sample to trap the analytes of interest, washing to remove interferents, and elution of the target analytes in a small volume using a strong solvent. At each stage, the nature of the solvent, solution pH, and ionic strength can be adjusted to promote analyte adsorption or release and as such the combination of phase choice and chemistry manipulation can lead to a highly selective extraction technique. The highest selectivity is typically obtained when analyte–sorbent interactions are electrostatic. Solid-phase extraction has been extensively employed across a wide of industries and applications.

A form of SPE in which the extraction media in powder form is dispersed into the sample solution rather than the sample liquid being passed through an immobilized bed of the extraction media. In its simplest form, dispersive SPE is used to retain matrix interferents on the extraction media, prior to it being centrifuged and the resulting sample solution aspirated from the supernatant. dSPE is most often used with intractable matrices or homogenates and is an integral step in the QueChERS workflow. QueChERS (Quick, Cheap, Effective, Reliable Safe—that acronym readers can have for free!) has become well established as an extraction technique following its introduction for the extraction of pesticides from fruits and vegetables. It is a two-step process in which the first step involves a salting-out extraction using buffered or unbuffered solvent (acetonitrile is typical), followed by dispersive SPE of an aliquot from step one to remove interferences and matrix compounds. Whilst the application areas for dSPE are diverse and still evolving, perhaps the most popular applications are in food and environmental analysis.

MIPSE: Molecularly-Imprinted Sorbent Extraction

A further extension to the SPE principle in which silica or polymer stationary phases are manufactured using one or more target analyte molecules as a template to form “lock and key”-type molecular recognition sorbents. This method of manufacture improves the specificity of the sorbent towards the analyte or structurally related analytes used as the original template and can yield very impressive levels of specificity and sensitivity. Whilst MIPSE is applicable to any situation in which SPE can be used, it is perhaps best used when the target analyte functional group chemistry or molecular volume is distinctly different from potential interferents. There has been a recent uptick in interest for MIPS extraction of target biologics from both endogenous and exogenous matrices.

A technique in which a small diameter liquid polymer or solid sorbent coated fibre is immersed into a liquid sample or suspended in the headspace above a liquid or solid sample to selectively extract either dissolved or volatile target analytes, respectively. Depending upon the nature of the polymer coating, analytes will diffuse into the coating until equilibrium is reached. The first in our list of microextraction techniques used where a high degree of analyte enrichment is required; in these techniques the acceptor phase has a lower volume than the donor phase or the acceptor phase possesses the ability to concentrate analytes for direct introduction into the separation or detection equipment. Optimization of the SPME extraction technique involves selection of the appropriate fibre coating chemistry, coating film thickness, sample extraction time, extraction temperature, sample agitation, and sample to headspace volume ratio, alongside several other variables. In gas chromatography (GC), the fibre is usually placed into the inlet and the analytes thermally desorbed onto the column. Cryo‑focusing can be used to reduce analyte band widths if required. The technique can also be used with LC by desorbing the fibre with a liquid extractant, although this approach is less popular. A recent development is the so-called 

, in which a metal rod of larger diameter than the traditional fibres is used and is coated with a thicker and larger surface area sorbent layer. SPME Arrow is purported to result in a 10-fold increase in sample sensitivity compared to the fibre-based technique. SPME has found applications in a wide range of applications including flavours and fragrance, forensics, toxicology, environmental, and biological analysis.

A version of SPME in which the liquid polymer or solid sorbent is coated onto a stir bar that is used to agitate the sample whilst target analytes are adsorbed to the stir-bar surface. SBSE is also an equilibrium technique, the advantage being the increase of surface area and therefore capacity of the sorbent compared to SPME. Extraction and equilibration times are in the tens of minutes, similar to those for the SPME technique. In GC, the stir-bar is typically placed into a thermal desorption unit for analyte introduction to the GC column. In LC, analytes are desorbed by washing or immersing into a suitable solvent prior to sample introduction. The SBSE technique is argubably more popular than SPME for LC applications and is particularly suited to the extraction of low- to medium-polarity analytes. Applications include food and beverage, biological samples, environmental matrices, and pharmaceutical products.

A name given to SPME when sampling the headspace above a liquid or solid sample. Also known as 

HS-SPME—headspace solid‑phase microextraction

A name given to SPME when the extraction takes place within the sample. This name can also be applied to a newer development of the SPME technique in which a metal “blade” of larger surface area is used for liquid samples or to penetrate a solid sample and allowed to equilibrate, primarily in the fields of bioanalytical chemistry and in vivo diagnostics. The larger surface area of the sampling device is combined with ultra-thin films to allow rapid equilibration and a significant enrichment factor of the target analytes. The blade may be wetted with solvent and can act as an electrospray ionization emitter source when a high voltage is applied or the blade can also be used for ambient or direct sampling into the mass spectrometer.

DI-SDME (SDME): Direct-Immersion Single-Drop Microextraction

A technique in which a small amount of immiscible solvent (acceptor phase, typically 1–3 μL) is suspended (typically from the end of a syringe) within a liquid sample to extract target analytes. The same syringe is then used to introduce the solvent and extracted analytes into the chromatography system. As the acceptor phase–donor phase ratio is very small, the degree of enrichment is high and good analyte sensitivity can be achieved. The technique can be optimized by altering the chemical nature and volume of the acceptor solvent, temperature and degree of agitation, and pH and ionic strength of the donor solvent. Of course, the degree of agitation should be limited to a rate that does not cause the hanging drop to be stripped from the syringe tip. A further implementation of this technique, also known as 

, involves an intermediary immiscible solvent of lower density and volume that is placed on top of the donor solvent. The hanging drop is then immersed within it. Typically, the donor phase will be aqueous, the intermediary phase an organic solvent, and the hanging drop aqueous—the analytes first partitioning into the organic intermediary and then back extracting from the intermediary solvent into the acceptor solvent. This approach allows extraction of ionogenic analytes through the manipulation of pH and ionic strength. A dynamic version of this technique has also been developed in which an organic plug within a microsyringe barrel is withdrawn into the syringe as the aqueous sample is aspirated. A thin film of acceptor solvent remains on the syringe barrel inner wall whilst the remainder of the syringe barrel is filled with donor solvent. The high interfacial area provides good extraction efficiency and this operation is repeated several times to increase the enrichment factor of the technique. SDME techniques have been used in environmental analysis and for the analysis of food and beverage for BTEX (benzene, toluene, ethylbenzene, and xylene) and priority pollutants, as well as flavour and fragrance applications. SDME has also been investigated for the analysis of contaminants and pollutants in pharmaceutical formulations.

HS-SDME: Headspace Single-Drop Microextraction

The headspace version of DI-SDME in which the acceptor phase is suspended above a solid or liquid sample to extract and enrich the volatile species that are evolved from the sample under ambient or heated/agitated conditions. The sample manipulation options for SHE such as pH, ionic strength, heating, and agitation are all also available in HS‑SDME.

HF-LPME: Hollow-Fibre Liquid-Phase Microextraction

A hollow microporous fibre of typically 1.5 cm is suspended from the end of a microsyringe and the acceptor phase is drawn into the lumen (channels) and the wall pores of the fibre. The technique then proceeds in the same fashion as HS-SDME. The high interfacial area and relatively low acceptor phase volume result in good sample enrichment factors. The advantage of the hollow fibre technique is that the acceptor phase is more mechanically stable, allowing a greater degree of sample agitation and the possibility to re-immerse the fibre into a liquid sample in the dynamic form of the technique.

A version of the single-drop microextraction technique in which a single drop of acceptor solvent is immersed into a flowing stream of donor solvent, usually driven by an HPLC or peristaltic pump, within a microextraction chamber.

DLLME: Dispersive Liquid–Liquid Microextraction

An extraction technique based on a ternary solvent system in which the donor and acceptor solvents are immiscible and the third solvent (often called a 

 solvent) is fully soluble in both phases. The acceptor and dispersing solvents are rapidly injected into the donor solvent and a fine mist of acceptor solvent droplets is formed, which presents a very high interfacial area for analyte extraction. The ternary mixture is then centrifuged to separate the two immiscible phases and the acceptor solvent harvested by aspiration using a microsyringe. The acceptor–donor solvent ratio is typically very low to promote high analyte enrichment factors and this often requires the use of a conical vial to enable efficient harvesting of the acceptor solvent from below or above the donor solvent layer post centrifugation. If a dispersing solvent is not used, then the acceptor solvent can be dispersed using ultrasonic assistance and the technique is then abbreviated to 

. The technique has applications in water and environmental, pharmaceutical and clinical, industrial chemistry, and the food, flavour, and fragrance industries.

HLLME: Homogeneous Liquid–Liquid Microextraction

An extraction technique similar to DLLME, but the three-phase system containing the donor, acceptor (immiscible), and dispersing solvents are fully mixed (homogenized) at the beginning of the experiment using agitation or sonication. Extraction occurs across the very high interfacial contact area as the immiscible phases are separated by adding salt or an ion-pair reagent, a surfactant, or changing temperature or pH value. The phase separation is often assisted by centrifugation. Typically, the acceptor–donor solvent ratio in HLLME is higher and hence enrichment factors lower than in the DLLME technique.

MASE: Membrane-Assisted Solvent Extraction

A small-scale liquid–liquid extraction technique in which the donor phase is separated from a lower volume acceptor phase through the use of a semi‑permeable membrane (typically low-density polyethylene). The analytes pass from the donor phase through the semi-permeable membrane into the immiscible acceptor phase, which is usually held within a cone of membrane suspended in the donor phase. The technique is seen as a convenient way of reducing the amounts of solvent used in traditional liquid–liquid extraction and the donor phase pH and ionic strength may be manipulated to promote extraction. Any application suitable for liquid–liquid extraction can be adapted for use with the MASE technique.

The acronym MASE is also used for matrix solid-phase extraction in which a bonded phase particulate sorbent is used as an abrasive to disrupt a solid sample matrix and is blended to facilitate extraction from complex solid samples. The sample and sorbent are transferred to a column and analytes eluted using appropriate solvents.

MESI: Membrane Extraction with Sorbent Interface

An adapted version of the dynamic headspace technique in which a hollow fibre or flat sheet polymer membrane is placed in the headspace environment of the sample. An inert gas is passed through the membrane and analytes permeable to the membrane flow from the headspace through to an adsorbent trap. Once accumulated, the adsorbed analytes are thermally desorbed into the chromatographic system.

Phew… that’s a lot of sample extraction options! I’m also convinced that I’ve missed some of the techniques and I haven’t included many of the emerging techniques used to extract analytes such as peptides, proteins, and amino acids—the article would simply have been too long. Whilst researching the options for my application development, I came across the following acronym CA-HS‑HF-LPME-GC-FID (cooling assisted headspace hollow fibre liquid phase microextraction gas chromatography flame ionization detection), which in my 35+ years of chromatography experience had me scratching my head! With all of these techniques and their sub-variants I wonder how on earth any of us ever feel empowered to make sample extraction choices beyond the realms of those with which we are already familiar. I wanted to produce this article because there was no good reference that gave a reasonable summary of all major techniques with an indication of the various pros and cons with respect to selectivity, enrichment factor, and automation capability. I’ve discovered so many new methods or adaptations and extensions of those with which I was already familiar, and it is striking how many new methods (and acronyms) have been introduced since I began my chromatography career, when liquid–liquid extraction was by far the norm and solid‑phase extraction was the emerging new sample preparation technique.

Hopefully this summary will serve the purpose of helping less experienced folk to make informed sample extraction choices when developing analytical methods. I leave you with the thoughts of the psychologist Barry Schwartz from his book, 

, “when people have no choice, life is almost unbearable…But as the number of choices keeps growing, negative aspects of having multiple options begin to appear....the negatives escalate until we become overloaded. At this point choice no longer liberates, but debilitates.”

 (Harper Perennial; New York, USA, 2004).

Incognito explores the overwhelming array of sample preparation 			techniques (and acronyms).

Entering the Sample Preparation Acronym Maze

Spring is in the air. A new crop of scientists sees graduation on the horizon. Recruiters are swarming the (frequently virtual) halls of higher education and conferences. One common question I get when performing the recruiting road show is, “What are you looking for in a candidate?” For context, I work in Bristol Myers Squibb’s Chemical Process Development department, where (to grossly oversimplify) our remit is to transform discovery medicinal chemistry syntheses into scalable, safe, economical, and sustainable commercial pharmaceutical processes. For my analytical colleagues and me, our day-to-day work can span the gamut of identifying new chemical impurities, inventing and optimizing analytical methods, troubleshooting synthetic hiccups, shaping process control strategies, and researching new ways to speed development timelines. Our dominant tools are separations‑based, with liquid chromatography (LC) (specifically, reversed-phase) being by far the most widely utilized. Hence, one would assume we would look for the best chromatographers, either in the fields of fundamentals or applications.

My personal observation is that, at least within North America, candidates lacking industrial experience (which is the vast majority of soon-to-be graduates) predominantly have not pursued in-depth chromatographic research. Indeed, there are a shrinking number of university groups studying the theory of separations (but if anyone reading this is writing an update to Giddings’ Unified Separation Science [1] I know many who would pre-order it). Perhaps the field is too mature, everything has been studied already, or a fancy software package can develop all of my separations methods for me. I hope that we can agree none of these are true.

To not limit our potential talent pool, we must expand beyond attempting to find plug-and-play scientists already possessing keen chromatographic skills. This is not to say job seekers should be totally naïve, because separations are a cornerstone of any quality chemistry education. Indeed, one component to the “What are we looking for?” question is a comprehensive analytical chemistry foundation, either learned formally or acquired experientially. As my American Chemical Society Subdivision on Chromatography and Separations Chemistry (ACS SCSC) colleague James Grinias noted a few blogs ago (2), the topics that could be covered under the analytical umbrella far outpace the time afforded to it in an undergraduate or graduate education. Do we skip burettes in favour of capillary electrophoresis, drop graphite furnaces for charge-coupled device (CCD) optics, study specific types of instrument or statistical moments, and on and on?

We hope that students get broad exposure to all facets of chemical measurements, as well as opportunities to delve deeper into a select few (typically a natural result of hands-on research). The most bang for the buck will be topics that transcend subfields. Sampling theory, data evaluation, error analysis, and basic laboratory skills (meaning the ability to weigh, pipette, and use glassware—and do all these things correctly) cannot be underemphasized. Electronics, programming, or in silico modelling experience are not only valuable for more advanced courses and research, but can also help differentiate otherwise similar candidates.

Nothing can replace hands-on research experience. Even at an undergraduate level, time in an academic laboratory or interning in industry will yield enormous dividends. This exposure will not only afford an opportunity to hone one’s investigative and technical skills, but it will also provide an environment that will require working within a team and being able to communicate effectively. Hopefully, it’s more than a resume builder and gives broader insight into the world of research, either igniting a passion for scientific exploration or a realization that another field may be more fulfilling.

As a personal example, like many undergraduates, I had no clue what I wanted to be when I grew up. I started in journalism, drifted towards pre-medical, and somehow landed a chance to work
in M. Bonner Denton’s Raman spectroscopy laboratory as a sophomore at the University of Arizona. I was paired with a senior graduate student and, after demonstrating that I could be trusted not to break (too many) things, I was allowed to come up with my own research proposal. I don’t know if it was seeing lasers dance across open optics benches, learning I could use software to transform dreams into instruments, or seeing my name in print (3), but I fell for analytical chemistry hard.

The techniques I learned in the Denton laboratory served me well when I joined Robert T. Kennedy’s graduate group. True, his research had zero projects focused on spectroscopy. However, the ability (and desire) to create new instruments, from the hardware to the software, translated across research groups. Hence, the goal of building a microfluidic immunoassay sensor for in vitro hormone monitoring from scratch did not seem so far-fetched. Not being afraid to fail and understanding what grit really means transcends specific projects. Being able to create a proposal, a presentation, or a manuscript also all made graduate life a lot easier, and, to this day, I rely on these skills almost daily.

If I had to distill it all down to address the question of “What am I looking for in a candidate?”, my answer would be “a passionate, innovative, analytical chemist”. If you do not believe me, some of my more recently hired colleagues’ PhD research areas were as varied as:

fundamentals and applications of nanopipette electrospray emitters

single-molecule fluorescence microscopy studies of speedy DNA robots

development and studies of novel bioanalytical stationary phases for LC and supercritical fluid chromatography (SFC).

If our department had limited its hiring scope to “trained separations scientists”, we would have missed out on two out of three of these successful scientists. It is not too difficult to learn how to prime a pump or light a flame ionization detector (FID) (4). Passion, innovation, and problem-solving skills backed by a solid chemical education is where the real gold lies.

 (John Wiley and Sons; New York, USA, 1991).

J.G. Shackman, J.H. Giles, M.B. Denton, “Pharmaceutical reaction monitoring by Raman spectroscopy,” in M.B. Denton (Ed.) “Further Developments in Scientific Optical Imaging, The Royal Chemical Society,” Cambridge, UK,
186–201 (2000).

One can even learn from home using online courses such as the ACS Analytical Division approved curricula at www.CHROMacademy.com.

American Chemical Society Analytical Division Subdivision

 on chromatography and separations chemistry.

is an associate scientific director in the Chemical Process Development department at Bristol Myers Squibb (BMS) and is based in New Jersey, USA. He earned his two B.S. degrees at the University of Arizona and his Ph.D. in chemistry from the University of Michigan under the direction of Robert T. Kennedy. Prior to joining BMS, he held a National Research Council position at the National Institute of Standards and Technology (NIST) and was a professor of chemistry at Temple University in Philadelphia, Pennsylvania, USA. To date he has authored over 30 manuscripts, two book chapters, presented over 30 oral or poster presentations, and holds one patent in the field of separation science. Jonathan has proudly served on the executive board of the ACS Subdivision on Chromatography and Separations Chemistry (SCSC) for two terms.

Is there a secret formula to getting hired into a pharmaceutical analytical chemistry group? What skills are prized above all others?

What I am Looking for When Hiring a Candidate: One Pharmaceutical Analytical Chemist’s Perspective

Researchers have carried out a critical analysis of high-throughput proteomic and metabolomic studies on the pathobiology of SARS-CoV-2 in humans and performed a meta-analysis of significantly altered biomolecular profiles in COVID-19 patients (1).

In any human infection the efficiency of the host’s immune system and the pathogen’s infectivity contribute equally to the effectiveness of the infection. Understanding the host immune response, viral mode of transmission, and the alterations that occur to specific biological pathways can provide valuable insights into the pathobiology of the virus and, ultimately, lead to improved survival outcomes for those affected.

The COVID-19 pandemic has led to urgent and intensive investigations into the responsible SARS-CoV-2 virus; however, the diversity of alterations to pathophysiological pathways, the diverse conditions, and the degrees of severity seen between patients as well as the diverse outcomes have contributed to uncertainty, with numerous multiomic investigations completed and ongoing attempts to unravel the intricacies of the virus. Researchers collated the results of these studies so far and analyzed them critically to bring about a greater understanding of the disease.

One of the key findings when interactomic results were analyzed was the range of cellular housekeeping functions, such as nucleic acid metabolism or protein trafficking, affected by COVID-19. The array of changes the virus was capable of inflicting in the most severe infections goes some way to explaining its possible fatal outcome.

Several studies on the varying grades of severity of COVID-19 found that the disease’s progression was mediated by commonly dysregulated pathways of the innate immune response. Furthermore, most COVID-19 patients exhibit up-regulation of the inflammation axis, with a strong antiviral interferon response causing the fever found in COVID-19 patients. The chain reaction caused by this ultimately leads to the well‑documented lung damage found in cases of severe COVID-19 infection, which ultimately exacerbates the symptoms of COVID-19.

Similarly, as a result of the excessive inflammation in the lungs, the blood coagulation pathway is disturbed. The consequences of which may be the exposure of the liver to pro-inflammatory molecules. Researchers noted that the Shu et al. recommendation of targeting these molecules could be a therapeutic option to manage COVID-19-related complications.

Proteomic analyses concluded that the most damage during severe COVID-19 infections was brought about through hyperinflammatory milieu coupled with tissue hypoxia that develops into acute respiratory distress syndrome (ARDS).

Results from the meta-analysis of significantly altered biomolecule profiles in COVID-19 patients revealed alterations in the immune response, fatty acid, and amino acid metabolism, as well as other pathways. These in turn manifest in symptoms such as hyperglycaemia and hypoxic sequelae.

Researchers have carried out a critical analysis of high-throughput proteomic and metabolomic studies on the pathobiology of SARS-CoV-2.

COVID-19 Metabolomic and Proteomic Analysis

Waters Corporation founder James L. Waters, a legendary inventor and pioneer in liquid chromatography whose work spurred a multi-billion-dollar industry and helped advance numerous scientific breakthroughs, passed away peacefully on May 17th. He was 95 years old.

Born October 7th, 1925, in Nebraska, Jim Waters relocated with his family to Framingham, Massachusetts, following a career opportunity for his father. It was from this new home that Jim Waters would launch his company, Waters Associates, in 1958 in the basement of the Framingham Police Department, with the intent to build scientific instruments. With just five employees, the company broke through in 1965 when it licensed a refractometer from Dow Chemical for analyzing plastics. Jim built an instrument around the refractometer, sales skyrocketed, and the company was on the road to success.

Seven years later, Jim’s biggest triumph came when the company solved a problem for Harvard Professor Robert Woodward, who was working on the synthesis of vitamin B

. Woodward had already won a Nobel Prize in Chemistry in 1965 for being the first to synthesize chlorophyll, and at Woodward’s request, Jim Waters accompanied a team to Woodward’s laboratory with a liquid chromatograph and solved the problem in the span of a few weeks. The Waters liquid chromatograph, and ones that came later, now help drive pharmaceutical drug discovery, development, and manufacturing throughout the world.

“While we mourn his passing, those who knew and worked alongside him remember Jim Waters as a brilliant and spirited scientist and businessman, who propelled the discipline of separations science with his revolutionary work in liquid chromatography,” said Udit Batra, CEO and President of Waters Corporation. “Alongside his loving family, our company and industry celebrate the legacy of this special man who always sought to ‘deliver benefit’ and whose work continues to catalyze innovation across the life, materials, and food sciences, and today contributes to the fast-evolving science on COVID-19 vaccine development and disease research.”

A symposium in his name is held annually at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon), an analytical instrument industry trade show and conference.

Waters has released a tribute video to Jim Waters, which is available to watch here: 

Waters Corporation founder James L. Waters, a legendary inventor and pioneer in liquid chromatography, has passed away.

Waters Corporation Celebrates the Life and Legacy of its Founder, Jim Waters

Sciex (Framingham, Massachusetts, USA) has announced an agreement with Waters Corporation (Milford, Massachusetts, USA) to offer customers fully configured and interoperable Waters ultra-performance liquid chromatography (UPLC) instruments as part of their purchase and deployment of Sciex mass spectrometry (MS) systems. The companies hope this collaboration will make it easier for customers to choose the most suitable tools to address their analytical needs for both large and small molecules.

“We strive to provide the best products and services, and live by the mantra, ‘when customers talk, we listen,’” said Dom Gostick, Vice President and GM LC–MS and CTO, at Sciex. “Through this collaboration, we are broadening the variety of LC instruments available to our growing customer base.”

Instrument control drivers have been developed that have been fully tested to ensure compatibility and compliance, with customers also benefitting from the commitment of both vendors to deliver coordinated service. Sciex will support the full customer relationship, while Waters will provide direct UPLC service and support.

“This agreement provides for a smooth, integrated experience for customers with the coordinated support of Waters’ expertise and service,” said Jon Pratt, Senior Vice President, Waters Division.

For more information, visit www.sciex.com or www.waters.com.

Sciex has announced an agreement with Waters Corporation.

Sciex and Waters Announce Collaboration

Scion Instruments NL BV (“Scion Instruments”) (Goes, The Netherlands) has announced an agreement to acquire the Teledyne Tekmar Headspace product Lines HT3 and Versa from Teledyne Instruments, Inc (California, USA).

Both parties have started work on the transfer of the production of the Teledyne Headspace systems from the Teledyne Ohio facility to Scion Instruments production facility in Goes, which will take about four months and is scheduled to be completed by 1 September 2021.

As part of the agreement, Scion Instruments has assumed immediate responsibility for the global supply of spare parts and technical and commercial support for the two Headspace products. USA customers with existing Teledyne service contracts will continue to receive service from Teledyne Tekmar for the duration of their contract. Scion Instruments has a large existing install base in the USA and is strengthening its service team to meet customer expectations going forwards, as well as opening a new office in Maryland in June 2021.

For more information, visit: https://scioninstruments.com

Scion Instruments NL BV has announced an agreement with Teledyne Instruments, Inc.

Scion Instruments Acquires Teledyne Tekmar Headspace Product Lines

Groundbreaking performance improvements for the popular flame ionization detector (FID) were achieved by incorporating a post-column reaction with a three-dimensional (3D)-printed two-stage microreactor. Sensitivity improvements for compounds, such as carbon oxides, alcohols, aldehydes, ketones, chlorocarbons, and especially the new capability of the detector in achieving carbon universal response, are amongst the key highlights of the analytical strategy.

Gas chromatography (GC) is one of the most well-practiced chromatographic techniques in analytical chemistry (1–3). This ubiquitous technique delivers critical analytical solutions in many areas, from targeted compound analysis for indoor pollution monitoring to characterizing toxic chemicals in forensic investigations. Some of the reasons for the popularity of GC are that it is relatively inexpensive to implement, simple to operate, easy to maintain, and has a high degree of reliability. Despite more than 50 years of existence, the role of GC has not diminished or been replaced. Instead, the use of GC increases with continual developments in hardware and techniques to tackle a steady increase in sample complexity, catalyzed by societal transformations and initiatives such as increasing recycling and sustainability efforts.

Unlike other analytical chemistry disciplines, GC was blessed with many detector choices (4–6). Without any doubt, one of the most important innovations in GC was the invention of the flame ionization detector (FID) that was almost simultaneously and independently reported by I. McWilliam and associates (Australia) and J. Harley and associates (South Africa) in 1957 (7–10). Since its invention, the FID has become a choice detector in GC. Justifiable reasons for the FID to be in such a dominant position are as follows: It has a respectable detection limit around 0.5 pg C/sec, which allows the detector to meet many contemporary and demanding applications, and it is relatively inexpensive to construct, operate, and service, providing one can have access to fuel gases. Also, the detector can tolerate harsh chromatographic conditions of up to nearly 500 

C and highly corrosive gases, such as hydrogen chloride or sulfur hexafluoride. However, despite its wide-spectrum application and popularity, the FID is not a panacea for all chromatographic applications. The detector has some critical constraints. For example, compounds that can not generate CHO

 ions in a hydrogen flame, such as carbon monoxide, carbon dioxide, carbonyl sulfide, and formaldehyde, can hardly be detected. Also, the FID has a reduced response for some oxygenated or functionalized species. Another important issue critical for accurate quantification is that the FID has a highly variable response throughout many organic carbon-containing compounds and, in some instances, even within the same class of compounds like short-chain aldehydes or alcohols. Reference standards must be synthesized or procured to perform the analysis. Also, it is not possible to avoid handling compounds that are highly volatile, chemically unstable, or with an elevated level of toxicity.

Several attempts to improve the performance of the FID detector for targeted compounds have been reported in the literature. For instance, the measurement of trace carbon dioxide (CO

). This application attracts attention as CO

 is a major greenhouse gas that contributes to climate change. Catalytical reduction of CO

 to methane for detection by FID was first described by Porter and Volman (11), who demonstrated that carbon oxides could be converted to methane with an appropriate catalyst, such as nickel at an elevated temperature in a hydrogen atmosphere. Analytical devices based on this approach, commercially known as the “methanizer”, have been made available for many decades as a stand-alone or integrated chromatographic tool by various instrument suppliers and manufacturers (12). Although the device functions adequately in targeting carbon oxides in a relatively clean gas matrix, such as ambient air, it is susceptible to catalyst fouling because of poisoning by matrix constituents such as sulfur or halogenated compounds. Also, integrating the methanizer as an adjuvant device into the analytical system reduces the flexibility of the chromatograph while increasing the overall chromatographic void-volume of the system. The outcome of both effects is not desirable. A new reincarnation to this approach involves incorporating a more robust catalyst inside an FID jet (Jetanizer) to minimize void-volume and increase the system’s ease of service and platform flexibility (13–16). Although this approach improves chromatographic performance and robustness, the quest for carbon compound independent response capability by FID which is highly sought after by chromatography innovators, remained an elusive and moving target.

The Post-Column Two-Stage Reaction and Metal Three-Dimensional (3D)-Printing Technology

The post-column two-stage reaction has the potential of being a viable solution to carbon compound independent response capability for FID. In this reaction process, organic compounds are first oxidized to CO

 with a catalyst such as platinum and subsequently reduced to methane with another catalyst like nickel for detection as described in the literature (16–18). Although this strategy has merits, the extra column band broadening effect (σ

) from having two packed bed reactors between the column outlet and the detector can be quite excessive and may negatively affect the overall chromatographic performance. A typical chromatography time domain of peak width in the range of 10 to 20 s was observed, making the technique not suitable for high-resolution gas chromatography.

A novel approach to attain the same objective involves using metal 3D-printing technology for the fabrication of metal microstructure devices that are augmented with the appropriate catalysts in this microreactor embodiment (Polyarc). The assembly integrates two catalytic chambers in series where carbon-containing molecules are first converted to CO

 through a combustion process immediately followed by the methanation process (19,20). Figure 1 shows the relative responses to methane of model compounds from many classes of volatile and semivolatile organic compounds with the technique described. Several key enhancements are noted. First, the employment of the post-column reaction strategy expands the capability of the FID in that critical compounds of industrial significance with low or no response by FID alone, such as carbon monoxide, carbon dioxide, carbon disulfide, and formic acid, can now be detected with excellent sensitivity in the range of 0.5 pg to 1.0 pg C/sec with a contemporary FID. Second, as shown in Figure 1, sensitivity improvements were also observed for popular oxygenated compounds and chlorinated hydrocarbons, such as formaldehyde, methanol, carbon tetrachloride, and chloroform. In formaldehyde, an improvement of sensitivity of more than ten times compared to FID alone was observed. Finally, except for acetylene, where a reduced but reproducible response was noted, a universal carbon response was achieved. It was postulated that the reduced response of acetylene was caused by selective irreversible adsorption of the molecule by the catalysts. The universal carbon response resulted from the catalytic conversion of a carbon-containing molecule to methane, combined with the inherent wide linear range of the flame ionization detector for a compound such as methane affords compound independent calibration with a high degree of accuracy within ±5%. This new and convenient analytical capability has the potential of eliminating the need for an analyst to handle highly toxic compounds and thereby improving industrial hygiene and laboratory safety. The technique reduces or eliminates the need for multicomponent and multilevel calibration as well as enables the measurement of analytes with no known commercially available reference materials (19,20).

FIGURE 1: Universal carbon response and sensitivity enhancements for FID. Conditions are described in the literature (20).

The suitability of the strategy with chromatographic techniques having the most stringent requirements on band broadening, such as high-resolution gas chromatography with columns having an internal diameter of less than 150 μm and GC×GC was demonstrated (19,20). Figure 2 shows a color plot of the separation of a Canadian winter diesel by GC×GC-FID with the microreactor incorporated. An average peak width at half-height (PWh) of 200 ms was achieved. As described earlier, with the added benefit of the analytical system’s universal carbon response, the sample constituents can be expediently quantified using a single compound standard.

FIGURE 2: Color plot of a commercial diesel fuel by GC×GC-microreactor-FID. Conditions are described in the literature (20).

Apart from the reduced response of acetylene, constraints associated with this technique include chromatographic peak tailing for silicon-containing compounds. This is because of the formation of active silica in the catalyst chambers. Although the peak tailing does not negatively impact equimolar response for carbon, the technique is contraindicated if trace analysis is required for said class compounds.

The incorporation of a post-column reaction using a 3D-printed, two-stage microreactor is showing groundbreaking performance improvements for flame ionization detection in many gas chromatography applications—and delivers carbon universal response.

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