In many applications of liquid chromatography (LC), excellent peak shapes are observed with simple, easy-to-use mobile phases and operating conditions. However, some specific combinations of analyte and LC column chemistry can lead to terrible peak shapes and even total adsorption of analytes to components of the LC system such that no peaks are observed at all. In this installment of “LC Troubleshooting”, I discuss two specific cases where an understanding of specific interactions between particular analyte functional groups and LC instruments or column materials is critical to achieving successful separations.
Despite best efforts in 2004 to ban their use, persistent organic pollutants (POPs) remain prevalent across the globe, including in soil. To protect human health, agricultural and environmental soil require careful investigation, but preparing samples for gas chromatography–mass spectrometry (GC–MS) analysis is time-consuming, and mostly done manually. Accelerated solvent extraction (ASE) has been the preferred preparation method for the past few decades, and while we have seen advancements, the method remains manual. Now, new technology offers parallel sample processing, combined extraction and evaporation, and automation—leading to faster analysis, reduced risk of error, and freed-up time for personnel.
Retention and selectivity in gas chromatography (GC) occur because of the thermodynamics of the phase transition from the vapor phase following sample injection to the solution phase of being dissolved in the stationary phase. Analytes then move along the column because the vapor phase is flowing. In this installment, we quickly review the basic thermodynamics underlying separation in GC, and we see how this impacts selectivity and retention. Last month, we saw how seemingly large differences in column polarity may still generate little, or even the opposite, effect on retention and generate confusion and difficulty in method development. This month, we explore this challenge more in-depth using thermodynamics and a freely available online GC simulator.
Peter Schoenmakers and Emanuela Gionfriddo are the winners of the 16th annual LCGC Lifetime Achievement and Emerging Leader in Chromatography Awards, respectively. The LCGC Awards honor the work of leading separation scientists for lifetime achievement and emerging potential. The award winners will be honored in an oral symposium at the Pittcon 2023 conference in March 2023 in Philadelphia, Pennsylvania, USA.
There are as many measurement values of the true column hold-up volume, V0, as techniques applied to evaluate this most important property in liquid chromatography (LC). The relative errors made on V0 measurements using conventional “non-retained” markers—such as acetone, uracil, or thiourea in reversed-phase liquid chromatography (RPLC), or benzene or acenaphthene in hydrophilic interaction chromatography (HILIC)—can be as large as ±30%. This situation is extremely confusing for LC users who wish to classify and predict the retention behavior of LC columns. In this work, along with advances in mass spectrometry (MS) instrumentation, the hold-up volume of any LC column—including, but not limited to, RPLC, HILIC, ion exchange chromatography (IEX), and mixed-mode columns—is accurately measured by injecting labeled deuterated acetonitrile (CD3CN) molecules and detecting them selectively by MS-single ion reaction (m/z = 45) using non-labeled and pure acetonitrile (CH3CN) as the eluent. This proposed harmonization of all conventional V0 measurement methods is illustrated and successfully applied to RPLC, HILIC, anion exchange (AEX), and RP-AEX mixed-mode chromatography, irrespective of the mobile phase composition selected.
Partition coefficients, log P or Kow, are often used in introducing and describing new extraction techniques. They can also be used in developing new separation methods. An understanding of partition coefficients, along with diffusion, is necessary to more completely guide separation processes. As with the application of any theoretical concepts, several assumptions may be applied. This month, we take a dive into the practical applications of partition coefficients as they are commonly applied to chemical extractions.
The pharmaceutical industry develops and manufactures life-saving medicines and is regulated by government authorities to ensure drug products’ safety, efficacy, and quality before reaching patients. This article provides a high-level overview of pharmaceutical regulations and associated public quality standards.
Chlorinated paraffins (CPs) are an emerging and ubiquitous group of environmental pollutants associated with adverse effects on human health, including endocrine disruption and possible carcinogenicity. In this study, supercritical fluid chromatography (SFC) and ultrahigh-performance liquid chromatography (UHPLC) — both coupled with high-resolution mass spectrometry (HRMS) — methods for the analysis of short-, medium-, and long-chain CPs in fish oil-based dietary supplements were developed and validated at concentration levels of 0.6 and 3.0 µg/g lipid weight (lw). The recoveries were in the range of 80–96% and repeatabilities, expressed as relative standard deviations, were <19%. The limits of detection for the UHPLC–HRMS method (from 0.03 to 0.05 µg/g lw) were 5 to 10 times lower than those obtained by SFC–HRMS (from 0.13 to 0.50 µg/lw).
Accurate quantification of per- and polyfluoroalkyl substances (PFAS) in food is necessary to understand potential dietary exposure. While beneficial for most applications, quantification of PFAS using low-resolution triple quadrupole instruments can be complicated by the presence of co-eluted interferences, which can result in false positives or inaccurate PFAS concentrations. This article discusses the use of high-resolution mass spectrometry (HRMS) to distinguish between matrix interferences and PFAS compounds in a variety of food matrices, and incorporation of these interferences into routine triple quadrupole methods for monitoring.
Over the last several years, our definition of biotherapeutics has evolved from solely protein-based therapeutics, such as monoclonal antibodies (mAbs), to include such product classes as cell and gene therapies. Cell and gene therapies are unique products that require innovative and new approaches to formulation, specifically related to drug delivery. One such delivery modality that has gained significant traction is lipid nanoparticles (LNPs), which, as their name suggests, are nanoparticles comprised of lipids. The first LNP product approved by the United States Food and Drug Administration, was the liposome-encapsulated Doxil, whereas, in 2018, the United States Food and Drug Administration approved the small interfering RNA (siRNA) product Onpattro, using lipid nanoparticles as part of its formulation. Since then, LNPs have gained more international attention in their role in drug delivery because of the mRNA-based COVID-19 vaccines. LNPs in the biopharmaceutical space are still in their infancy related to our understanding of them and how to characterize them, presenting both challenges and opportunities. In this column, we discuss some of those challenges and opportunities and look at the current and future landscape of the most common analytical tools used to analyze and characterize LNPs.
Enumerable types of gene therapies are collectively one of the fastest-growing areas of biopharmaceutical products today. Gene therapies are generally defined as treatments, perhaps even cures, for diseases through the transfer of genetic material to host cells. The analytical technologies that exist to characterize these novel therapies are rapidly advancing. In this column, we will briefly present some of the basic concepts related to this novel product class, specifically related to formulation and delivery, focusing on adeno-associated virus (AAV) formulations. We will then briefly discuss the analytical technologies and approaches most commonly used to characterize these products, specifically on the characterization of AAV-related products. We will briefly discuss what many consider the gold standard in analytical tools, analytical ultracentrifugation (AUC), and then specifically discuss liquid chromatography-mass spectrometry (LC–MS) tools and methods (such as the multi-attribute method, MAM) that are becoming more commonly used in characterization.
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, LCGC 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.
Phytotoxins, the secondary metabolites synthesized by plants, are now recognized as a new category of environmental micropollutants. So far, only a limited number of phytotoxins have been detected and reported in terrestrial and aquatic environments, partially due to the analytical challenge involved. In this article, a robust, reliable, and efficient approach, namely Source Supported Suspect Screening (4S), is introduced for high-throughput analysis of phytotoxins from their plant origin to the downstream environmental compartments. The approach was established on an ultrahigh-performance liquid chromatography–electrospray ionization–quadrupole‑time‑of‑flight-mass spectrometry (UHPLC–ESI-QTOF-MS) analytical platform where optimal sample preparation and chromatographic conditions were investigated and a new suspect screening protocol was suggested.
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 installment, we explore the impact of green chemistry on analytical chemistry and gas chromatography. We define green chemistry, and discuss what it is and what it is not. For example, it is not "chemistry lite." We then briefly review relevant literature on green analytical chemistry, and begin a conversation on green gas chromatography and the many areas in which gas chromatography-based methods can be made "greener.
Detectors in gas chromatography (GC) are unique in all analytical science. The detector performs several difficult functions that make sensitive and selective analysis possible. In this installment, we will explore the challenges involved in detecting the effluent from a capillary gas chromatographic column, and summarize the many detector options. We will then consider basic ideas and tasks that allow detectors to function properly. Finally, we will examine some specific troubleshooting and preventative maintenance tips for common detectors. We will see that, with some simple preventative thinking and action, detectors in gas chromatography can provide excellent sensitivity and selectivity for years of use.
Molecular spectroscopy techniques, most commonly ultraviolet-visible (UV-vis), are usually thought of as analyzing liquid-phase samples, with techniques such as high-performance liquid chromatography (HPLC). Although not as common as in HPLC, molecular spectroscopy can be used with gas chromatography (GC) as well. In this installment, we examine molecular spectroscopy in combination with GC. The two most common techniques used with GC today are Fourier transform infrared spectrometry (FT-IR) and vacuum ultraviolet spectroscopy (VUV). Both provide structural and quantitative information and can be used in complementary fashion with the more common GC–mass spectrometry (GC–MS) and classical detectors. We will discuss the basics of GC–FT-IR and GC–VUV, when and when not to use them, and how they compare to and complement classical detectors.
