Liquid Chromatography (LC/HPLC)

This article gives a brief overview of just some of the chiral environmental studies carried out recently that cover the differing enantiomeric activity of pesticides, their environmental transformation, and the degradation of pollutants in general. They highlight some of the recent advances in chiral stationary phases (CSPs) that have enabled higher efficiency and faster separations than previously seen in this area.

With many new biopharmaceuticals now being developed, robust analytical methods are needed to ensure that these protein-based drugs are of high purity and safe with a minimum amount of side effects. Size-exclusion chromatography is an important technique in investigating purity and is useful to identify and monitor protein aggregation, which can have economic and immunogenicity effects. This article discusses those column parameters that are most important in the selection of the optimum phase for SEC separations.

This installment describes high performance liquid chromatography (HPLC) instruments and related products introduced at Pittcon 2016 held in Atlanta, Georgia, or in the year prior. We highlight innovative features and benefits of new HPLC systems, modules, software, and product extensions.

Our annual review of new LC columns and accessories introduced at Pittcon and through the previous year.

In proteomics studies, proteins are digested into hundreds of thousands of peptides, thus creating very large and complicated mixtures. Simultaneous electrospray ionization of these complex mixtures, however, results in suppression of ion formation. Therefore, it is essential to have effective chromatographic methods to separate the peptides before analysis with mass spectrometry, to relieve ion suppression and to allow the mass spectrometer sufficient time to collect tandem mass spectra of peptide ions. The challenges involved in developing such separations are great, however. This is the first of a series of articles exploring topics that will be addressed at the HPLC 2016 conference in San Francisco, from June 19 to 24.

How suitable is the column plate number for system suitability testing?

The silica-based packing in reversed-phase columns is not inert. Here we consider what happens when the mobile phase pH is too high or too low.

Pavel Jandera discusses building his own LC instrument, the birth of modern HPLC, the importance of exploring (and understanding) earlier research papers, and current trends in contemporary chromatography. He also offers inspiring advice for young chromatographers.

Glycosylation of monoclonal antibody (mAb) therapeutics is widely recognized by the regulators and the industry as a critical quality attribute (CQA). Hence, it is necessary that glycosylation is measured and adequately controlled during production. This installment reviews the various process parameters and raw material attributes that affect glycosylation, as well as the different analytical tools that are used for characterization, with greater emphasis on the chromatographic methods of analysis. Key recent advancements that have occurred in the past five years are also discussed briefly. While significant progress has been made in the monitoring of glycosylation, its real time control has yet to be demonstrated.

An unusual source of contamination was found in the reversed-phase LC analysis of a 0.4% (w/w) drug product during the development stability studies at levels much greater than the 4 ppm limit of quantitation. System-related sources (mobile phase, injector carryover and contamination), formulation components (drug substance, excipients), and the sample-handling procedure were studied by a systematic approach. The evaluation of the sample-handling procedure identified extractables from the sample vial (different from that specified in the analytical method) and the rubber-lined vial cap, and incidental transfer of residue from commonly used nitrile gloves as the sources of unintended contamination in sample analysis.

Affinity separations offer great potential for pharmaceutical analysis.

During the course of my scientific career beginning in the 1960s, I have grown up with the birth of modern LC column technology, the refinements of the instrumentation, and the development of widespread application of this most powerful separation and analysis technique. In this installment, I would like to share with you some of my observations and experiences with the beginning, the growth period, and the maturation of HPLC columns, where I have focused nearly 33 years of writing for this magazine. I will explore some of the early column breakthroughs beginning with the development of large superficially porous particles (SPP), the porous irregular and spherical microparticulate particles, inorganic and organic polymeric monoliths and the rebirth of the current generation of SPP. In next month’s installment I will look into my crystal ball and see what the future of HPLC and UHPLC holds.

How to get started in the process of identifying the problem source for column-related problems.How to get started in the process of identifying the problem source for column-related problems.

So you think you know all about UV detection? Double check your understanding with this short primer on the principles and key variables of ultraviolet visible detection.

The capability to separate and analyze a wide range of proteins in complex systems remains a prime requirement in the biochemical sciences. Intact protein separations are especially difficult as these large molecules can present different conformations, association states and amphoteric features with chromatographic surfaces. Combining high performance liquid chromatography (HPLC) and ultrahigh pressure liquid chromatography (UHPLC) with mass spectrometry (MS) has proven to be an effective approach for solving difficult problems involving protein analyses. Considerable effort has been made to develop columns for separating proteins with high efficiency for reversed-phase, ion-exchange, size-exclusion chromatography, hydrophilic interaction liquid chromatography (HILIC), and hydrophobic interaction chromatography (HIC). Even so, many situations still exist where insufficient resolution is available for accurate protein analysis even when high-resolution MS is available. This presentation provides a brief overview of new approaches being investigated in the author's laboratories for obtaining superior protein separations. This includes new approaches for obtaining better protein separations with columns of highly-efficient superficially porous silica particles and techniques using MS-friendly mobile phases with effective methods for changing protein selectivity (band spacings) by column type and organic mobile phase modifiers.

This article provided guidance for working with the low-dispersion, small-volume columns that were gaining popularity in 2003. These considerations are still appropriate today with the short, narrow HPLC and UHPLC columns now in vogue. Anatomy

Advances in Liquid Chromatography–Tandem Mass Spectrometry (LC–MS–MS)-Based Quantitation of Biopharmaceuticals in Biological Samples

Analyzing Host Cell Proteins Using Off-Line Two-Dimensional Liquid Chromatography–Mass Spectrometry

Multi-Toxin Determination in Food – The Power of "Dilute and Shoot" Approaches in LC–MS–MS

Solvent costs too high? Here are some ideas on how to decrease the amount of mobile phase you use.

Miniaturized separation techniques are advantageous for the analysis of illicit drugs and new psychoactive substances.

I recently returned from a tour of teaching liquid chromatography (LC) classes to users in Minnesota, the United Kingdom, Poland, and Malta. One thing that always impresses me on such trips is that no one group has a corner on the LC problem market. The same problems pop up in most laboratories, no matter where they are located, the role of the laboratory (for example, analytical, forensic, production, research), what industry is involved, or the brands of instrumentation used.

HPLC 2015, chaired by Gérard Hopfgartner was held in Geneva, Switzerland, from June 21–25. This installment covers some of the highlights observed at the symposium including stationary phase developments, particle technology, and areas of growing application of HPLC. In addition, trends and perspectives on future developments in HPLC culled from the conference are presented.

This article presents a method for comparing the levels of baseline interference arising from common laboratory mobile phase contamination sources and assesses different approaches for removing dissolved contaminants to generate interference-free chromatogram baselines. The authors demonstrate that recirculating mobile phase through a semi-preparative scale column using a reagent delivery pump has advantages over previously published mobile phase decontamination methods.

The 42nd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), chaired by Gérard Hopfgartner was held 21–25 June in Geneva, Switzerland. This instalment covers some of the highlights observed at the symposium including stationary-phase developments, particle technology, and areas of growing application of HPLC. In addition, trends and perspectives on future developments in HPLC culled from the conference are presented.

Biopharmaceutical Candidate Screening with Automated Dynamic Light Scattering

The potential causes are considered for peak distortion for the first two peaks in the chromatogram.The potential causes are considered for peak distortion for the first two peaks in the chromatogram.

Here, we concentrate on one particularly useful equation that allows us to make changes to an analytical system to improve throughput or efficiency, while retaining the selectivity of the original method.

Tips & Tricks GPC/SEC: Answering Common Questions About GPC/SEC Columns