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We have been working to develop the use of liquid chromatography–triple quadrupole–mass spectrometry (LC–MS/MS) for quantification of intact proteins from biological fluids.

To answer the question "Is there a good flow diagram I can use for GC method development?" please see below for our first attempt at something suitable, in the form of stepwise decision trees and flow diagrams.

As we close in on a little more than a month to go before the 43rd International Symposium on Capillary Chromatography and the 16th GCxGC Symposium (ISCC & GCxGC 2019; www.isccgcxgc.com), May 12 – 17 in Ft. Worth, Texas, my excitement burgeons. All of the groundwork has been laid to provide forums for presenting and discussing the latest advances in capillary and comprehensive separations science.

In the late “noughties” we couldn’t avoid the webinars, seminars and online calculators which were being released by HPLC column manufacturers to extol the virtues of the “new” core-shell particle morphology that promised high performance at lower back pressures.

Virtually exclusively, liquid chromatography–mass spectrometry (LC–MS)-based assays for protein quantitation rely on bottom-up strategies, where the protein is initially digested into constituent peptides during sample preparation. Top-down intact protein quantitation, especially using affordable, low resolution triple quadrupole (QQQ) mass spectrometers, has been largely unexplored.

Just as medical practitioners are able to discern worrying features from a variety of medical physics devices (electrocardiogram, electroencephalogram, ultrasound, for example), we need to develop the skill to identify worrying symptoms from our HPLC instrument output.

If you have a method or process that involves a number of different variables, multivariate optimization approaches can provide a faster route to optimum conditions and can lead to a more reliable outcome than using a one-factor-at-a-time approach. With a little study and practice, students and researchers can apply these optimization techniques, even if a complete understanding of the underlying statistical treatments is not immediately apparent.

You may be one of the many analytical scientists who look with envy at those laboratories who are equipped with sophisticated automated HPLC method development systems. These systems are indeed very nice and can be very efficient in narrowing down choices, however, they aren’t a universal panacea and one can achieve a lot with a simple, paired down approach.

In 2009, the founders of VUV Analytics, Inc. approached my group about the potential of coupling a vacuum ultraviolet absorption spectroscopy detector to a gas chromatograph (GC–VUV).

UV detectors are undoubtedly the most frequently used type of detector with HPLC systems, and I predict this will continue for many years, despite the rise of the modular mass spectrometric detector.

From May 12 – 17, 2019 at the Hilton Ft. Worth in Ft. Texas, I will be co-chairing (together with my colleague Prof. Dan Armstrong) the 43rd International Symposium on Capillary Chromatography (ISCC) and the 16th GCxGC Symposium. I am writing to tell you this not only because organizing a meeting is quite an undertaking, but also because I am really excited about how this event is shaping up.

I think its fair to say that there has been a paradigm change in the way we approach mobile-phase design for HPLC in recent years.

Unconventional oil and gas (UOG) extraction is a multistep process that involves horizontal drilling, hydraulic fracturing, and massive infrastructure to handle fossil fuel resource recovery and associated wastewater generation.

In my previous blog, I discussed the possibility of backflash in splitless GC injection and its effect on quantitative reproducibility and carry-over.While much is written in the literature on optimization of splitless injection conditions, little is available on the implementation and optimization of increased head pressure (pressure pulsed) injection, so we will concentrate on this aspect of injection optimization.

For an international conference, highlighting the diversity of research, and the people performing it, is important. Diversity comes in a lot of different flavors: Industrial vs. academic; different cultures, values, needs, and resources; age and gender; among others. Representatives from all different backgrounds and experiences should be given a voice.

Let’s first properly define carry-over in the context that I’d like to discuss here. An injection is made and a chromatogram obtained. On injecting a “blank” as the next injection, one or more of the components of the previous injection appear in the “blank” chromatogram.

I just finished a 10-month stint as Interim Associate Dean for Research and Development in the College of Science at The University of Texas Arlington. I was afforded that opportunity when some restructuring in another college left a temporary vacancy, which I was asked to fill. I certainly considered it an honor to be asked to serve in that role, but the temporary nature of that role also piqued my interest. For me, it seemed like a chance to do an internship in administration, to see if I liked it or not. I worked with great people, I did not really like the role.

Chromatographic methods often require that the analyte response is calibrated (and validated) over a wide concentration range when the analyte concentration in the sample is either unknown or is expected to vary widely. Bioanalysis, environmental, and clinical applications are just a few examples of where this may be the case.

For several years, our group has been working on a concept that we have termed multipath liquid chromatography (LC). The main idea is to target multiple classes of compounds following a single injection of a sample, the components of which are segregated on-line and directed to separate appropriate paths for simultaneous separation; the streams are then recombined for detection. I believe that this approach would be powerful for biomarker quantitation, where it would be more informative to track both metabolite and protein biomarkers to better define a disease state, or in the case of antibody–drug conjugate (ADC) development, where the metabolism of the ADC might involve understanding both the levels of the released drug and the remaining protein.

It is often possible to achieve better sensitivity and lower limits of detection and quantitation using standard gas chromatography (GC) equipment-here I’m referring to a standard split/splitless injection port and a Flame Ionization Detector (FID). Paying attention to some of the fundamental variables as well as some of the more esoteric considerations can lead to much improved method performance.

If given the need to determine drug A or its metabolite in blood, 99% of the time I would choose to start with liquid chromatography–mass spectrometry (LC–MS).

HILIC is not straightforward and there may be a number of mechanisms in play which need to be considered.

What type of mass spectrometry (MS) instrumentation provides the best specificity during trace quantitative analysis from complex mixtures?

It’s excellent to see that compendial authorities are considering updating “allowable change” regulations, and the updates to allow changes to gradient profiles certainly open up a whole new world, but there are some reservations around the new restrictions on allowable changes to stationary-phase chemistry.

One of the initiatives that the SCSC oversees is the nomination process and awarding of the Satinder Ahuja Award for Young Investigators in Separation Science. Where are all of the young investigators in separation science? Certainly, those that have been honored to date have been worthy; however, there must be more eligible parties out there.

What is the chemistry of this phase? What are the mechanisms of interaction with the analyte and hence how is retention and selectivity gained from this phase? How can we troubleshoot separation problems or develop suitable methods without a good knowledge of the bonded phase chemistry?

I do not remember the application, but I remember very clearly Professor McNair telling us that soil is one of the most challenging sample matrices, if not the toughest, from which to perform analytical determinations. Sources indicate the composition of soil ideal for growing plants to be 25% air, 25% water, 45% minerals, and 5% organic matter. That does not seem like a daunting makeup, but the reality is that the relative proportion of the constituents can vary dramatically.

The overriding majority of articles on problems with the technical transfer of HPLC methods ultimately focus on differences between HPLC dwell volumes. However, as the title suggests, there are many more issues which can cause problems in the transfer of HPLC methods, and I wanted to highlight some common issues that come across my desk, in the hope that it will help you avoid these problems in your own practice.

Several years ago, I would have held the stance that environmental analysis was fairly boring. How complicated can water be? I am not ashamed to say that was a naïve view. It is clear from our research and related research by others on similar topics that much more work in these areas is needed. Standard methods cannot solely accommodate the growing list of targets and the multitude of unknowns associated with complex samples taken from the interface between the petroleum industry and the environment.

I'm often asked to help with the development of column "screening" platforms and automated development systems. While this covers a large amount of analytical science there are some common elements to this type of approach, perhaps the most important of which is column selection. Unsurprising given that "selectivity" is the most powerful tool we have in chromatography and we all know that the best way to optimize selectivity is to choose the most appropriate stationary phase.