Columns | The LCGC Blog

I always think of a conference presentation to be like a rock band concert. Sure, the band is going to play some of their biggest hits, but they also want to propagate their new stuff. More importantly, they want to put on a show so that people are entertained. I do think there should be more emphasis on entertaining the audience during oral presentations.

Sometimes troubleshooting a separation can rely upon the end user spotting subtle clues within the chromatogram, and at other times the visual signs can be much more obvious. To start the New Year, I wanted to share some of the most common issues that we see with peak shapes in gas chromatography in the hope that if you spot some of these in your own work, you may be able to intercept problems and deal with them more effectively.

Two things were surprising about some recent research we reported. First, with regard to chemically impaired groundwater quality, it may not always be the chemicals that are most worrisome for human health impacts. Second, the primary methodology we used in that work for microorganism identification, matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS), is a vastly underappreciated tool, especially outside the clinical realm in areas such as environmental monitoring.

It’s very easy to be comfortable with what you have. It’s only when we realize what could be, that we become interested in changing things.

When I want to hear some humorous stories, there are few friends in the instrument manufacturing and sales business I can contact. If I ask them about their recent experiences with the cannabis industry, their stories will cover topics ranging from instruments purchased using duffel bags of cash (cue images of large men in suits and sunglasses packing heat) to recent college graduates who cleared $25 million in their first year of business selling cannabis butter (cue images of large men at breakfast laughing uncontrollably).

I hear the words “struggling for sensitivity” so often when speaking to folks using LC–MS for bioanalysis, environmental analysis, metabolomics, proteomics, and a host of other applications where target analytes are present at low concentrations in complex matrices. We spend fortunes on MS/MS instruments to increase specificity of detection in order to improve sensitivity. Some of us go to great lengths to optimize sample extraction and HPLC conditions in order to minimize matrix suppression effects and improve specificity and hence sensitivity.

The presence of capable students and researchers, each with a technical niche to offer, prepares us for future opportunities. On top of that structure, we also aim to maintain this mindset for our instrument selection before beginning projects. The installment of core labs at UT Arlington (www.uta.edu/sirt), making a wide range of instruments available to all research groups, has allowed our students to brainstorm about which instrument is the most appropriate for a specific analysis rather than how to make a measurement work with a given instrument.

Data validity and a thorough understanding of the results that we produce should be of great interest to us all. Here's why.

Internal standards (IS) are commonly incorporated into quantitative methods to increase accuracy and precision. An IS is a compound that is different than the analyte of interest, has similar physicochemical properties to the analyte, and is added to samples, calibration standards, and quality control samples in a known quantity. It should not be present in the sample, it should be available in high purity, and it should be easily differentiable from the analyte of interest.

The ability to rapidly screen stationary phases through column-switching capabilities provides significantly greater efficiency in method development than was previously possible. The approach does require some additional hardware and software. And, while such capabilities may limit the ability to expand one’s literary knowledge during excessive months in the laboratory developing separation methods, real progress to key decision points for method optimization can be realized instead.

With the increasing proliferation of UHPLC, with its very much reduced extra column volumes, is the concept of dwell volume still relevant? Indeed, in your world, was it ever relevant at all?

As the fall semester starts, I begin my 13th year as a chemistry professor. Let me get right to the point-if you do not love this job, you will hate it!

The ability to rapidly screen stationary phases through column-switching capabilities provides significantly greater efficiency in method development than was previously possible. The approach does require some additional hardware and software. And, while such capabilities may limit the ability to expand one’s literary knowledge during excessive months in the laboratory developing separation methods, real progress to key decision points for method optimization can be realized instead.

Until recently, I hadn’t heard of ternary or quaternary gradients being used for many years. They have gained a reputation for being somewhat difficult to reproduce-less robust, if you like.

Hydrophilic Interaction Chromatography can be a very useful tool in our analytical armory, however there are some practical peculiarities to this chromatographic mode which need to be understood in order to ensure success. 

I believe that the term “top-down proteomics” holds a particular connotation with respect to the use of ultrahigh-resolution mass spectrometers in people’s minds. And rightfully so. If one is to determine with confidence the sequence and charge state of a particular fragment ion generated in the gas phase, then high mass accuracy is a must. From the discovery side of things, where qualitative analysis is most important, this is not likely to change. However, when you turn to quantitative analysis, where you want to now monitor levels of a particular protein biomarker for the purpose of disease diagnosis, prognosis, or treatment, then invariably bottom-up strategies are the norm. Protein quantitation using top-down strategies, especially on low-resolution triple-quadrupole systems, have been largely ignored, until recently .

I’m often asked “what reproducibility should I expect to get from my [insert instrument manufacturer and model]?” So, most folks are referring to the repeatability aspects of precision, as in: “what relative standard deviation (usually expressed at %RSD) for peak area or quantitative result should I be able to achieve from repeat injections from a single vial of sample?”

Precise and accurate quantitative analysis based on chromatographic measurements has historically relied very heavily on careful peak integration. Seasoned analysts know that while automated algorithms exist in modern chromatography software, it is a best practice to manually check that the integration points-the points at the beginning and end of a peak, between which the peak will be integrated to obtain a peak area-are appropriately specified.

I guess we have all had the issue at some time or other. A blank injection of eluent (or a zero volume dummy injection) gives rise to discreet peaks on the chromatographic baseline which may interfere with analyte peaks or increase the complexity of the chromatogram obtained, especially when performing trace analysis.chromatogram obtained, especially when performing trace analysis. Of course this is an undesirable situation and one which can be difficult to understand or troubleshoot as, if we didn’t inject anything, how on earth do we end up with discrete beaks in the blank chromatogram? The secret to understanding the phenomenon lies in understanding the mechanisms of gradient of HPLC.

As part of the Earth Day celebration in Dallas, Texas, last month, the Collaborative Laboratories for Environmental Analysis and Remediation (CLEAR) at U.T. Arlington hosted the first annual Responsible Shale Energy Extraction (RSEE) symposium (www.shalescience.org). We had an exceptional range of speakers who conveyed all sides of the issue, including U.S. Secretary of Energy Rick Perry and atmospheric scientist Dr. Katherine Hayhoe from Texas Tech University, one of Time’s top 100 most influential people. We had representatives from major oil producers, environmental groups, land management groups, water recycling service companies, and scientists conversant on many key issues related to unconventional oil and gas (UOG) extraction. Even though we have been very involved in this conversation for the past several years, several points stood out.

As I’ve written previously, our Nirvana in HPLC column selection would be to enter the structures of our analytes and have a database tell us the column and mobile phase conditions that we should use to carry out the separation successfully. Although I know of several groups who are working on this, in practice we are still a long way from realizing this goal.

I have written previously about occupying the middle ground in the debate over the environmental implications of unconventional oil and gas extraction operations. It seems we are just in the right place, when we are criticized and praised at separate times by proponents of both extreme views-namely those who think UOG is perfectly safe and those who think it cannot be done without ruining the environment.

A short treatment of what to consider when choosing the appropriate sample solvent so you can be better informed when developing, optimizing, transferring, or troubleshooting your GC methods.

Students who have an internship on their resume, and are seeking jobs in a particular sector, are doing so with an informed opinion. While an internship comes in many forms, that real-world experience has provided a clear touchstone of understanding of what it would be like to work in a given sector.

This time I’m going to be looking at how to get the most from your GC–MS (Gas Chromatography–Mass Spectrometry) system, and I intend to keep things as simple as possible, however I also make no apology in the fact that some of the concepts may be beyond your current understanding. My intention here is to explain some of the basics so that you will be able to use your instrumentation to best effect.

For the most part, we are still instructing undergraduate students in the same way as when I went to school, and I think this is a disservice to the students and to the nature of chemistry. No wonder chemistry programs have trouble attracting students compared to other science disciplines, like biology and psychology. Students will take general chemistry, but they cannot see where it may lead. I want to change that.

As I wrote the title of this LCGC Blog instalment, I could not help but wonder where the cliché “more than one way to skin a cat” came from. Turns out it is from Mark Twain in his 1889 work, A Connecticut Yankee in King Arthur’s Court. I have never read that book, but I certainly have heard this saying used more than once - even if it might offend some cat lovers. Of course, it means simply that there is more than one way to do something.

Many of our instrument techniques rely on a calibration in order to relate the detector response to the amount of analyte within our sample.

In order to contemplate and test your knowledge, Kevin A. Schug provides a sampling of some of his chromatography true–false questions from his senior-level Instrumental Analysis course.

Our technical support center deals with many issues regarding irreproducibility of retention and selectivity in reversed phase HPLC. Very often, the problem lies in poor equilibration of the HPLC column between injection, which in gradient HPLC can affect the separation selectivity as well as analyte retention.