Solvent, Chemistry, and Other Myths in LC–MS -

Solvent, Chemistry, and Other Myths in LC–MS

Dec 1, 2009
By: Michael P. Balogh
LCGC North America

Michael P. Balogh

Sometimes we can be surprised to find we have overlooked details in areas where we are otherwise so well versed. Then, forced to have another look, find we have opened a wealth of understanding. The art and science of chromatography often seems to offer opportunity for passionate debate. Over the past seven years, since CoSMoS conferences began, the chromatography discussion sessions have been the most highly rated, often inciting the most vigorous debate. A now-famous quote from former U.S. Secretary of Defense Donald Rumsfeld might apply well: "There are known knowns. These are things we know that we know. There are known unknowns. That is to say, there are things that we now know we don't know. But there are also unknown unknowns. These are things we do not know we don't know." (This quote was taken from a press briefing February 12, 2002, on the increasingly unstable situation in Afghanistan.)

A recent discussion at CoSMoS 2009 (Boston, Massachusetts, see http://www.CoSMoScience.org/) proves Rumsfeld's observations aptly describe the state of much of the underlying, and often misguided, assumptions we bring to bear when applying our understanding of chromatography to liquid chromatography–mass spectrometry (LC–MS) practice. The inherent value of the charter principle of CoSMoS since its founding in 2003 is crafting open discussion workshop sessions to encourage a free exchange of ideas, leading to enhanced practical knowledge.

One might, for example, approach LC–MS with a wealth of condensed phase knowledge based upon years of chromatography practice and accept the prospect that protonation (and therefore sensitivity) is enhanced at least in positive-mode electrospray by ensuring the analyte is more basic than the solvent. While at odds with accepted practice previous to widespread MS use, adding a volatile modifier such as acetic acid in place of the usual, less-volatile mobile phase components found in LC practice becomes commonplace. In many ways, our solvents of choice have followed similar paths.

Marketing literature — for example, a trade column written by a scientist employed by industry — while typically designed to be useful, can nonetheless be biased, seeming to further confound understanding. The same can be true for conference presentations. A speaker who brings a lifetime of interest, effort, and achievement in a given area will necessarily color the presentation and typically place too much burden of preparation and prior knowledge on listeners much less familiar with the topic. The members of the CoSMoS planning board believe providing a platform for ideas is, of itself, not enough and those ideas need "rounding out" if not debunking outright.

For example, the commercial success of a new technology such as ultrahigh-pressure liquid chromatography (UHPLC) is often debated by what in political terms might be called the "loyal opposition." Such a debate requires a neutral, public arena for discussion on the merits with as many different facets of the argument as possible in full view. Arguing the merits of even common practice such as heating the mobile phase or the number of plates one needs to achieve, while clearly important and enduring topics, requires more than a single voice to achieve understanding. I stumbled upon a comment when preparing material on monolithic columns from the thesis of Jennifer Smith (1), someone I met briefly when she was a student of Harold McNair (Virginia Polytechnic Institute, Blacksburg, Virginia). "The term 'fast HPLC' is a relative one. Analysis time of itself is a poor measurement of chromatographic performance; rather the important parameter is the number of peaks separated per unit time. For example, a 10-component run in 10 min is more time efficient than a two-component run in 10 min. Nevertheless, it should be noted that the terms "fast LC,", "fast HPLC," "high-speed HPLC," and "ultrafast HPLC" are commonplace in the literature without formal definition."

Smith's observation conveys needed clarity. The work of Uwe Neue and others (2) describes the possibility of extremely efficient separations under fast-flow conditions when pressure is not a factor. Today, when continuing this discussion, we also should be cognizant of work done by Jorgenson, Lee (3), and others who advanced other time-honored practices such as high temperature along with high pressure because it benefits our analytical interests. Lee's work published in 2003 shows an initial 4-min separation for an eight-component barbiturate mixture with a uracil marker performed at 10,000 psi and 220 °C shortened to less than 40 s at 35,000 psi and 800 °C. Of course, when the mixture is too complex, simply making a run faster is clearly not of value nor is this impressive capability within reach of most practitioners without specialized equipment.

The value of conferences such as CoSMoS lies in discussing topics and concepts that are often difficult to comprehend. The conference is not the byproduct of a gathering but is instead the primary purpose for practitioners to gather. This year, for instance, attendees were fortunate to hear Georges Guichon and Pavel Jandera speak on the advantages and costs of two-dimensional chromatography (the presentations are available to download at http://www.cosmoscience.org/archive_2009.htm). Both speakers were featured at other conferences as well but being a small discussion-focused event, CoSMoS attendees also got to participate in the ensuing discussions.

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