Columns | The LCGC Blog

When was the last time you reported your results with an estimate of the error associated with the data? You don’t need to because your method is performing within the levels defined by various agencies and which were confirmed by your validation and your daily QC checks. The person for whom you are producing the data is aware of these tolerances and therefore inherently appreciates the associated precision of the data and can make judgements based on this. Not in the world I work in!

If you use SPE in your work, then most likely it’s very important to the success of your applications and it’s proper implementation will be key to the performance of your analyses. However, SPE protocols are “variable in quality” (I’ve been as I kind as I can there!) and this variability appears to come from some common issues, misunderstandings and, frankly, ignorance of the mechanisms which are in play.

I got into a discussion with a learned colleague recently regarding the relationship between peak height and flow rate in gradient HPLC. We haven't really resolved the discussion, there are suggestions regarding "peak focussing," the number of column volumes in relation to the gradient volume (number of column volumes per minute), increases in efficiency etc.

What if we could make the troubleshooting poster on the laboratory wall come to life? What if we could build an engine which figured out the most likely causes of groups of symptoms and offer these up as a prioritised list for folks to work through and give them supporting information on each problem, why it occurred, how to fix it and, crucially, how to avoid it happening next time?

There are many aspects of analytical science which abound with myth and legend – but gas chromatography – mass spectrometry (GC-MS), and more specifically the electron ionization (EI) process, stands out as the technique which has given rise to the largest number of ‘urban myths’ and misunderstandings.

I have had enough conversations with experts in the field of high performance liquid chromatography (HPLC) stationary-phase supports to know that there is more to the increased efficiency provided by the use of superficially porous particles (SPP) compared to fully porous particles (FPP) than simply mass transfer effects. Yet, I would argue that this is still one of the biggest misconceptions propagated by some members of the chromatography community.

In this issue – I hope to outline some 'small bolts' that are commonly ignored in everyday chromatography and mass spectrometry - instrument settings that I have to come call 'lock and leave' parameters. We can generate data without changing them or paying them any attention, but how much better could our data be if we bothered to optimise them.

Much has been written about options for increasing efficiency in HPLC ? primarily through the use of core shell and sub 2 ?m particles, which have been used to increase efficiency, speed up separations or increase peak capacity. However, many separations can only be effectively improved, by optimising selectivity ? resolution is a function of selectivity, efficiency and retention, with selectivity being the most effective at achieving good resolution.

I will preface this blog installment by saying that I am not an expert on separation of proteins, but I am learning. What I do understand are the fundamentals of chromatography for small molecules. This information abounds, but it is much more difficult to find the same level of information for biomacromolecule chromatography.