Tony Taylor

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?

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.

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.

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.

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.

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.

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.

So, what’s not in your standard operating procedure? Documenting details can prevent headaches associated with method transfers between laboratories.

What’s not in your standard operating procedure? Documenting details can prevent headaches associated with method transfers between laboratories.

So, what’s not in your standard operating procedure? Documenting details can prevent headaches associated with method transfers between laboratories.

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

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?

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’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?”

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 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.

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.

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.

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

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.

As we enter the Generalization phase of the industrialization of Analytical Science, we find ourselves striving for the generic in as many areas as possible.

I started my career working in a Quality Control laboratory. I'm familiar with the constraints that must be imposed to ensure consistent global standards and the pace at which change can be implemented without loss of control either within a business or across an industry. OK-scene set.

I’ve often written about the “lazy” chromatography which has swept through our industry, whereby 0.1% (w/w or w/v!) TFA or Formic acid is used to “buffer” the eluent system well away from the pKa of analyte molecules, leaving most acidic analytes in the ion suppressed form and most basic analytes in the ionized form. This approach avoids having issues with retention time drift and effectively eliminates pH as a variable used to control retention or selectivity for separations involving ionogenic analytes.

I’ve done that thing where I’ve stated a very interesting title-I hope I can deliver something which lives up to it. I dislike it when people “overstate” their talk or poster titles at conferences to draw me in and then don’t deliver against the promise-I’ll let you judge how we go here.

The title of this piece may have put you off reading it – in which case you won’t be insulted when I say that even the most experienced gas chromatographers often fail to install columns in the best way possible.

Not every method is as we would hope it would be. Some methods come to us in imperfect form and we have to live with them, while others are difficult separations and, by necessity, need to be developed using close control of several variables such as eluent pH, eluotropic strength, ionic strength etc.

In 2009 I wrote an article on the emerging field “Bio Chromatography,” which for a small molecule analyst such as myself, perfectly described the situation. I realize that the separation of biomolecules had been happening for many years, but the expansion and development of protein based therapeutics from that point onwards has seen an avalanche of developments in instrumentation, sample preparation, and column technologies the like of which I have not seen in my 30 year career.

I often get asked about the other important aspects of “screening” in HPLC, which include the mobile phase composition, gradient parameters and flow rate – so that’s the theme of this installment.

I’ve been talking about ‘ToolKit’ method development platforms a lot lately and I have to say I’m not sure we are making the most of this approach.