I’m sure that is a question that many cannot answer. How do you know if you are missing analytes if you’ve never detected them previously? I am confident, though, that most would wish to have the confidence that every possible analyte in the sample is detected because think about the consequences; missing an impurity in a food or drug product could affect its safety, missing an analyte in a diagnostic test could result in a wrong diagnosis being made or missing something critical in an experiment could take you down a different route. It’s a bit like missing that mouldy orange in your fruit bowl and within days having to throw it away as it has now spoiled the rest of the fruit.
Luckily, there are several adjustments and tricks that you can employ in your liquid chromatography that can help increase the chances of not missing any analyte in your sample. In this article, I will briefly describe some of the options available to you.
An obvious place to start is with sample preparation, because if you don’t extract your analytes or get them into solution for separation then you fail at the first hurdle. Sample preparation is an enormous topic and too much to cover here, so I recommend reaching out to my colleague Petra Gerhards if you have specific sample prep questions. However, the one area that is often overlooked is the vial. Something so trivial can be the source of your missing analyte as analytes can bind to the free silanol groups on the surface of the glass vial preventing their separation and detection. As they say, ‘you can’t detect what you don’t inject’. If you are interested, more details can be found in this short article.
The second option to ensure you are not missing analytes is by increasing the peak capacity, or separation efficiency. By increasing the peak capacity, you increase the opportunity to detect peaks that might otherwise be masked by other peaks, especially things like trace impurities. There are several little tricks to do this:
Mobile phase – by adjusting or changing the mobile phase you change the selectively which alters your separation to improve peak capacity (it works both ways though, you may end up reducing the peak capacity, so you need to do your research!). One nice example of this is switching from a salt gradient to a pH gradient for the ion-exchange separation of monoclonal antibodies as described in this article.
The column – this area gives many options to super-charge your separations and increase resolution. First, the array of chemistries available gives you the power to experiment and select the column chemistry and mobile phase combination that offers you the best resolution – if you need help in selecting the right column, this resource page can assist. The second option with the column is to use UHPLC columns with small particles, typically under 2 µm, to achieve increased peak capacity and peak shape. The only caveat here is that you need a UHPLC system able to cope with the high back-pressure that such columns generate. The final option is to simply use a longer column as the more opportunity for interactions between the stationary phase and the analytes, the greater opportunity for separation and increased resolution; although the principle does not work for every application (see the pH gradient example above).
Two-Dimensional Liquid Chromatography (2D-LC) – As the name suggests, this technique uses two complementary separation parameters to increase the resolution. Separation is carried out on a particular column chemistry in the first dimension and fractions collected on- or off-line. These fractions are then separated on a second, complementary chemistry to improve the resolution. For more details on 2D-LC and the HPLC system configurations required to perform this technique, please visit this resource page.
The final area for consideration if you are missing analytes is detection. To visualise any analyte, you first must be able to detect it, but that is not always as easy as it sounds. All analytes are different, and detectors can have limitations and biases for different analytes. If you would like to read a short overview of the different detector technologies and their pros and cons, then please take a look at this previous article I wrote. The detector must be matched to your application, level of sensitivity required and analyte properties – obviously, there is no benefit to using a fluorescence detector if your analytes don’t contain fluorophores! In liquid chromatography, the most common detection mechanism is UV, however, some analytes lack chromophores which means they are not detected by UV-based detectors. In these situations, a universal detector, such as a charged aerosol detector, is recommended because they can detect analytes irrespective of their chemical structure. The added bonus is that a charged aerosol detector also gives you quantification data. Still, looking for complete confidence that you are detecting every analyte? Then the solution is to use a multi-detector approach for comprehensive sample coverage and described in this application note which use a combination of UV, charged aerosol and single quadrupole mass spectrometer detection to ensure analyte detection.
In summary, you have many options available to you to increase your chances of not missing critical analytes in your sample, but which one(s) you employ is dependent on your application and requirements. It is likely that you will need a combination of approaches, but whichever you choose there are likely to be trade-offs in the terms of speed/throughput and/or resources, be that increased technology costs or method development time. However, the costs of missing analytes often outweigh the cost of implementing new methods and technologies.
For those wishing to read more around topics in liquid chromatography, please visit the liquid chromatography website.
If you are considering Thermo Fisher Scientific for your liquid chromatography, then this infographic offers some guidance.
Watch this short whiteboard video to see how charged aerosol detection technology works.