Over the last several years there has been a slow and steady progression within the LC-MS community to move traditional high-flow applications to lower flow rates. In particular, moving into the microflow regime has proven to be a simple adjustment in methodology that can result in a lot of gain for only a little pain. Microflow chromatography can provide an instant boost in sensitivity because of the increased ionization efficiency at lower flow rates. Additionally, microflow chromatography can lower solvent consumption and reagent costs and reduce downtime spent on routine instrument cleaning.
In the recent webinar Microflow Chromatography: The Key to More Sensitive Met ID, we discuss the benefits of microflow chromatography for metabolite ID applications. We compare microflow versus high flow for the identification of metabolites from several well-characterized drugs. The results are clear. Microflow provides:
- More complete profiles
- Increased metabolite identification
- Better quality MS/MS spectra for more confident identifications
As a follow-up to the webinar, we wanted to take the opportunity to answer some questions we received about using microflow for metabolite ID. After watching the presentation and reading our answers, we hope you will be convinced to consider microflow chromatography for your metabolite ID applications, too.
- For people who are traditional analytical-flow chromatography users, how easy is microflow—given the perception that low-flow LC can be difficult to use? With the advancements in microflow pumps, ionization sources and the increasing availability of columns, implementing microflow should no longer be considered difficult.
- Met ID samples tend to be prepared with little sample prep to limit losses. How robust do you see microflow for these heavier matrices? Because of their smaller diameter, microflow columns can be a little more prone to clogging than larger diameter columns. Users should ensure that their samples are particulate free, and do not have components prone to precipitating during separation. With a little more care during sample preparation, a microflow system should be very stable.
- Do you see a continuing trend to move from analytical flow to lower flow rates with SCIEX customers? Yes. Our OptiFlow® ion source has now been on the market for over a year and a half, and customers who have evaluated it are now purchasing them for routine use.
- Is the increase in ESI sensitivity when going from high flow to micro flow a general phenomenon, or is it highly dependent on source design? I believe it’s a combination of both. At lower flow rates, electrospray ionization should be more efficient than it is at higher flow rates, assuming proper voltage and gas flows. If the source is not well designed, the transmission of ions into the mass spectrometer will not be as efficient as it could be.
- Have you assayed drug residues in extracts of animal tissues or plasma? What was your sample clean-up procedure? How was your signal-to-noise ratio (S/N) for the analyte? I have done plasma bioanalytical small molecule quantification using our OptiFlow ion source. The sensitivity gains were very compound dependent. I’ve used techniques from basic protein precipitation to well-developed SPE. As I recall, the lowest gains were for a compound analysis where high-flow LC was already very sensitive, and that showed a threefold improvement in S/N. For more difficult compounds, I’ve seen an over 20-fold improvement in S/N.
- For microflow applications where small ID columns are used, limited injection volumes are probably necessary, especially in isocratic elution mode. What type of autosampler is used and what are the lowest injectable volumes of sample? All of the microflow work I’ve done has been on an nanoLC™ 425 system (Eksigent™) operating in microflow mode with the low microflow exchangeable flow module. Large injections can be approached in a couple of ways. One is to do a standard direct injection and allow the full volume of the injection loop to flow onto the column, focusing analytes with a low-organic hold. But this can be slow, depending on the loop volume and flow rate. Alternatively, trap and elute with a generic trap column and a higher flow rate load can be done to speed things up. I have not personally done isocratic microflow separation, but I would think small injection volumes would be required for this to work well. Also, expect column lifetime to be compromised because of the lack of a high-organic wash. The lowest injectable volume will depend on the autosampler. I’ve never gone below 1 ml on this microflow LC system because there was no need.