Organic aicds and the 'BEH Amide column'

Dear fellow UPLC users.I am currently working on a method for the analysis of small polar molecules (e.g. organic acids). (UPLC-MS/MS)I have tested the ‘HSS T3' column however I obtain better peak shapes for the individual compounds using a ‘HSS C18' column (2.1 x 100mm). It is difficult to get good separation on all the analytes. Using the 150mm (‘HSS C18') column tends to give more broad peaks. I use MeOH and water with formic acid as eluents. (e.g. succinic- and fumaric acid co-elute with retention time 1.15 minutes, with good peak shapes though). When going through waters applications and this forum, I found this recent Waters application: http://www.waters.com/webassets/cms/library/docs/WA60096.pdfIt looks like this is the column (‘BEH amide') should give me better resolution. My experience in working with normal phase (HILIC) is limited though, so I have a few questions before I purchase this column.1) My samples are aqueous, however I might be able to dilute to 50% organic. Will this work well with this column using high organic as initial conditions?The samples to be analysed are filtered but contains quite high concentration of glucose. I'm concerned whether this will affect the detection (e.g. matrix effect) as the BEH amide' column is recommended also to retain carbohydrates. 2) I read that better sensitivity is usually obtained when working with HILIC. Should I expect the same when switching from ‘HSS C18' to ‘BEH amide'? If yes, how much should I expect in case of improved sensitivity? (Approximately).3) How well does this column retain underivatized amino acids? Best regards,/Rune

Answers

  • One of the first applications that we tested with the BEH Amide chemistry was small, organic acids such as those you describe. The result of this work is the Application Highlight that you highlight.

    To answer your questions:

    1) Diluting your samples with some organic modifier will likely give you the best peak shape. However, you may be able to still obtain very good peak shape with a high aqueous solvent strength injection solvent. For example, when analyzing carbohydrates, our scientists were able to injection 100% aqueous samples (beer) with no loss of peak shape due to solvent strength. So, I suggest you emperically test the maximum percent aqueous that you can use in your samples that does not compromise peak shape.

    2) HILIC can provide improved sensitivity as compared to reversed phase-LC when the detection mode benefits from higher organic solvent content (e.g., MS). If using UV, you'll see little to no sensitivity benefits when using HILIC vs. RP-LC.

    3) To my knowledge, we've not tested underivitized amino acids.

    I hope this information helps,

    --Doug

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