What Are the Considerations for Using Ion Pairing Reagents in My Mobile Phases?

日付: 01/24/2019

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For research use only. Not for use in diagnostic procedures.


Ion pairing reagents can greatly help with problematic chromatography, but any added ion pairing reagents also tend to stick to LC components, the column, and the MS source. This can leads to signal suppression in subsequent analyses, as well as diminished analytical column performance.

Here are some suggestions for a more successful experience when using ion pairing reagents and mass spectrometry:
1) Use the minimum concentration of ion pairing reagent that is effective.
2) Select a commonly used reagent that also volatile. TFA, TEA, HFIP, volatile alkyl quarternary amine salts such as TBuAA or TBuAF, and alkyl primary amines, such as n-hexylamine are recommended.
3) Match the ion pairing reagent to the analyte in question. (For example, positively charged species, such as peptides or proteins, will not likely respond to positively charged ion pairing agents such as TBuAA. Likewise, negatively charged species will not respond to negatively charged ion pairing agents.)
4) Flush the LC thoroughly after using an ion pairing reagent to remove traces amounts of the agent. Choosing which solvent and flow rate to use will depend on the IP reagent used. (For example, 50/50 ACN/Water is sufficient for TFA, but probably not TBuAA.)
5) Remove the column used with ion pairing reagents after analysis is complete and save for use with ion pairing applications only. (While the actual mechanism of IP agents is unknown and may vary, there are two different models that have been proposed, the partition model (IP agent complexing with the analyte) and the adsorption model (IP agent complexing with the column stationary phase). In either case, the IP agent can (and often does) complex with the stationary phase and slowly bleed off the column if it is re-used for other analyses.)

When switching to volatile ion pairing agents from an alternate method, note that the selectivity may not be the same as it is in the original method. While IP-LCMS methods have been successfully used in the past, these should only be tried if other approaches are not working. Hydrophilic interaction chromatography (HILIC) may be a good option to try instead of an IP-LCMS-based method. However, not all analytes are amenable to HILIC (e.g., therapeutic oligonucleotides). In these instances, IP-LCMS is a powerful tool, but care needs to be taken to prevent detrimental effects to other analyses run on the same LCMS system, and, therefore, dedicated columns and possibly dedicated LC systems are recommended for any IP approach.