TO ADD OR NOT TO ADD - HPLC BLOG, K.SYCHEV© 2020

– that is a question when it comes to TEA (triethylamine), one among the most often used mobile phase additives in the history of HPLC

Triethylamine mobile phase additives were widely used in the past. Many reversed-phase separations obtained with the use of TEA can be come across in pharmacopeia. So why was it so popular?

No more than a couple decades ago the quality of intact silica was very poor. Among other things, the silica was highly contaminated with metal impurities.

To a greater or lesser extent, such impurities are able to interact with all kinds of ionizable functional groups of analytes. The most unwanted type of interaction caused by the formation of labile n-d complexes occurs for basic and particularly chelating compounds.

In all HPLC modes, this usually results in a number of quite specific separation problems. They may include excessive peak broadening, tailing, or fronting, often in combination with unexpectedly high retention times, or even no analyte elution in situations where they definitely should elute.

Whereas most of the pharmaceuticals and natural compounds contain basic or chelating groups, the mentioned problems were causing much trouble for practicing HPLC scientists in the past. Many technologies of additional silica treatment (including excessive end-capping) were invented and implemented by HPLC column producers, but none of them could solve the problem completely.

Therefore, in those days, to obtain a narrow peak with a good peak shape for a basic drug, one had to use the mobile phase with some added triethylamine – usually 0.1-1% depending on HPLC mode used for the separation. The mechanism of TEA action is quite straightforward: it blocks metal impurities and high-acidity silanols, thus diminishing the formation of their complexes with basic/chelating analytes.

As a rule, this results in better peak shapes for such analyses, but not always. Sometimes analytes break through the triethylamine layer to the intact silica surface. For such compounds adding TEA to the mobile phase does not produce any benefits. Hence, the effectiveness of this approach is limited, anyway.

But a modern ultrapure silica-based stationary phase, whether nonpolar or polar, does not need the dynamic modification by TEA, because it is already chemically inert.

For a chemically inert silica-based HPLC packing, adding TEA would have no effect on plate count or peak symmetry regardless of the HPLC mode used for the separation.

Adding TEA can be useful only in case of exploiting outdated, dirty silica-based HPLC columns.

Thus, if adding TEA has a positive impact on plate count and peak symmetry for some basic/chelating analyte, it means that the packing used is not quite modern. In turn, in case it does not, the truly modern packing is used, but then the approach itself is unhelpful and senseless from the viewpoint of chromatographic separation.

Therefore, TEA added to the mobile phase clearly indicates that the method exploiting such an approach may be recognized as obsolete.

Besides, TEA used as the mobile phase additive may have quite a negative impact on the method’s validation characteristics, as well as on its cost-effectiveness.

In combination with UV detection, it presents the source of multiple system peaks and a higher noise; it is also barely compatible with the gradient elution because of a very sharp baseline drift often observed in such cases.

Adding TEA to the buffer and adjusting it to a certain pH are both time and labor-consuming operations since they are carried out in the fume hood. Besides, inaccurate buffer pH adjustment can lead to the retention time variation.

Lastly, HPLC grade TEA is quite expensive: 10-12 $/mL, or approximately 10-50 $/day. So, TEA should not be used just in case, anyway.

But the best way is not to use it at all. And fortunately, the state-of-the-art high-quality HPLC columns nowadays fully provide us with such opportunity.


Key Words: Outsourcing HPLC Method Development & Validation, Analytical Method Development Services, Pharmaceutical Outsourcing, CDMO, Pharmaceutical Analysis, ICH Method Development & Validation

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