Effect of Alternative Mobile Phase Additives on the Peak Shape of Basic Analytes in RP-HPLC

Abstract

Introduction: Reversed phase liquid chromatography (RPLC) is a central analytical technique in pharmaceutical and biopharmaceutical science, where it is essential to reliably detect, separate, quantify, and purify drugs, of which a majority contain basic groups.
Background: Basic analytes often exhibit poor peak shape, for example tailing, due to mixed-mode interactions with the stationary phase. Acidic additives are commonly used to lower pH and act as ion pairing agents, improving chromatographic performance. Unfortunately, strong ion pair agents often suppress the signal in mass spectrometry detection. Followingly, it is desirable to find alternative additives that fit the silver lining between good chromatographic performance and MS compatibility.
Aim(s): This study aims to evaluate additive effects on peak shape, mainly tailing factor (Tf), of amines and peptides in analytical and overload conditions on two columns.
Methods: Different additives of varying concentrations were used to analyse Tf and w50% for amines and peptides in both analytical and overload conditions on HPLC-UV and -MS. Tf was calculated with the help of an Excel template and statistical analyses such as ANOVAs were conducted in RStudio.
Results: PrA and GA generally yielded worse tailing for the amines, than all the other additives. Peptides exhibited charge-dependant tailing and increased additive concentrations mitigated the extent of that tailing in both analytical and overload conditions. TCA was found not to be a suitable alternative to TFA with regard to MS compatibility, as indicated by previous studies.
Conclusion: No universally best additive was found, and the importance of MS compatibility was highlighted. Additive effects are highly dependent on analyte type and column.

Popular Abstract

The Search for Alternative Additives
In 1957, a drug for nausea during pregnancy was marketed; Thalomide. This drug proved to have a devastating side effect, resulting in infants with serious birth defects. The cause was later proved to be the presence of a mirror-image of the active compound. This case effectively shows the importance of robust and reliable separation and detection techniques when it comes to drugs. Reversed-phase liquid chromatography (RPLC) is widely used for this matter.
RPLC separates compounds based on their ability to interact with the hydrophobic surface of the small particles present in the separation column. The mobile phase is the liquid in which the compounds travel through the column, and it is modulable. Heterogeneities of the particle surface (stationary phase) sometimes cause compounds to interact in mixed ways with the surface. This is especially problematic for compounds containing amino groups, which under acidic mobile phase conditions become positively charged. Acidic additives can be employed to improve the interactions, as they are negatively charged and can form ion pairs with the amines. Trifluoroacetic acid (TFA) is exceptional at this but causes problems when detection techniques such as mass spectrometry (MS) need charged compounds to be able to detect them, as well as TFA being proven to be accumulative in nature (PFAS-compound). Formic acid (FA) is more environmentally friendly and does not form as strong ion pairs and consequently yields better MS signal. In essence, there is a trade-off between the chromatographic performance and MS detection.
In this study, different additives are evaluated based on the effect they have on the peak shape (mainly tailing) of the analysed compounds. Both small molecule amines and peptides are used as test compounds, and they are separated on RPLC with UV- and MS-detection.
It was found that two additives (propionic acid and glycolic acid) were particularly poor performing. For peptides which contained different number of positive charges, the effect of the additives on tailing was mainly charge-dependant, but the magnitude depends on the additive type. Increasing the concentration improves the peak shape for both small and big loads of peptides on the column. One alternative additive (trichloroacetic acid) to TFA was tested for its MS compatibility and proved to be even poorer performing than TFA.
Conclusively, the additive effect on peak shape depends heavily on the compound analysed and on which column. The search for alternative additives is not over yet!

• author: Eriksson, Matilda
• supervisor: Firas Jumaah
• organization: Department of Chemistry
• course: KEMR10 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: additives, amines, peptides, RPLC, tailing factor, analytical chemistry
• language: English
• id: 9246434
• date added to LUP: 2026-07-03 11:28:07
• date last changed: 2026-07-03 11:28:07