Complementary Chromatographic Strategies for Enhanced Impurity Analysis and Purification of Peptides: A comparative study of HILIC, SFC, and RP-LC

Abstract

Introduction: Peptide therapeutics are becoming increasingly important across multiple disease areas, bringing increased structural diversity and a broader range of physicochemical properties. This diversity, together with more complex synthetic- and product-related impurities, raises the bar for liquid chromatography methods that should be efficient and sustainable. Reversed-phase liquid chromatography (RP-LC) is widely used; however, it may have limited resolving power for closely related impurities. Hydrophilic interaction liquid chromatography (HILIC) and supercritical fluid chromatography (SFC) offer complementary selectivity that may improve impurity resolution and workflow efficiency.
Background: Despite recent studies on the topic, systematic guidance for applying HILIC and SFC to peptide samples is limited; the influence of organic modifier, buffer conditions, and stationary-phase chemistry on selectivity and robustness remains greatly unexplored.

Aim(s): To compare HILIC and SFC with RP-LC for peptide analysis and purification, establish orthogonality, and identify practical method-development guidelines to apply to diverse peptide profiles.

Methods: A set of nine commercially available peptide drugs were analysed by RP-LC, HILIC and SFC for comparison. The effects of chromatographic variables on retention, elution order, peak shape and resolution were investigated.

Results: HILIC exhibited distinct and often inverted elution orders relative to RP-LC, consistent with their different retention mechanisms. Some of the HILIC methods resolved several synthesis-related deletion impurities that co-eluted in RP-LC.
In SFC, additive identity governed retention via ion pairing: average retention followed TFA < ESA < MSA for most peptides. The columns CN, GSBC and GSFS provided the most balanced SFC performance across additives; highly basic and polar peptides (e.g., ACTH, liraglutide) retained strongly across phases.
Conclusion and future perspective: HILIC provided robust, orthogonal selectivity to RP-LC for peptide mixtures, potentially improving impurity detection and purification. In SFC, peptide retention and peak quality were influenced by the additives, as well as peptide and column chemistries.

Popular Abstract

Cleaner peptides, faster: how HILIC and SFC complement RP-LC to find and purify impurities
Peptides are promising drugs, but they often come with structurally similar impurities that are hard to spot and remove. This project compares three analytical methods used to analyse and purify peptides: the standard reversed-phase liquid chromatography (RP-LC), and two complementary methods - hydrophilic interaction liquid chromatography (HILIC) and supercritical fluid chromatography (SFC). The aim was to show when and how HILIC and SFC can give different separation of peptide mixtures (orthogonal selectivity).
Why this matters:
● Peptide mixtures can contain almost-identical impurities. If we can separate them better, medicines can be made more safely and efficiently.
● RP-LC is the standard method, but it sometimes leads to co-elution of impurities and the target peptide. HILIC and SFC can offer different separation mechanisms providing complementary merits in removing impurities.
● HILIC and SFC allow replacement of hazardous solvent and easier post-purification processing, which helps the environment, safety, and costs.
What was done:
● Nine peptides were analysed by RP-LC, HILIC, and SFC to compare different elution profiles.
● In HILIC, acetonitrile (ACN) and methanol (MeOH) were tested as solvents.
● HILIC and RP-LC selectivity was compared by analysing degradation- and synthesis-related impurities elution relative to the target peptide of the samples.
● In SFC, a CO2-rich mobile phases were used along with three acids (trifluoroacetic acid, ethane- and methanesulfonic acids), that change how peptides travel through the column.
Key findings:
● HILIC often separated peptides in a different order than RP-LC. In several instances it was able to separate impurities from the target peptide when RP-LC failed to do so.
● In HILIC, ACN usually gave cleaner separations than MeOH, which typically led to early elution of the tested peptides.
● In SFC, the choice of acid mattered: ethane- and methanesulfonic acids usually improved the analysis process when compared to trifluoroacetic acid.
Take-home message:
● HILIC and SFC are complementary methods to standard RP-LC for peptide analysis and purification. The results offer a starting point for future exploration of this topic.

• author: Alessi, Octaviano
• supervisor: Margareta Sandahl
• organization: Centre for Analysis and Synthesis
• course: KAKM01 20261
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Chemistry
• keywords: Hydrophilic-interaction liquid chromatography (HILIC), Method development, Orthogonal separation method, Peptide analysis and purification, Supercritical fluid chromatography (SFC), Analytical chemistry
• language: English
• id: 9247423
• date added to LUP: 2026-08-10 09:59:35
• date last changed: 2026-08-10 09:59:35