• Application Note

Utilizing the Mixed-Mode Atlantis PREMIER BEH C18 AX Column for the Analysis of Cationic Surfactants Under Reversed-Phase Conditions

Utilizing the Mixed-Mode Atlantis PREMIER BEH C18 AX Column for the Analysis of Cationic Surfactants Under Reversed-Phase Conditions

  • Chris Henry
  • Paul D. Rainville
  • Waters Corporation

This is an Application Brief and does not contain a detailed Experimental section.


This application brief demonstrates a simple method to provide an analytical solution for a selection of polar cationic surfactants using reversed-phase solvents and the mixed-mode Waters Atlantis PREMIER BEH C18 AX Column.


The analysis of quaternary ammonium compounds has long presented a challenge analytically, due to the high polarity of these compounds resulting in poor retention under reversed-phase conditions. The development of the mixed-mode Atlantis PREMIER BEH C18 AX Column allows us to demonstrate the separation of three cationic surfactants using reverse phase and mobile phase.


Surfactants are used extensively in the cosmetic, pharmaceutical, and agricultural industries covering a diverse range of uses, from antimicrobial agents to anti-static agents. Due to their amphiphilic nature (i.e., possessing both a polar hydrophilic and non-polar hydrophobic region), they represent a challenge analytically.

Cationic surface-active agents often contain nitrogen atoms carrying a positive charge (mainly in an amine or quaternary ammonium group), which are coupled with one or several long chains of the alkyl type. Quaternary ammonium compounds (QACs) are one of the widest applied groups of cationic surfactants.¹

HPLC is commonly employed for surfactant analysis, however using a conventional reversed-phase (RP) separation can struggle to retain these compounds due to high polarity. Ion-pair chromatography (IPC) and gas chromatography (GC) are also used but IPC suffers from long equilibration and run times, while GC often relies on analyte derivatization, which is time consuming and has the potential to introduce sample preparation errors and/or variation.


Table 1. Instrument parameters.

Results and Discussion

The challenge of those strategies has led to the development of mixed-mode RP/anion-exchange (AX) columns, which contain stationary phases having both hydrophobic and AX functionalities.

However, many mixed-mode RP/AX columns suffer from poor batch-tobatch reproducibility and poor hydrolytic stability. To overcome these issues, Waters has developed a new stationary phase based on hybrid organic/ inorganic particles that has excellent batch-to-batch reproducibility and is stable over a wide pH range. This material is packed into new MaxPeak High Performance Surfaces (HPS) column hardware, which is designed to increase analyte recovery by minimizing interactions with stainless steel that can lead to analyte loss.²

In this technical brief, we analyze a mixture of three cationic surfactants (detailed in Figure 1) with the goal of achieving good retention, baseline separation, and good peak shape.

The samples were run on an ACQUITY UPLC H-Class PLUS System equipped with an evaporative light scattering detector (ELSD), as some of the compounds tested exhibited weak/no chromophore.

Analytical standards of all compounds detailed in Figure 1 were sourced from Sigma-Aldrich (Poole, UK). A solution containing all three compounds was prepared in water and injected directly. The samples were analyzed using the method conditions detailed in Table 1.

Figure 1. Cationic surfactants analyzed using the mixed-mode Atlantis PREMIER BEH C18 AX Column.

Good retention was observed for all three compounds with retention factors (k) of 3.8, 5.1, and 7.8, and tailing factors of 1.16, 1.33, and 1.39 for 1-dodecylpyridinium chloride, benzyldimethyldodecyl ammonium chloride, and benzyldimethylhexadecyl ammonium chloride, respectively (Table 2).

Table 2. Results obtained for the three compounds tested.

The Atlantis PREMIER BEH C18 AX Column successfully separated the QACs with full baseline resolution, while exhibiting excellent peak shape in a 10-minute runtime (Figure 2).

Figure 2. Quaternary ammonium compounds analyzed using the mixed-mode Atlantis PREMIER BEH C18 AX Column (25-85% B gradient) using the ACQUITY ELSD Detector.


The mixed-mode Atlantis PREMIER BEH C18 AX Column has proven its effectiveness for the analysis of a mixture of three high polarity QACs using reversed-phase solvents without the need for derivatization or ion-pairing reagents.

This simple method demonstrates how this column can provide a straightforward analytical solution to a traditionally challenging group of compounds.


  1. Olkowska, E; Ruman, M.; Kowalska, A.; Polkowska, Z. Determination of surfactants in environmental samples; Part 1 cationic compounds. Ecol. Chem. Eng. S. 2013, 20, 331–342.
  2. Zhang, K. and Liu, X. Mixed-mode chromatography in pharmaceutical and biopharmaceutical applications. J. Pharm. Biomed. Anal. 2016, 130, 19–34.
  3. Walter, T.H.; Alden, B.; Boissel, C.; Field, J.; Lawrence, N.; Osterman, D.; Patel, A. A New Mixed-Mode Reversed-Phase/Anion Exchange Stationary Phase Based on Hybrid Particles. Waters Corporation Application Note, submitted for publication, 2020.

720006746, February 2020

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