Working in Tandem: The Benefits of Sequential Elution Liquid Chromatography in Pharmaceutical Analysis

LCGC International spoke to Lauren Lovejoy, principal scientist in the Drug Substance and Product Analysis group at GSK (Philadelphia, Pennsylvania), about the advantages of using tandem columns in sequential elution liquid chromatography (SE-LC) to investigate weak acids, neutral compounds, and permanent anions in pharmaceutical applications.

Q: What is sequential elution liquid chromatography (SE-LC), and how does it differ from conventional high performance liquid chromatography (HPLC)?

SE-LC is a technique where multiple elution strategies are applied in a series to separate different groups of compounds. In this approach, a sample containing various compound groups is introduced into a chromatographic system using a weak mobile phase that causes strong retention of all compounds. These compounds are then sequentially exposed to selectively strong mobile phases, each designed to facilitate the elution of a particular class of compounds.

SE-LC offers several benefits compared to conventional HPLC, particularly because of the use of sequential elution modes, denoted as “r.” These modes represent each distinct set of conditions designed to elute specific compounds, regardless of whether a single column or a tandem setup is used. SE-LC can enhance peak capacity by a factor of r and reduce separation disorder (entropy) by log r. As each elution mode in the sequence isolates and elutes only one group of analytes, the overall peak capacity of SE-LC is cumulative. This results in a significant increase in peak capacity for SE-LC compared to conventional HPLC, leading to a higher likelihood of achieving successful separations as a result of the reduced disorder and enhanced peak capacity.

Q: What are the primary stationary phases used in this study (1) for SE-LC?

In this particular study, a tandem column approach was used with a superficially porous C18 (reversed phase) column coupled with a strong anion exchange (SAX) column. For this separation, the tandem column approach was required to retain and separate the three classes of compounds: weak acids, neutrals, and permanently charged anions.

Q: What are the key advantages of using SE-LC in pharmaceutical analysis?

The increased peak capacity and reduced separation disorder are the key advantages of SE-LC in pharmaceutical analysis over conventional HPLC (2,3). Importantly, the same conventional HPLC instrumentation can be used to perform SE-LC separations. For separation of complex analytes that cannot be achieved by conventional HPLC, an SE-LC approach should be considered. The SE-LC method that I developed showed great promise related to analyte peak area precision, retention time precision, and resolution for three replicate injections. Further validation work was also successfully performed (4).

Q: What were the observed retention time and peak area precision in this study?

System precision was assessed by calculating the percent relative standard deviation (%RSD) for the peak areas and retention times for three injections of the analyte test mix. The repeatability of the peak areas of the analytes was 0.0–0.9% RSD. The repeatability of the retention times of the analytes was 0.01–0.12% RSD. The ideal peak area and retention time precision of not more than 2% RSD was achieved in this study.

Q: How does the tandem column configuration contribute to the success of SE-LC separations?

A tandem column approach is not required for an SE-LC separation. There are other researchers who have performed SE-LC with individual columns. I would have preferred this approach for my test analyte mix because it would have been a simpler system. However, the analytes I chose for the application were not amenable to a single column. I ran into issues with the neutral compounds being essentially unretained on the SAX column and the permanently charged anions likewise essentially unretained on the C18 column. Therefore, I used a tandem column approach, which required development of the separation of individual analyte groups on a single column and then optimization once the columns were coupled. Understanding the column chemistry and interactions with the analytes in various mobile phases was an integral part of the success of the separation developed.

Q: What role does mobile phase selection play in the success of SE-LC?

As with all liquid chromatography, the conditions of the mobile phase are one of the two essential factors for successful separation, with column chemistry the other crucial factor. I was fixed on the column chemistries of SAX and C18 and so I chose selectively strong mobile phases to optimize separation of each analyte group. Mobile phase pH and ionic strength were critical to achieve success for this separation because weak acids are affected by both factors and permanently charged anions are affected by ionic strength. In my work, the weak acids performed better at low pH (in their almost “neutral” state). There appeared to be some retention on both columns for the weak acids but more so on the C18 column because the weak acids were predominantly in their uncharged state, behaving more similarly to a neutral compound. I knew that the permanently charged ions and the neutral compounds would be unaffected by mobile phase pH, so the choice to elute the weak acids first was easy. I thought that eluting the permanently charged anions second would also be a simple task because I knew the neutral compounds would be unaffected by the change in ionic strength that would be required to elute the anions. However, I could not achieve the elution of the anions as a group between the weak acids and neutrals. Through further development, I learned that the permanently charged ions were not greatly affected by high percent acetonitrile. I redirected my focus to eluting weak acids -> neutral compounds -> permanently charged anions using a low pH isocratic elution -> acetonitrile gradient -> ionic strength gradient to elute those groups in that order. I shared my step-wise approach and optimization of the mobile phases in more detail in my paper (1).

Q: Why is SE-LC particularly suited for pharmaceutical applications?

SE-LC shows great promise for pharmaceutical applications over conventional HPLC because of the increased peak capacity, the decreased separation disorder, and the resulting higher probability of a successful separation (R_s ≥ 1.5 for all analytes). The SE-LC method is flexible and not restricted to specific stationary phases such as reversed-phase liquid chromatography (RPLC), anion exchange chromatography (AEX), or cation exhange chromatography (CEX), nor to particular mobile phases; alternative conditions can be applied based on the characteristics of the analytes. SE-LC can be used throughout the development life cycle of drug substances or products in the chemical and pharmaceutical sectors, particularly when traditional HPLC separations fall short in resolving complex mixtures. Analytical chemists can really appreciate when compounds elute in an ordered fashion, reliably, and with great precision!

References

(1) Lovejoy, L. K.; Foley, J. P. Separation of Weak Acids, Neutral Compounds, and Permanent Anions Using Sequential Elution Liquid Chromatography with Tandem Columns. J. Chromatogr. A 2024, 1731, 465178. DOI: 10.1016/j.chroma.2024.465178

(2) Socia, A.; Foley, J. P. Sequential Elution Liquid Chromatography Can Significantly Increase the Probability of a Successful Separation by Simultaneously Increasing the Peak Capacity and Reducing the Separation Disorder. J. Chromatogr. A 2014, 1324, 36–48. DOI: 10.1016/j.chroma.2013.11.016

(3) Little, E. L.; Jeansonne, M. S.; Foley, J. P. Sequential Multimodal Elution for Pseudomultidimensional Liquid Chromatography on a Single Column. Anal. Chem. 1991, 63 (1), 33–44. DOI: 10.1021/ac00001a007

(4) Lovejoy, L. K.; Foley, J. P.; Grinias, K. M. Validation of a High-Performance Liquid Chromatography Separation of Weak Acids, Neutral Compounds, and Permanent Ions Using Sequential Elution with Tandem-Column Liquid Chromatography. J. Sep. Sci. 2025, 48, e70124. DOI: 10.1002/jssc.70124

About the Interviewee

Lauren Lovejoy is a principal scientist in the Drug Substance and Product Analysis group at GSK. Her professional endeavors concentrate on analytical activities that facilitate the development and manufacturing
of injectable suspension pharmaceuticals. Additionally, she is pursuing a part-time PhD at Drexel University under the guidance of Joe Foley, focusing her research on sequential elution liquid chromatography with ion exchange columns. Since earning her B.S. in biochemistry from Temple University, she has spent the past 12 years developing separation methods for pharmaceutical analysis and leading analytical projects within the pharmaceutical industry.