Chromatography is Full of Ups and Downs!
At Emery Pharma, our team of expert bioanalytical chemists routinely provides R&D support and GLP-compliant analytical services for our clients. Some of the services we offer include: high-performance liquid chromatography (HPLC) method development, method validation, method transfer, and analysis of biological samples, raw materials, formulated products, and environmental samples. Sample analysis is conducted using HPLC in common modes, paired with a variety of detection techniques, such as UV, PDA, DAD, ELSD, CAD, and Mass Spectrometry (MS).
One of the most important aspects of any chromatographic method development is selecting a suitable stationary phase relevant to the application. Unfortunately, screening various stationary phases is often tedious and time-consuming. At Emery Pharma, we use a systematic stationary phase selection approach to choose the ideal column for the method of interest—this strategy significantly accelerates the method development process.
The first step in stationary phase selection is choosing a chromatographic mode. In HPLC, most analytical methods are developed using the common modes of chromatography: Reversed-Phase Chromatography (RP), Hydrophilic Interaction Liquid Chromatography (HILIC), Normal Phase Chromatography (NP), Ion Exchange Chromatography (IEC), and Gel Permeation/Size Exclusion Chromatography (GPC/SEC). The figure below represents the types of stationary and mobile phases used in each HPLC mode, as well as the general retention behavior of analytes.
The tables below outline the HPLC mode selection approach according to physicochemical properties of the target analytes, including molecular weight and solubility [1]:
| Molecular Weight ˂ 2000 | Solubility | Type of Solvent | HPLC Mode |
| Organic | Non-Polar | Normal Phase | |
| Water Miscible | Reversed Phase | ||
| HILIC (for weak retention in RP) | |||
| THF | Gel Permeation (small molecules) | ||
| Aqueous | Non-ionic | Reversed Phase | |
| HILIC (for weak retention in RP) | |||
| Ionic | Ion Exchange | ||
| Reversed Phase with Ionization Control | |||
| Reversed Phase with Ion-Pair Agent |
| Molecular Weight ˃ 2000 | Solubility | HPLC Mode |
| Organic | Gel Permeation | |
| Aqueous | Ion Exchange with Wide-Pore Material | |
| Size Exclusion Chromatography | ||
| Reversed-Phase with Wide-Pore Material |
A variety of HPLC stationary phases are available within each chromatographic mode, depending on the interactions with the analytes. Here, we provide examples demonstrating how selecting the right stationary phase enhances separation performance and overall chromatographic efficiency.
In Reversed-Phase Chromatography, different alkyl chain lengths of hydrophobic silica packing materials are used, including C₁₈, C₈, and C₄, along with phenyl or diphenyl functionalized silica. The chromatograms below demonstrate the separation of a group of analytes using these stationary phases in RP mode². The retention time and separation efficiency can be modulated—for example, C₄ or C₈ phases are better suited for highly hydrophobic analytes that exhibit excessive retention on C₁₈.
For a hydrophobic compound with an aromatic moiety, a phenyl or diphenyl-modified C₁₈ phase provides enhanced selectivity compared to a regular C₁₈ phase. As illustrated in the chromatogram below, positional isomer separation is significantly improved with a phenyl-functionalized stationary phase [3]:
In Normal Phase Chromatography, several polar stationary phases are available, including bare silica, amino-bonded, diol, and cyano-modified silica. The choice of bonding chemistry and polarity influences separation performance. As shown in the chromatogram below, a diol-bonded silica phase yields superior separation due to hydrogen bonding interactions with analytes [4]:
It’s important to note that the best separation of target analytes may require combining multiple HPLC modes. Many analytical scientists select stationary phases based on analysis time, resolution, and column availability.
For example, Size Exclusion Chromatography (SEC) and Ion Exchange Chromatography (IEC) are common techniques used in protein and peptide analysis. SEC separates molecules by size using porous particles. Larger protein molecules bypass the pores and elute faster, while smaller molecules enter the pores and elute later. The chromatogram below shows albumin protein separation in the presence of its aggregates and dimers [5]:
In Ion Exchange Chromatography, proteins are separated based on surface charge and electrostatic interactions with the ion exchange stationary phase. As shown in the chromatogram below, a cation exchange column is used to separate basic proteins [6]. The method involves a salt gradient, where positively-charged sodium ions displace bound proteins on the negatively charged resin:
At Emery Pharma, we are committed to helping you overcome complex challenges in drug development, medical devices, clean tech, solar energy, and more. If you are interested in our chromatography and HPLC method development services, please contact us online or call us at +1 (510) 899-8814!
About the Author
Originally authored by Dr. Charles Francavilla. This article was reviewed and updated on July 11, 2025 by Dr. Ryan Cheu, current Director of Chemistry.
References
- www.agilent.com/cs/library/primers/Public/LC-Handbook-Complete-2.pdf
- “Reversed-Phase Liquid Chromatography (RP-LC).” http://www.chromatographyshop.com/
- Comparison of Selectivity Differences Among Different Agilent ZORBAX Phenyl Columns using Acetonitrile or Methanol, Agilent Technologies Publication, 5990-4711EN
- www.ymc.co.jp/en/download/pdf/pdf03.pdf
- www.agilent.com/cs/library/eseminars/Public/Column%20Choices%20for%20proteins_037214.pdf
- DeLeenheer, A.P. et al. J. Pharm Sci., (1991) 80, 11.
