Using Temperature to Improve Terpenoid Separations with Cogent Bidentate C18™ and UDC‑Cholesterol™ Columns

Temperature is a powerful yet often underutilized variable in HPLC method development. Adjusting column temperature can change analyte retention, enhance or diminish selectivity, improve efficiency, and significantly influence resolution.

This study compares how temperature affects the separation of three structurally related terpenoids—bexarotene, tretinoin, and tazarotene—using two different Cogent TYPE‑C™ columns: Bidentate C18™ and UDC‑Cholesterol™.

Understanding how these phases respond to thermal changes allows analysts to fine‑tune retention and resolution for challenging separations.

Why Temperature Matters in Terpenoid Separations

Temperature influences chromatographic behavior in several ways:

• Selectivity changes as molecular interactions shift
• Efficiency increases or decreases depending on viscosity and diffusion
• Retention times shift due to altered partitioning behavior
• Shape-selective phases (such as UDC‑Cholesterol™) respond differently at various temperatures

Together, these variables make temperature a valuable tool for optimizing separations.

Tested Stationary Phases

1. Cogent Bidentate C18™ Column

• Traditional reversed‑phase interactions
• Does not exhibit shape selectivity
• Selectivity improved at higher temperatures for the critical peak pair

2. Cogent UDC‑Cholesterol™ Column

• Exhibits shape‑selective behavior due to rigid cholesterol moieties
• Selectivity improved at lower temperatures
• More sensitive to molecular geometry than Bidentate C18™

Experimental Overview

Three terpenoids—bexarotene, tretinoin, and tazarotene—were analyzed across multiple temperatures on both columns.
Retention times were plotted against temperature to observe trends.

The goal was to determine how thermal changes alter:

• Retentive behavior
• Elution order
• Resolution of the most critical peak pair
• Efficiency and peak shape

Key Findings

1. Temperature Alters Elution Order (UDC Only)

The UDC‑Cholesterol™ column showed a different elution sequence than the Bidentate C18™, attributed to shape‑selective interactions not present in C18.

2. Selectivity Behavior Opposes Between Columns

• Bidentate C18™:

  • Better selectivity at higher temperatures

• UDC‑Cholesterol™:

  • Better selectivity at lower temperatures

This difference is rooted in the rigidity of the UDC phase, which enhances shape recognition more strongly at cooler conditions.

3. Low‑Temperature Limitations (C18)

At 15 °C, where the UDC column delivered its best separation, the Bidentate C18™ performed poorly—bexarotene appeared as a shoulder peak on tazarotene, indicating insufficient resolution.

4. Temperature and Efficiency

Efficiency improved with increasing temperature until reaching a maximum (35–40 °C), then decreased as temperature increased further.
This effect was more pronounced on the UDC column, suggesting stronger temperature dependency due to its structural rigidity.

5. Selectivity vs. Efficiency Trade‑off

• UDC provides best selectivity at 15 °C

• UDC provides best efficiency at 35–40 °C

Because selectivity and efficiency peak at different temperatures, analysts must choose the optimal balance based on method goals.

Method Conditions (Table Summary)

The article includes specific method conditions for both columns, covering:

• Mobile phase
• Temperature ranges
• Analyte details
• Gradient conditions

These parameters support reproducible evaluation of temperature effects.

Conclusion

Temperature significantly influences terpenoid separations on Cogent TYPE‑C™ columns.

• The Bidentate C18™ benefits from higher temperatures for selectivity.
• The UDC‑Cholesterol™ benefits from lower temperatures, where shape selectivity is strongest.
• Peak efficiency generally maximizes at 35–40 °C, but selectivity may not.

Method developers should evaluate both selectivity and efficiency when choosing column temperature—the optimal value is often a compromise rather than a single “best” point.

Figure 3. Efficiency of tazarotene as a function of temperature on both columns.