How Does Column Temperature Affect HPLC Resolution?

HPLC Column temperature affects retention time, selectivity, peak shape, and pressure. Let’s look at this in more detail.

As Temperature Increases, Retention Time Decreases

Retention measures the time that the sample component resides in an HPLC column’s stationary phase, relative to the time it resides in the mobile phase. In most applications, separation is done at about 40 °C. At higher temperatures, retention decreases and faster elution is possible. At lower temperatures, retention often increases, especially in reversed phase separations. In UHPLC applications, higher temperatures are used to decrease solvent viscosity and lower the backpressure caused by small particles and high flow rates.

Temperature Can Affect Selectivity

Unfortunately, there is not a hard rule on how temperature affects selectivity, except that changes in temperature can affect selectivity of some analytes. With complex samples that have difficult separations, temperature can be used to achieve the desired selectivity and separation.

Temperature Gradients Affect Peak Shape

John Dolan wrote a great article in LCGC about “The Importance of Temperature;” I encourage you to read this article in full, as it will describe in more detail how column temperature is a powerful tool for chromatography separations. In this article, he shows how a temperature gradient in your system will affect the peak shape. When an analyte is injected into an HPLC system, it enters the column in a narrow band, gradually getting broader as the analytes interact with the stationary and mobile phase, and should be eluted with a nice gaussian-shaped peak in a well-controlled HPLC system. If the mobile phase is at ambient temperature (cold) and the column is heated to elevated temperatures (warm), the cooler mobile phase would cool the column’s inlet, creating a temperature gradient in the column and peak distortion. Because of this phenomenon, we recommend pre-heating the mobile phase by using a HPLC inline solvent heat exchanger.

Why Temperature Control Is Important in HPLC

As scientists, we learn to make one change at a time. As we already discussed, temperature affects HPLC retention and selectivity, which determines resolution. Therefore, controlling the temperature of an HPLC column will limit your experiment’s variables.

We recommend using a column sleeve to “heat” the column 5°C above room temperature, even when you don’t need the elevated temperature for resolution. Controlling th e temperature of the column vs. the fluctuation of lab’s ambient air limits the variables in your experiment. As many scientists know, the temperature swings in the laboratory can vary between day shifts/evening shifts, difference in seasons, or differences in where one HPLC System is in the lab, compared to an identical HPLC system (Windows, Doorways, HVAC vents, etc.).

As mentioned above, when heating your column above 40°C, it is important to preheat the mobile phase so that a temperature gradient does not occur inside the column. Add 10 cm to your column length when ordering your Hot Sleeve and the HX-038-EXP preheater (3.8 µL preheater coil) will fit inside the heated column jacket.

Temperature control for separations is important for long-term retention reproducibility, one factor of method ruggedness. Controlling temperature above ambient (~35°C) is normally sufficient for good method reproducibility and ruggedness. In addition, the use of elevated temperature can have other benefits. First, it reduces the system operating pressure by reducing the mobile phase’s viscosity. Second, it will reduce analysis time, which can substantially increase productivity. Third, temperature may change the selectivity of a separation. Not all compounds have the same response to temperature, so the selectivity of a separation can change dramatically when temperature is increased or decreased.

A heated HPLC column in a column compartment oven or in a column sleeve with a solvent heat exchanger is recommended to use to minimize the variabilities in your analysis. We realize that laboratories are tight on space, and the Column Sleeve is a great addition to a current HPLC System, as it takes up minimal bench space. Reach out to Chrom Tech for a Quote for a Column Sleeve.