The Importance of Temperature in Liquid Chromatography

Liquid chromatography is a commonly used technique for separating and purifying complex mixtures of chemical compounds. The success of this technique relies on several factors, including the choice of stationary and mobile phases, the column dimensions and the operational parameters, such as flow rate, pressure and temperature.

Specifically, temperature plays an important role in HPLC & UHPLC. As a result, maintaining stable temperatures throughout the chromatographic run can help achieve reproducible and robust separations. However, temperature variations can affect the physical and chemical properties of the stationary and mobile phases, causing changes in retention times, selectivity, resolution and peak shape.

Temperature affects the chromatographic separation in several ways. Firstly, it influences the thermodynamic properties of the solvents and solutes, such as pressure, viscosity and diffusivity, which affect the equilibria between the mobile and stationary phases.

Secondly, temperature affects the adsorption and desorption kinetics of the solutes on the stationary phase, which can affect the retention times and peak shapes. Thirdly, temperature affects the chemical reactions that may occur during the separation, such as hydrolysis or oxidation. Maintaining the temperature within a narrow range throughout the chromatographic run is essential to ensure reliable and reproducible chromatographic results.

What effects can column temperature have in liquid chromatography?

The column temperature is an important parameter in liquid chromatography and can affect the analyte separation process. The column temperature influences the viscosity and diffusivity of the mobile and stationary phases, affecting analyte retention, selectivity and peak shape.

The effects of temperature on analyte retention and selectivity differ with each analyte and depend on the properties of the mobile and stationary phases. Generally, increasing the column temperature decreases the retention time of polar compounds, while nonpolar compounds have longer retention times.

Additionally, temperature affects the distribution coefficient of the analytes between the stationary and mobile phases, which, in turn, affects the selectivity of the separation. For instance, in reversed-phase chromatography, decreasing the temperature increases the retention of polar analytes relative to nonpolar ones, leading to better separation.

The Role of Column Temperature in Method Development

Temperature is a vital parameter to explore for method development in liquid chromatography. Chromatographers can optimize the separation of analytes and achieve better resolution by varying the column temperature.

For instance, changing the column temperature can help adjust the retention factor and selectivity of analytes, leading to better separation. Additionally, incorporating temperature into the design of experiments (DOE) can help reduce the time required for method development by quickly identifying the optimal temperature conditions for the separation.

How column temperature affects method reproducibility

Changes in column temperature can affect the separation and elution of components. If temperature is not tightly controlled, it can lead to variations in retention times, peak shapes and peak areas, resulting in poor reproducibility and inconsistent chromatography.

The Effect of Variation in Ambient Temperature

Talk about how variations in ambient air temperature can affect consistency in chromatography results. Variations in ambient air temperature can affect the consistency of chromatography results if column temperature is not controlled. Ambient temperature fluctuations can cause column temperature changes, ultimately affecting sample component separation and detection.

For example, in places where the air conditioning system is not adequately maintained, fluctuations in the ambient temperature can cause temperature variations in the column, leading to changes in retention times and peak shapes. Additionally, ambient temperature changes can affect the viscosity of the mobile phase, altering the components’ pressure and solute elution time (retention).

In cases where column temperature control is not possible, for example in preparative chromatography, more robustness testing should be done to ensure you are working within a robust design space where fluctuations in ambient temperature do not cause loss of selectivity and separation.

How Column Ovens Can Solve Issues With Liquid Chromatography

In all modern HPLC and UHPLC instruments, column ovens are commonly used to control the column temperature and ensure consistency in chromatography results. These ovens provide a stable and controlled environment for the liquid chromatography column to minimize the effect of ambient temperature variations.

The ovens are designed to maintain a constant temperature within the liquid chromatography column, most commonly by circulating heated air around it. The temperature can be set according to the method requirements, and the oven will maintain the temperature throughout the chromatographic run.

Temperature effects on retention time and selectivity

Generally, an increase in temperature can decrease the retention time of compounds, as the solutes have greater kinetic energy and interact less strongly with the stationary phase. This effect is more pronounced for nonpolar or weakly polar solutes than polar solutes, which usually interact more with the stationary phase.

Besides affecting retention time, the temperature can also affect the selectivity of the separation by changing the interactions between the solutes and the stationary phase. This tends to be compound-specific and also differs between different chromatographic modes.

For instance, a polar stationary phase may become more hydrophobic at higher temperatures, increasing polar compound retention and decreasing nonpolar compound retention.

Elevated Temperatures Can Increase Throughput

Elevated column temperatures can help increase chromatography throughput by reducing the time required for the separation. At higher temperatures, the solutes move through the column more quickly, causing shorter retention times and faster elution of the compounds. This effect is particularly useful for applications that require high sample throughput.

Besides reducing the time required for each separation, elevated temperatures can also allow an increase in the maximum flow rate that can be used without causing the chromatography column to overpressurize. This increase in flow rate can further enhance the throughput of the chromatography system.

Note: The elevated temperatures can also have drawbacks, such as decreased separation efficiency, peak broadening and detrimental changes in selectivity. Therefore, the temperature must be carefully optimized to achieve the desired balance between separation efficiency and throughput.

Temperature effects on peak width

Temperature can significantly affect band broadening and peak width in liquid chromatography. As the column temperature increases, the viscosity of the mobile phase decreases, leading to lower resistance to mass transfer. This reduction in resistance can cause faster diffusion of analytes to produce narrower peaks and reduced band broadening.

However, it is essential to note that temperature has a complex effect on the separation performance and is compound-specific. While elevated temperature can improve peak symmetry and reduce peak width, it can also increase the baseline noise and decrease the separation efficiency. Additionally, excessively high temperatures can cause degradation or adsorption of analytes (some biomolecules, for example), affecting the results negatively.

Other considerations when elevating column temperatures

The column’s thermal stability, the system’s pressure rating and the sample’s compatibility with elevated temperatures are other factors or considerations when elevating column temperatures. The column material’s thermal stability and the stationary phase should be considered before increasing the temperature to prevent column damage or degradation.

Determining the ideal column temperature for your applications

Correct and consistent column temperature plays a critical role in the success of the method. Finding the ideal column temperature is essential for achieving optimal separation and detecting analytes. Here are some steps to follow to find the ideal column temperature for your application and analytes:

Start With the Recommended Temperature

Most column manufacturers provide recommended temperature ranges. These recommendations are based on the stationary phase used in the column and the analyte properties. The recommended temperature range can provide a good starting point for your experiments.

Perform a Temperature Gradient

Temperature gradients involve running the chromatography method at different temperatures and observing the effects on analyte separation and detection. The temperature gradient can be performed manually by changing the column temperature each time (2 or 3 runs will be necessary) or using a specialized temperature program. A temperature gradient can help determine the temperature range where the analytes are well separated and have good peak shapes.

Optimize the Temperature

Once the temperature range where the analytes are well separated is identified, further optimization can be performed to find the optimal temperature for separating the analytes. This involves running the chromatography method at different temperatures within the identified range and comparing the results. The optimal temperature is the one that provides the best resolution and peak shape for the analytes of interest. A robustness window should be identified and care should be taken that the final method does not sit on a knife edge — check temperatures either side of the optimum to ensure any slight changes in temperature do not lead to immediate method failure.

It is also essential to note that different analytes have different optimal temperature ranges. Therefore, it is recommended to perform a temperature gradient for each analyte or group of analytes to find the ideal temperature for each component.

Learn more about the effect of column temperature in liquid chromatography

To learn more about the effects of column temperature in liquid chromatography, read this knowledge note from Avantor: The Importance of Column Temperature in Liquid Chromatography.

Avantor is a leading supplier of ultrahigh-purity materials and solutions for the life sciences and advanced technology industries. We offer a wealth of resources for scientists to learn more about chromatography and its applications, including the effects of column temperature in liquid chromatography.

This resource provides an in-depth analysis of the effects of temperature on chromatographic separations and provides practical advice on optimizing column temperature for specific applications.