Temperature is a critical parameter that significantly influences the efficiency, selectivity, and overall performance of column chromatography techniques. Whether it’s liquid chromatography (LC) or gas chromatography (GC), understanding the impact of temperature is essential for achieving optimal separations and obtaining accurate results. Therefore, maintaining a stable temperature throughout the chromatography run helps achieve reproducible and stable separations. However, temperature changes can affect the physical and chemical properties of stationary and mobile phases, resulting in changes in retention time, selectivity, resolution, and peak shape. In this blog post, we will delve into the effects of temperature on column chromatography, exploring its influence on retention, resolution, and method development.
If the column temperature is higher than the maximum operating temperature of the stationary solution, the stationary solution will be lost with the carrier gas, which will not only affect the life of the chromatographic column, but also the stationary solution will enter the detector with the carrier gas, contaminating the detector and affecting the analysis results.
If the column temperature is too high, the distribution coefficient of each component will become smaller and the separation will be reduced; if the column temperature is too low, the mass transfer rate will be significantly reduced, the column efficiency will be reduced, and the analysis time will be prolonged.
The higher the column temperature, the faster the peak will elute and the shorter the retention time will be. Changes in column temperature will cause poor reproducibility of retention times, thus affecting the qualitative results of sample components. Generally, if the column temperature changes by 1%, the retention time of the component will change by 5%; if the column temperature changes by 5%, the retention time of the component will change by 20%.
When the column temperature increases, under normal circumstances, the half-peak width will become narrower, the peak height will become higher, and the peak area will remain unchanged. However, when the component peak height becomes higher, the analysis results may change when the peak height is used for quantitation; when the column temperature decreases, the opposite is true.
First of all, it should be ensured that the column temperature is not higher than the maximum operating temperature of the stationary solution, that is, the maximum withstand temperature of the chromatographic column, to avoid loss of the stationary solution and affecting the efficiency and service life of the chromatographic column.
Secondly, choose the appropriate column temperature. The column temperature should be selected so that the two components that are difficult to separate can achieve the expected separation effect. The peak shape should be normal and the analysis time should be short. Generally, the column temperature should be higher than the average of each component in the sample. The boiling point is 20~30℃ lower and is determined experimentally. For samples with a wide boiling point range, programmed heating should be used to increase the temperature linearly or nonlinearly with time at a predetermined heating rate. Generally, the heating rate is linear.
Finally, it is especially important to ensure the stability and uniformity of the column temperature control of the instrument, as well as the consistency between the actual temperature and the preset temperature. Generally, the column temperature control accuracy of gas chromatograph is ±0.1℃, and some manufacturers can reach ±0.01℃.