Effects of temperature in HPLC

It is not possible to set generally valid rules about the influence of temperature on HPLC separations. At increased temperature the performance of a column often increases because of the decrease of mobile phase viscosity which improves mass transfer however, it is also possible that performance decreases. The separation factor can increase or decrease. An advantage is the shortening of analysis time due to the possibility to use higher flow rates of the mobile phase due to the increase in diffusion coefficients. If the eluent or the sample solution are viscous it is even necessary to work at higher temperatures less pressure is needed to pump the mobile phase or it is possible only under these circumstances to inject the sample, respectively. 

An extreme example of this has been given by M. Vecchi, G. Englert, R. Maurer and V. Meduna, Heir. Chim. Acta, 64, 2746 (1981). It describes the separation of /3-carotene isomers on alumina with a deli tied water content (hexane as mobile phase). The separation alters significantly with a change in temperature of 1 C  

Thermostat control is no problem in the more expensive compact units with an incorporated column oven. However, a glass or metal jacket fitted around the column, which is then connected to a thermostat bath, is an effective self-help measure. The mobile phase must also be thermostatically controlled by the same bath prior to entry into the column, by means of suitable equipment, e.g. a long spiral. Pumps with thermostatically controlled heads are also available as well as detectors with thermostated cell. 

The maximum temperature is around 120 °C for silica columns and should not exceed 80 °C for chemically bonded phases. 

The fact that the mobile phase is heated by flow resistance as it passes through the column should not be overlooked. A rule of thumb is a 0.1 °C temperature increase per 1 bar pressure drop or 10 °C per 100 bar (0.025 °C per bar or 2.5 °C per 100 bar for water).It may be advisable to cool the column and work with low volume flow rates when very low-boiling mobile phases such as pentane (boiling point 36 °C) are involved. 

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