Question

Why should (High performance liquid chromatography) HPLC equipment be kept in low temperature rooms during operation?

Answer 1

High performance liquid chromatography (HPLC), which is an instrumental analytical method that gained increased acceptance mainly because it met two basic factors: (1) the need for a wide range of rapid analyses for nutrients, and (2) the need for methods that can be easily automated. HPLC is applied to several categories of substances: carbohydrates, lipids, vitamins, additives, synthetic colorings, natural pigments, contaminants, and amino acids. HPLC is a form of liquid chromatography, where separation occurs between a mobile phase and a stationary phase. Depending on the nature of the stationary phase, the separation process can be of four different modes: adsorption chromatography, partition chromatography, ion-exchange chromatography, and size exclusion chromatography. The choice of the solvent depends on the nature of the operation mode, that is, isocratic or gradient elution. The polarity for such an elution medium can, therefore, vary from buffered aqueous solutions to hydrocarbons.

The SandraSelerity Polaratherm is designed to enhance the analysis of chemical mixtures through HPLC at elevated and sub-ambient temperatures. Although often neglected, temperature plays an important role in HPLC since the majority of the chromatographic properties are a function of temperature. Nevertheless, to this date the possibilities of temperature to improve LC separations have not been fully investigated and the majority of liquid phase separations are performed in the temperature region between 20 to 40°C or without thermostatting. The use of elevated temperature has proven effective for improving the overall chromatographic performance on conventional equipment. The main advantages of using temperature in LC are:

1. Speed Increase speed by increasing the temperature and flow rate.

2. Efficiency and resolution Increase efficiency by using longer columns/smaller particles at elevated temperature.

3. Selectivity Change selectivity with temperature.

4. Lower consumption of organic solvents - Green chromatography Higher temperature requires less solvent.

5. Improved detectability Improve peak shape. Use only water.

6. Temperature programming Replace solvent gradients with a temperature program.

Since the temperature of the column and mobile phase influences the retention, programming the temperature in time can be used to elute compounds from the column. If the system is capable of covering large temperature ranges, a temperature gradient can be used as in gas chromatography and in many instances, can replace the solvent gradient. This enables the use of temperature programmed elution on detectors that are restricted to isocratic operation such as a refractive index detector.