The Use of Temperature to Eliminate Organic Solvent in Analytical HPLC Methods

Increased temperature has been used to assist the elimination of organic modifier required in the mobile phase, to achieve analyte separation in pure aqueous mobile phases.

Generally, LC separations are run at temperatures between 25 and 40 °C. At higher temperatures, solvent viscosity is lower, which allows the use of higher flow rates to increase speed, without losing efficiency. Reduced mobile phase viscosity results in an increase in analyte diffusivity, which improves mass transfer, consequently the chromatographic peaks obtained are sharper, improving the sensitivity of the analysis.

Temperature can also be used to modify the elution strength of the mobile phase. If the temperature of water is increased its elution strength also increases due to a decreased dielectric constant. At 190 °C, the dielectric constant of water is similar to that of acetonitrile at 20 °C, indicating that the use of 100% water mobile phase can potentially be used as an eluent in analytical HPLC.

Although these positive temperature effects on HPLC separations have been understood for many years, the use of ultra-high temperatures has not been fully exploited. One significant reason for this is the poor temperature stability of silica based stationary phases. Hypercarb (Porous Graphitic Carbon) columns have no bonded phase and can withstand temperatures in excess of 200 °C. This article describes the retention of a mixture of purines and pyrimidines at high temperatures on a Hyercarb column using a 100% aqueous mobile phase.

Results and Discussion

The retention and separation of a mixture of purines and pyrimidines, obtained with a mobile phase of water, acetonitrile, and formic acid, at 50 °C is shown in Figure 1a. By removing the organic component (acetonitrile) and the electronic modifier (formic acid) from the mobile phase, elution strength is greatly reduced, and at conventional temperatures the mobile phase is too weak to elute the solutes from the PGC column.

Figure 1: 1) Cytosine, 2) Uracil, 3) Thymine, 4 Hypoxanthine, 5) Guanine, 6) Xanthine

In Figure 1b, the purines and pyrimidines are separated with a mobile phase of pure water at 190 °C. The same elution order is maintained, although there is a slight change in the separation selectivity obtained with 100 % water at 190 °C. The lower solvent viscosity allows a higher flow rate to be used, reducing analysis time and improving mass transfer. The overall peak symmetry is improved at this high temperature with asymmetry values for peaks 3 and 4 (measured at 10 % height) being reduced from 1.37 and 1.56 under conventional conditions to 1.22 and 1.19 respectively at 190 °C.

Conclusions

The use of ultra-high temperature can be used to eliminate the requirement for organic solvents in a HPLC mobile phase. The separation of purines and pyrimidines is achieved in less time and with improved peak shape.

Thermo Fisher Scientific

112 Chadwick Road, Runcorn WA7 1PR, UK

tel. +44 (0) 1928 588089, fax: +44 (0) 1928 581078

Website: www.thermo.com