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The combination of several gradient modes (solvent, temperature, and flow programming) is rarely used in HPLC analysis. In this work, the separations obtained utilizing simultaneous flow and temperature gradient in capillary column and microcolumn HPLC were compared with the separations performed under isocratic, isothermal, and isorheic (constant flow) conditions. When the mobile phase flow rate and the column temperature were changed simultaneously during the separation run, the analysis time was shortened up to 50%, while the separation efficiency was preserved. The separations obtained with combined temperature and flow gradients show high reproducibility (relative standard deviation <2.0%), comparable to the reproducibility normally seen with a mobile phase gradient. For capillary HPLC, simultaneous temperature and flow programming is the method of choice because of the great technical difficulties involved in performing solvent gradient elution.

In HPLC, temperature programming and isocratic separation were investigated for resolving a mixture of nucleotide polymers. A mixture of short oligonucleotides pd(A)(12)(-)(18) was resolved in less than 4 min by utilizing isocratic separation. The method is sensitive to organic modifier concentration; a 0.5% change of organic modifier in the mobile phase leads to more than doubling of the total analysis time. Temperature programming was used to optimize the separation of larger oligonucleotides pd(A)(25)(-)(30) and pd(A)(40)(-)(60). When the column temperature was changed by programming during the separation, the analysis time was 75% less than for an isocratic/isothermal run. The low amounts of ion-pairing agent in the mobile phase (between 5 and 10 mM) make these methods suitable for electrospray ionization mass spectrometry.

The use of ultrahigh column temperatures, up to 110 degrees C, in micellar electrokinetic capillary chromatography was investigated. The number of plates generated per unit time increased from 0.22 to 12.8 plates/s for separations at 15 degrees C and 110 degrees C, respectively. Ultrahigh-temperature micellar electrokinetic capillary chromatography was used for the separation of cyclic undecapeptides (cyclosporins). A minimum resolution of 1.39 was calculated for a critical peak pair at 110 degrees C, which is more than a 50% increase over resolution generated at 40 degrees C. During a run time of more than 90 min at 110 degrees C and at pH 9.3, no sample degradation or solvent boiling was observed.