Temperature-assisted on-column solute focusing: A general method to reduce pre-column dispersion in capillary high performance liquid chromatography

Abstract

Solvent-based on-column focusing is a powerful and well known approach for reducingthe impact of pre-column dispersion in liquid chromatography. Here we describe an orthogonal temperature-based approach to focusing called temperature-assisted on-column solute focusing (TASF). TASF is founded on the same principles as the more commonly used solvent-based method wherein transient conditions are created thatlead to high solute retention at the column inlet. Combining the low thermal mass of capillary columns and the temperature dependence of solute retentionTASF is used effectivelyto compress injection bands at the head of the column through the transient reduction in column temperature to 5 °C for a defined 7 mm segment of a 6 cm long 150 μm I.D. column. Following the 30 second focusing time, the column temperature is increased rapidly to the separation temperature of 60 °C releasing the focused band of analytes. We developed a model tosimulate TASF separations based on solute retention enthalpies, focusing temperature, focusing time, and column parameters. This model guides the systematic study of the influence of sample injection volume on column performance.All samples have solvent compositions matching the mobile phase. Over the 45 to 1050 nL injection volume range evaluated, TASF reducesthe peak width for all soluteswith k’ greater than or equal to 2.5, relative to controls. Peak widths resulting from injection volumes up to 1.3 times the column fluid volume with TASF are less than 5% larger than peak widths from a 45 nL injection without TASF (0.07 times the column liquid volume). The TASF approach reduced concentration detection limits by a factor of 12.5 relative to a small volume injection for low concentration samples. TASF is orthogonal to the solvent focusing method. Thus, it canbe used where on-column focusing is required, but where implementation of solvent-based focusing is difficult.

Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing

“…Under these conditions, TASF is able to limit the increase in FWHM to 5% for injection volumes up to nearly 100% of the column volume (450 nL) for methylparaben and 165% for ethylparaben and propylparaben. These data are similar to our published data [9] confirming, as expected, that technical changes did not alter the essential function of the TASF device.…”

“…Therefore selection of a thin (low thermal mass), high power TEC is desirable for rapid temperature changes from T 1 to T 2 . The TEC is rated at 10.9 W, significantly more powerful than the TEC (6.3 W) used in our previous work [9] and is capable of changing temperatures of the focusing segment by approximately 60–70 °C ( T 2 − T 1 ). The operator can choose actual T 1 and T 2 values according to the need.…”

“…Typically in reversed-phase liquid chromatography retention increases as temperature decreases [7,8]. Thus, as we have recently shown [9], temperature-based on-column solute focusing (TASF) can be used to minimize the volume overload problem. TASF works by maintaining a temperature of a short segment (e.g., 1 cm) at the head of the column well below the rest of the column temperature for the time period during the injection.…”

“…In our previous work [9], we demonstrated that TASF decreases volume overload significantly when the sample is prepared in a liquid with the same composition (chromatographic strength) as the mobile phase. Here, we demonstrate that TASF is also effective in isocratic elution: 1) when the sample solvent is weaker than the mobile phase, and 2) when the sample solvent is stronger than the mobile phase.…”

Improving the Sensitivity, Resolution, and Peak Capacity of Gradient Elution in Capillary Liquid Chromatography with Large-Volume Injections by Using Temperature-Assisted On-Column Solute Focusing

“…57,58 With TASF, the head of the column is cooled to subambient temperatures during injection. This results in focusing of solutes into narrow bands at the head of the column.…”