Monitoring gradient profile on-line in micro- and nano-high performance liquid chromatography using conductivity detection

• We determined the relationship between specific conductance and organic-solvent content in a gradient.
• Based on the above relationship we established a new method for on-line gradient profile monitoring.
• We observed the effect of pressure on gradient conductivity and
• We determined the above effect and corrected it when a gradient profile was monitored.

In micro- or nano-flow high performance liquid chromatography (HPLC), flow-splitters and gradient elutions are commonly used for reverse phase HPLC separations. When a flow splitter was used at a high split-ratio (e.g., 1000:1 or higher), the actual gradient may deviate away from the programmed gradient. Sometimes, mobile phase concentrations can deviate by as much as 5%. In this work, we noticed that the conductivity (σ) of a gradient decreased with the increasing organic-solvent fraction (φ). Based on the relationship between σ and φ, a method was developed for monitoring gradient profile on-line to record any deviations in these HPLC systems. The conductivity could be measured by a traditional conductivity detector or a capacitively coupled contactless conductivity detector (C4D). The method was applied for assessing the performance of an electroosmotic pump (EOP) based nano-HPLC. We also observed that σ value of the gradient changed with system pressure; a = 0.0175ΔP (R2 = 0.964), where a is the percentage of the conductivity increase and ΔP is the system pressure in bar. This effect was also investigated.