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

Abstract

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 (C(4)D). 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 (R(2)=0.964), where a is the percentage of the conductivity increase and ΔP is the system pressure in bar. This effect was also investigated.

High‐performance liquid chromatographic cartridge with gradient elution capability coupled with UV absorbance detector and mass spectrometer for peptide and protein analysis

“…5. The separation efficiencies and resolutions were comparable to experimental results we obtained before [16], while separation profiles compared well to profiles previously recorded using a commercial Agilent HPLC system. However, the F I G U R E 5 Digest separations.…”

“…The total volume of eluent solutions in GL was ∼18 uL. After V2 was switched in W and a voltage of +1.5 kV was applied to EOP-2, the partially mixed eluent solutions were pushed out to W. A C 4 D detector (TraceDec Contactless Conductivity Detector, Innovative Sensor Technologies, Austria) was fixed on the connection capillary between V2 and waste reservoir to monitor the gradient profile as we did previously [16]. Figure 4 presents typical reproducibility results of a gradient profile.…”

“…This was to achieve a gradient from 0 to 60%. A contactless conductivity detector was used to monitor the gradient change range for separating both myoglobin and BSA digest as shown in our previous studies [14,16,17].…”

Recent advances in miniaturization of portable liquid chromatography with emphasis on detection

“…On-column ECD has also been exploited for portable chromatographic systems to minimize challenges associated with replacing or activating electrodes within flow cells [48]. For example, capacitively-coupled contactless conductivity detectors (C 4 D) have been extensively used with miniaturized electrophoresis systems (EC), capillary ion analysis of various formats, and monitoring of the profiles of gradient programs in LC systems [50,51].…”

A Performance-enhanced Electroosmotic Pump with Track-etched Polycarbonate Membrane by Allylhydridopolycarbosilane Coating

“…Moreover, it has the characteristics of continuously and accurately transporting micro-liter and nano-liter fluids, no moving parts, no pulsation, no mechanical wear and avoiding micro-leakage from valves and conveying process. EOP has been applied in flow injection analysis (FIA) 1 , micro-, nano liquid chromatography (HPLC) separation [2][3][4] , femto-pipette 5,6 , electroosmotic droplet switch 7,8 and micro-cooling systems 9 .…”