Miniaturized LC Optimized Using HPLC-Based System

Scientists from Nano Separations Technologies in São Carlos, Brazil recently developed a new high-performance liquid chromatography (HPLC) system to address issues with miniaturized liquid chromatography (LC). Their findings were published in the Journal of Chromatography A (1).

High-performance liquid chromatography (HPLC) is a chromatography-based technique for separating, identifying, and quantifying components in a mixture (2). Transitioning from conventional liquid chromatography (LC) to miniaturized scales (capillary/nano-LC) offers not only analytical and methodological benefits, but also significant advantages in environmental, toxicological (related to analysts' health), and cost. Miniaturized LC enhances ionization efficiency when integrated with mass spectrometry. At low flow rates, the mobile phase’s influence on the electron ionization process lessens allowing capillary/nano-LC to be coupled with electron ionization mass spectrometry.

Despite advances in miniaturized LC instrumentation, its implementation for routine analysis remains quite limited, the scientists wrote. Capillary and nano-LC instruments are typically more expensive than conventional ones, in addition to being more delicate and costly when using and maintaining them. Additionally, many facilities using HPLC have invested heavily in conventional LC technology, often making the transition to capillary/nano-LC instruments infeasible.

For this study, the scientists developed a cost-effective, active two-channel flow splitter capable of delivering capillary flow rates from conventional HPLC pumps, for the recycling and reuse of the split solvent. Additionally, a capillary ultraviolet–visible (UV–vis) detection cell compatible with conventional HPLC detectors was developed and evaluated based on its performance. The system employs an Arduino Uno board to monitor a flow sensor and control a stepper motor that automates a split valve to achieve capillary-scale flow rates from a conventional pump. A capillary UV–vis cell compatible with conventional detectors was developed to address the detection challenges at this scale and minimize extra column band broadening. The system performance was assessed by a lab-packed LC capillary column with 0.25 mm x 15 cm dimensions packed with 3.0 µm C18 particles. Model compounds, namely polycyclic aromatic hydrocarbons (PAHs), were employed to assess the functionality of all developed components in terms of theoretical plates, resolution, and band broadening.

The Arduino system continuously monitored the sensor readings and based on precalibrated functions, actuated the motor to maintain the flow rate at the user-defined value. The system demonstrated satisfactory performance in delivering capillary flow rates (2–7 µL/min) from conventional HPLC pumps designed for larger scales (> 0.1 mL/min). Similarly, a capillary UV–Vis detection cell was developed and optimized based on the conventional LC detection cells. The positions and inner diameter of the capillary used to construct the optical path length were crucial factors that could affect the miniaturized cell's performance. Utilizing a 75 µm i.d capillary in the optical path length construction struck a balance between detectability and minimal contribution to detection band broadening.

The injection system can be a critical determinant of miniaturized system performance, with miniaturized LC necessitating injection volumes in the nanoliter range. Adapting conventional HPLC instruments for miniaturized operation may require appropriate injection valves with rotors designed for nanoliter injections or exploring alternative adaptation strategies. Further research into these aspects is warranted.

References

(1) Contreras, E. M. C.; Tanis, E.; Lanças, F. M.; Medina, D. A. V. Exploring a Reversible Adaptation of Conventional HPLC for Capillary-Scale Operation. J. Chromatogr. A 2024, 1730, 465021. DOI: 10.1016/j.chroma.2024.465021

(2) Koester, V. What is HPLC? Wiley-VCH GmbH 2016. https://www.chemistryviews.org/details/education/9464911/What_is_HPLC/ (accessed 2024-8-5)