title: Portable and modular liquid chromatography for pharmaceutical industry
Creators: name: Lam, SC
Publisher: name: University of Tasmania
Year: 2020

description: A state-of-the-art miniaturised and field-portable liquid chromatography (LC) system has been developed for use in the pharmaceutical sector. Developments on the instrumentation included (1) a small-footprint high-pressure pumping system that allows fast LC separation, (2) integration of an auto-sampling interface that enables automatic injection and cleaning, (3) utilisation of high-efficiency LC columns for fast separation, (4) adoption of robust and versatile detection approaches with superior sensitivity. The first phase of development involved the integration and performance testing of a fully automated hand-portable capillary LC system with a total weight of ~2.7 kg. This portable LC integrated multiple small footprint LC components within a 3D-printed enclosure, including a modular microfluidic system with automatic injection, column heater, and low-UV light emitting diode (LED) based Z-cell absorbance detector. This portable LC when used with a 5 µm C18column (100 mm × 300 µm I.D.) delivered highly reproducible chromatograms with retention time and peak area relative standard deviations (RSDs) of &lt;0.7% and &lt;3.3% in isocratic mode (n = 10), as well as of &lt;0.1% and &lt;2.3% in gradient mode (n = 10), respectively, with sub-4 min run times. Limits of detection (LODs) for four test small molecule pharmaceuticals ranged from 65 to 101 µg·L\(^{-1}\) based on a 316 nL injection volume, with separation efficiencies between 18,000 and 29,700 N·m\(^{-1}\). In addition, the portable LC was coupled to a small footprint mass spectrometer (MS) to demonstrate compatibility and ‘point-of-need’ LC-MS capability. The limitations and improvements of low-UV LED and Z-cell based absorbance detection were addressed in the second phase. Commercial low-UV LEDs produced limited spectral outputs with low external quantum efficiencies (EQEs), which affected the detection sensitivity, especially when light was transmitted through narrow capillary-format optical paths. In order to achieve higher emission intensity, the LED was operated at high input currents. However, this led to a stressed LED as a result of excessive heat generation. A solution proposed and investigated herein was to apply active cooling for effective heat dissipation, which recovered the performance of the affected LED. Hence, a simple 3D-printed liquid cooling interface was developed for low-UV LED based absorbance detection. This active cooling interface improved LED performance by reducing noise and LOD by a factor of 2 as well as shortening equilibration time by 6-fold. Another solution was to use a high-power UV-LED in conjunction with collimating lenses to increase photon throughput. A novel surface mount device (SMD) LED emitting at 235 nm with improved output power and EQEs, was used in capillary-scale UV detection. This SMD had extremely low light transmission (~0.001%) through the 100 × 100 µm channel of the Z-cell detector, so the optical alignment was optimised by the simulation and integration of focusing lenses into the optical path. A 9-fold increase of light transmission with a dual-lens setup yielded an enhancement of detector sensitivity by a factor of 2.2. In the third phase, an upgraded portable LC with higher pressure capability was deployed as process analytical technology (PAT) for use within an industrial pharmaceutical setting, namely reaction monitoring prototype (RMP). For at-line analysis, a self-packed 3.5 µm XBridge C18 capillary column was used for separation in the RMP system. The difference in quantitation of the starting compound between the RMP and a full-sized benchtop LC was minimal, which is not statistically significant. To cope with the sample degradation issue within a highly aqueous environment, a normal-phase LC was attempted using a 1.7 µm BEH HILIC packed capillary column on the RMP system. A successful attempt at developing an on-line coupling of the RMP system with a sample dilution cart demonstrated its high potential and flexibility as a robust PAT approach.