High-temperature ultrafast ChipHPLC-MS

Abstract

Herein, we present a miniaturized chip-based HPLC approach coupled to electrospray ionization mass spectrometry utilizing temperature to achieve high-speed separations. The approach benefits from the low thermal mass of the microfluidic chip and can form an electrospray from the pre-heated mobile phase. With the help of this technology, isothermal and temperature-programmable operations up to 130°C were pursued to perform reversed-phase separations of pesticides in methanol and ethanol-containing eluents in less than 20 s.

Keywords: Chip chromatography; High-temperature chromatography; Mass spectrometry; Microfluidics.

Fig. 1

Overview of the microchip design and experimental setup of the HTchipHPLC-MS approach. A Photographic image of the functionalized HT-HPLC microchip. B Schematic drawing of the external fluidic circuitry in injection mode. It includes the microfluidic design of the microchip (top view) and fluidic components, such as HPLC pumps, valves, and a pressure sensor. The flow paths of the sample stream (green), pinch stream (blue), and eluent stream (black) are colored. Since the sample and pinch streams leave the chip at the waste outlet to flow into one of the restriction coils (R), the corresponding tubing is colored cyan. Capillary tubings not connected to a pumping device in this fluidic situation are colored in gray. In addition, arrows indicate the direction of flow. C Insight of the post-column layout of the HT-microchip. D Photographic image of the microchip thermostat with the functionalized HT-HPLC microchip installed. More details about the experimental setup used in the presented study, including the fluidic circuitry for elution mode, visual inspections of the microfluidic cross injecting a fluorescent sample, and pressure data, are given in the Electronic Supplementary Material Fig. S1 and S2

Fig. 2

A HTchipHPLC under isothermal conditions with MS detection (from ϑ_µ-column =30°C to 130°C, whereas ϑ_column =130°C is displayed within insight view), column length: 35 mm, material: XBridge C18 BEH, dp=2.5 µm, maximal elution pressure: 132 bar, pesticide mixture containing fenuron (1, c=50 µM), cyanazine (2, c=50 µM), diuron (3, c=50 µM), fluometuron (4, c=50 µM), and metolachlor (5, c=50 µM) dissolved in 50/50 v/v MeOH/H₂O, 0.1% FA was injected. All phenyl urea pesticides were detected as [M+H]⁺ can be found in Electronic Supplementary Material Fig. S4. B Stability of the MS baseline signal under isothermal conditions (listed from ϑ_column =30°C to 130°C), intensity of the normalized total ion current (nTIC) and its corresponding deviation depicted as σ_{TIC(ϑµ-column)} in % are displayed, no sample injected, 50/50 v/v MeOH:H₂O, 0.1% FA was used as an eluent. C Electrospray formation within the developed HTchipHPLC MS interface using heated eluent (50:50 v/v MeOH/H₂O, 0.1%, ϑ_column =110°C, MS inlet: 3.5 kV). D Thermographic image of the HTchipHPLC assembly at column temperatures of 130°C. E Insight of the temperature distribution in the post-column area of the microchip. A linear IR-radiation scale and an emissivity of ε=0.88 for the microchip were applied. F Illustration of the HTchipHPLC MS assembly installed in front of the mass spectrometer. The red dashed box points out the post-column region of the assembly.

Fig. 3

Dependency between column temperature and maximal peak intensity of selected analytes, data are withdrawn from separations illustrated in Fig. 2A

Fig. 4

Illustration of a HTchipHPLC ESI MS measurement utilizing a thermal gradient condition. 2-step thermal gradient from 60 to 140°C, column length: 35 mm, material: XBridge C18 BEH, dp=2.5 µm, maximal elution pressure: 133 bar, sample solution was identical to Fig. 2A. R_C, resolution of the critical peak pair

Fig. 5

Greening of HTchipHPLC ESI MS illustrated by A isocratic separation using 30% v/v EtOH:H₂O, both 0.1% FA under ambient conditions, linear velocity 2.21 mm/s, H=50934 plates m⁻¹ and B thermal gradient 60 to 110°C under isocratic conditions 30% v/v EtOH:H₂O, 0.1% FA, linear velocity 3.84 mm/s, H=54523 plates m⁻¹, column length 35 mm, material C18 BEH XBridge, dp=2.5 µm, sample fenuron (2, c=20 µM), diuron (3, c=70 µM), fluometuron (4, c=50 µM), tebuthiuron (5, 50 µM), metolachlor (6, 100 µM), and thiourea (1, 5 mM) as deadtime marker in 40:60 MeOH:H₂O, elution pressure: 133 bar