Innovations in Liquid Microjunction Surface Sampling Probes Louis Searcy1, Cuong Nguyen2, Timothy Garret3, Richard A. Yost1 1Department of Chemistry, University of Florida; 2Department of Veterinary Medicine, University of Florida; 3Department of Pathology, University of Florida, Gainesville FL Objectives Theta Capillary Liquid Microjunction Surface Sampling Probe Results Objectives: (1) Develop a liquid microjunction surface sampling probe (LMJ-SSP) with a 0.1 mm outer diameter (OD). (2) Apply a peristaltic pump to control flow rates before and after the LMJ-SSP. Methods: (1) Probes are made out of borosilicate theta glass capillaries. The capillaries are pulled to a point around 0.1 mm in diameter. (2) A Gilson Minipuls 3, peristaltic pump was tested to pump solvent to and from the LMJ-SSP. Conclusion: The innovations in this work show promise in reducing the spot size of LMJ-SSPs and allowing for an alternative pumping method that matches solvent flows before and after the LMJ-SSP. with the same pump. Theta Capillary Liquid Microjunction Probe a) b) Figure 3. (a) Image of the pulled theta capillary analyzing rhodamine 6G on a glass slide. The instrument parameters are shown in (b) for the spectrum of rhodamine 6G shown in (c). 443 is the M+ and 485 is the [M-H+Na]+. Theta Capillary LMJ-SSP Epoxy Extraction Solvent In Solvent Out Theta Capillary Liquid Junction Sample Glass Slide a) The theta capillary LMJ-SSP had difficulties sealing with the fused silica tubing. When pumping solvent through the probe to the surface, solvent would go around the fused silica and back up the unmodified end of the capillary. The current solution to this problem was to epoxy the fused silica in the theta capillary. The downside to the epoxy is reduced solvent compatibility. Capillary tip sizes less than 0.1 mm prevented the syringe pump from getting solvent to the surface; therefore all tips were cut to have a 0.1 mm OD. The designed capillary probe was capable of holding a liquid junction with a glass slide, shown in Figure 3(a). b) LMJSSP-MS Conditions Fused Silica m/z Range 100-800 Capillary Temp. 325 °C Nitrogen Pressure 25 psi Extraction Solvent Methanol c) d) Introduction Peristaltic Pump Driven Liquid Microjunction Probe LMJ-SSPs are a method of ambient ionization that use a liquid junction to extract analytes from the surface of interest. LMJ-SSPs are coupled to ionization sources capable of handling liquid samples, ESI and APCI.1,2 Most commonly, a syringe pump moves the extraction solvent to the surface and the nebulizing gas moves the same solvent to the ionization source.2 The size of LMJ-SSPs varies from commercial probes around 0.6 mm OD to individually produced probes around 10 μm.1,3 This work aims to analysis single cells with the miniaturized probe, while maintaining the convenience of using a commercial LMJ-SSP and investigates changes to the solvent pumping method by using the peristaltic pump in lieu of the nebulizing gas. Thermo Scientific Q Exactive MS Conditions Currently, the LMJ-SSP uses a syringe pump and the nebulizing gas from the ESI source to control the flow rate of solvent. The two-channel peristaltic pump controls the flow rates on either side of the LMJ-SSP allowing use of a large solvent reservoir, permitting longer analysis times between syringe refills, and less user interferences during sampling. Some contamination occurred from solvent compatibility issues with the peristaltic pump tubing made from polyvinylchloride. PharMed, a polypropylene tubing, proved to be resilient enough for use with methanol, and was used in these experiments. The PharMed tubing was 0.5 mm internal diameter (ID), which provided flowrates between 30 and 800 μL/min. The ID of the PharMed tubing was too large. With thinner tubing, the peristaltic pump could run at higher RPM without increasing the flow rate, thus decreasing the effects of pulsations. The use of a dual channel peristaltic pump showed potential to control the solvent flow rate in and out of the LMJ-SSP, but needs further optimization to reduce tubing contamination and pulsations. Instrumental Parameters Polarity Switching Full Scan Top10-ddMS2 AIF m/z Range 100-1000 70-1000 Resolution 35,000 70,000 17,500 AGC 3 x 106 5 x 106 IT (ms) 200 256 100 NCE   20 ± 5 Iso. Width (m/z) 1 Underfill (%) 0.1 Inten. Threshold 5 x 104 Apex Trigger (s) 5-20 Dyn. Exclusion (s) 6 443 485 c) Figure 2. (a) Pulled theta capillary (left) next to unmodified theta capillary (right). (b) Image of fused silica in one side of the theta capillary at 95x magnification. (c) Measurement of the pulled capillary tip (0.1 mm) at 95x magnification. (d) Pulled theta capillary with fused silica solvent lines. Figure 1. Schematic of the theta capillary LMJ-SSP. Methods Theta Capillary Liquid Microjunction Probe Peristaltic Pump Liquid Microjunction Surface Sampling Probe Theta capillaries, from Hilgenberg GmbH(Germany), were used to create a 0.1 mm OD LMJ-SSP. A Kopf Model 720 needle/pipette puller (Tujunga, CA) was used to create a 0.1 mm tip on the theta capillary. Fused silica with a 0.36 mm outer diameter and 0.15 mm inner diameter were inserted into both sides of the theta capillary (Figure 1 and 2). The fused silica were glued in place to prevent leaking. The probe was mounted to the existing Flowprobe stage, which is on a Thermo Scientific LTQ XL mass spectrometer (San Jose, CA). Solvent Reservoir ESI-MS Peristaltic Pump Channel 1 Peristaltic Pump Channel 2 LMJ-SSP Liquid Junction Sample Glass Slide a) c) Future Directions Future goals include capillary probe improvement for use in single cell analysis, and the use of the peristaltic pump to incorporate chromatography after the LMJ-SSP. References b) Figure 8. The blue lines are the signal intensity of methanol and the red dotted line is the moving average. Different pump speeds are shown: (a) 2 RPM, (b) 4 RPM, and (c) 10 RPM. (1) Gary J. Van Berkel, Vilmos Kertesz, 1 Kenneth A. Koeplinger, Marissa Vavrek, A.-N. T. K. J. Mass Spectrom. 2008, 43 (7), 854–864. (2) Berkel, G. J. Van; Pasilis, S. P.; Ovchinnikova, O. J. Mass Spectrom. 2008, 43 (7), 854–864. (3) Pan, N.; Rao, W.; Kothapalli, N. R.; Liu, R.; Burgett, A. W. G.; Yang, Z. Anal. Chem. 2014, 86 (19), 9376–9380. Peristaltic Pump Driven Liquid Microjunction Probe Figure 5. Schematic of the peristaltic pump in use with the LMJ-SSP. A two-channel peristaltic pump was used to control the flow rates on either side of the LMJ-SSP. A Gilson Minipuls 3 peristaltic pump (Middleton, WI) was used with 0.5 mm inner diameter PharMed tubing. The flow rates of the peristaltic pump were measured by running methanol through the tubing and collecting it in a 1 mL volumetric flask. The flowrates achieved with methanol are shown in Table 2. The total ion chromatograms were taken to assess the amount of pulsations in the peristaltic pump at 1, 4, and 10 RPM. A sample of rhodamine 6G was then analyzed at 7 RPM to test the peristaltic pump with an analyte. LMJ-SSP-MS Conditions Peristaltic Pump Flow Rates m/z Range 100-800 Capillary Temp. 325 °C Nitrogen Pressure 25 psi Extraction Solvent Methanol RPM Flow rate (μL/min) 1 34 4 69 6 137 8 195 10 250 18 645 48 870 Acknowledgements Table 2. The flow rates achieved for specific RPMs. Figure 7. The TIC and mass spectrum of rhodamine 6G using methanol in the peristaltic pump at 7 RPM. The TIC shows the signal suffers from pulsations caused by the pump. The Southeast Center for Integrated Metabolomics U24 DK 097209 CTSI Biorepository Prosolia, Inc. The Yost Group Members Table 1. Conditions used for LMJ-SSP with the peristaltic pump.