Download Innovations in Liquid Microjunction Surface Sampling

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
Extraction Extraction
Solvent In Solvent Out
LMJSSP-MS Conditions
b)
c)
m/z Range
Capillary Temp.
Nitrogen
Pressure
Extraction
Solvent
d)
Theta
Capillary
4
c)
Theta Capillary
LMJ-SSP
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.
100-800
325 °C
Methanol
Currently, the LMJ-SSP uses a syringe pump and the nebulizing gas
from the ESI source to control the flow rate of solvent. The twochannel 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.
2.5
2
1.5
485
0.5
0
100
200
300
400
500
600
700
800
m/z
ESI-MS
a)
10
8
c) 10
Intensity (x103)
Peristaltic Pump
Channel 2
6
4
2
0
0
Solvent Reservoir
b)
LMJ-SSP
Figure 5. Schematic of the peristaltic pump in use with the LMJ-SSP.
LMJ-SSP-MS Conditions
m/z Range
100-800
Capillary Temp.
325 °C
Nitrogen Pressure
25 psi
Extraction Solvent
Methanol
Table 1. Conditions used for LMJSSP with the peristaltic pump.
Peristaltic Pump Flow Rates
RPM
1
4
6
8
10
18
48
6
Future Directions
4
2
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
0
0.05
0.1
0.15
0.2
Minutes
Sample
Glass Slide
Liquid
Junction
8
0
Flow rate (μL/min)
34
69
137
195
250
645
870
0.25
0.3
0.35
0.4
0.45
0.5
Minutes
10
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.
8
6
4
2
0
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
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
(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.
Minutes
Table 2. The flow
rates achieved
for specific
RPMs.
3000
Intensity (103)
Peristaltic Pump
Channel 1
Intensity (x103)
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).
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).
Peristaltic Pump Driven Liquid Microjunction Probe
3
1
Theta Capillary Liquid Microjunction Probe
25 psi
443
3.5
Intensity (x106)
Sample
Glass Slide
Liquid
Junction
Theta Capillary Liquid Microjunction Probe
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.
a)
Fused
Silica
Methods
Peristaltic Pump Driven Liquid Microjunction Probe
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]+.
Epoxy
Introduction
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.
b)
a)
Intensity (x103)
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.
Results
Theta Capillary Liquid Microjunction Surface Sampling Probe
2500
2000
1500
1000
500
0
0
0.02
0.04
0.06
0.08
0.1
Minutes
0.12
0.14
0.16
0.18
0.2
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.
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Acknowledgements
The Southeast Center for Integrated Metabolomics
U24 DK 097209
CTSI Biorepository
Prosolia, Inc.
The Yost Group Members