Method Article
Production of packed nanospray emitters with integrated emitter-end frits for liquid-chromatography mass spectrometry
https://doi.org/10.1038/protex.2017.154
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This protocol describes how to frit the emitter-end of a pulled silica capillary and subsequently pack it to produce a robust integrated column. Integrating liquid chromatography separations directly into the emitter of a nanospray source for mass spectrometry offers improved sensitivity and resolution. This is achieved by reducing post-separation dead volume. The method described here provides an optimized setup to maximize sensitivity and separation.
The sensitivity of mass spectrometry-based proteomics is enhanced by limiting the diameter of the electrospray emitter to a few micrometers. Additionally, capillary liquid chromatography with small internal diameters (50 µm or less) enhances the detection of low-level species in complex samples. To maximize the benefit of this style of column, dead volume after the separation of compounds should be minimized. Current protocols for custom-packed fritted columns1 and pulled silica emitters with distal-end frits2 can be joined by a union to offer a setup appropriate for nanospray ionization mass spectrometry; however, this introduces dead volume post-separation. Ideally, the column should extend to the tip of the emitter. Emitters packed without a frit may be unsuitable for smaller particle size resins which can escape the opening or be prone to clogging. Consequently, we have developed a protocol to put a frit in the emitter-end of a pulled silica nanospray emitter.
Emitter fritting:
Polyimide-coated fused silica capillary with dimensions:
360 µm outer diameter (O.D.) / 50 µm internal diameter (I.D.)
Kimwipes
KASIL 1624 potassium silicate solution
Formamide
Hydrofluoric acid (HF)
Emitter packing:
Chromatography resin
Methanol
P2000 laser puller
Ceramic scribe for fused silica
Lighter
Diamond cutter for fused silica
Vortex mixer
Microcentrifuge
Microscope
Oven
Capillary packing pressure bomb with stir plate and appropriate gas hookup
Stir bar (3mm)
Erlenmeyer flask
Nanoflow capable HPLC
Fused silica emitter production
This section of the procedure is modified from Cifani, et al [2].
Cut a portion of polyimide-coated fused silica capillary that is twice the length of your desired final column using the ceramic scribe.
Burn away approximately 1-2 cm of the polyimide coating at the middle of the silica piece with a lighter.
Gently remove the charred residue of the coating using methanol and a Kimwipe.
Mount the piece of fused silica on the laser puller; carefully feed the middle section where the silica is exposed into the shielded mirror target of the puller.
Close the cover of the puller. TIP: Longer lengths of silica may extend past the cover, so take care not to crush them when closing it. You should be able to bend the silica into the cover so that it sits completely enclosed by the shield.
Enter the following program, then press “pull”: HEAT=250, FIL= , VEL=25, DEL=180, PULL=25
HEAT=250, FIL= , VEL=25, DEL=180, PULL=25
HEAT=250, FIL= , VEL=25, DEL=180, PULL=25
HEAT=250, FIL= , VEL=25, DEL=180, PULL=25
Fritting the emitter
Add 75 µL of KASIL 1624 to a 1.5 mL polystyrene tube.
Add 25 µL of formamide.
Vortex for 10 seconds.
Spin down in a centrifuge for 10 seconds.
Take the pulled silica emitter and dip the pointed end into the frit solution for ~5 seconds, keeping the capillary vertical. TIP: The emitter should barely touch the fluid’s surface and not be submerged in the solution any more than necessary.
Verify that the capillary has soaked up a small plug of frit solution under a microscope (figure 2a).
Put the emitter in an Erlenmeyer flask with the tip pointed upwards. Heat the flask at 100°C overnight.
Remove the emitter from the oven and let it cool before proceeding.
Check the emitter under a microscope to verify that a solid frit has formed in the tip of the emitter (figure 2b) NOTE: A thin film of frit solution on the exterior of the emitter may have formed to cause opacity. This will be removed in the following steps.
Caution: the next step utilizes HF. Take all necessary safety precautions and perform the next steps in a fume hood.
Emitter packing
Suspend the chromatography material of your choice in methanol in a 1.5 mL polystyrene tube.
Place a 3 mm magnetic stir bar into the vial.
Place the vial into the capillary packing pressure bomb and secure the lid.
Turn on stirring.
In a fume hood, insert the fritted emitter into a capillary packing pressure bomb. Tighten the nut to secure it in place.
Gradually pressurize the bomb to 500 psi.
Bend the emitter to dip the tip into a small vial of HF. Hold it in place for 10 seconds. NOTE: Buildup of the solid frit will likely have obstructed the small opening of the emitter. The emitter should have a small but visible solvent drop that forms at the tip after HF etching. Flow will also be indicated by slow movement of the packing material towards the tip.
If no flow is apparent, repeat step 7.
Increase pressure to 1500 psi.
Allow the column to pack. NOTE: Small particle sizes with small I.D. columns may take several hours.
Once fully packed. Close the valve from the gas canister and allow the column to depressurize slowly.
Remove the column from the packing bomb.
Connect the emitter to an HPLC and run the column at 80% ACN with an appropriate flow rate for the I.D. and packing material for 1 hour, or until the pressure stabilizes.
Emitter production: 30 minutes
Emitter fritting: 8.5 hours
Emitter packing: variable
The procedure results in an emitter with an integrated frit that is packed with chromatography material.
Thanks to the City of Hope Mass Spectrometry and Proteomics Core Facility for use of their equipment and instruments.
No relevant conflicts of interest.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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