By our own MS guru Jon Reed
A Thermo Engineer once told me that “Nano-LC is not for the faint-of-heart.” A 0.5µl bubble in a 10-port valve can make a mess of an experiment. Incomplete proteolytic digestions can clog up your brand new $600 column and render it useless. A few centimeters of post-column dead volume can cause significant peak broadening despite the fact that you have a $50,000 LC system. The list of problems goes on and on. This causes more than its share of heartache, which leads to this simple truth:
If the favorite pastime of proteomics researchers is bragging to one another about their latest and snazziest technological acquisitions, then the second favorite pastime is whining about how they don’t work.
Still, as time progresses, people chip away at old problems and find out how to do things better, faster, cheaper. So this week, I’ll leave you with one less thing to cry about and describe HOW TO MAKE A BETTER HOMEMADE FRIT FOR FUSED SILICA COLUMNS.
I had originally learned to make frits by dipping a piece of fused silica into 75% KASIL and 25% DMF, however these frits were lengthy, un-reproducible, and lead to high back pressure and inconsistent chromatography. That technique sucks. Sorry, stinks. No… wait… it sucks.
A new and improved protocol was first shared with me by Jennifer Busby and Valerie Cavett from Scripps. They’re very smart, and you should read some of their papers. Go on… log on to Pubmed and get to reading.
It’s an adaption of earlier work by Maiolica et al (Proteomics 2005, 5, 3847–3850), and takes a whopping 2 minutes, and $0.25 of reagents from start to finish. Well, that doesn’t include the ½ hour drying time, but if you’re saving that kind of time and money for a superior product, don’t complain!
OK, here goes…
- Make a Kasil/formamide mix (75/25) and use approximately 2 µL to wet a glass microfiber filter (GC/F, Whatman).
- Gently push and twist the end of a fused silica capillary onto the wetted filter.
- Dry the frit for 5 minutes at approximately 37ºC or at room temperature for approximately 20 minutes before packing. Note: times given for a 75 µm capillary; larger diameters may need longer to dry before packing.
Note: different column IDs may require a 2nd glass fiber plug (do not wet this one) to ensure you don’t blow the frit loose. To do this, first push the end of the fused silica tubing into a dry piece of filter and core out what you need, then repeat this on the wetted section of filter. You may want to try this using 2 wetted cores, but that may lead to increased back pressure. The only way to know is to try it out.
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