Nano-LC isn't easy but it's necessary so I’m chasing peaks like Tom chases Jerry

Man if I had a quarter for every time my Nano-LC system Failed. I mean it’s broken more than it works. And how many do I have 5 of them?

Instead of going all Office Space on my Nano-LC’s , I thought I’d praise the virtues of it and why you need to just bite the bullet, and live with it.

Everyone who has done it knows how painful it is, and if it isn’t painful, you are either not pushing it to its limits or maybe just injecting clean standards and not real dirty biological samples.  If Nano-LC wasn't an issue, you would not have companies touting their Easy-LC (which is anything but Easy), their chip based systems or their direct spray systems like Bruker’s acquisition (and ultimate destruction) of Michrom’s Captive spray system (RIP). I’ve tried all of them and they generally are not worth it.

Let’s get to the virtues of nano-LC  first.

1. You really get a sense of accomplishment whenever it works.
2. You can Make most of the parts yourself (and you really should)
3. It really is more sensitive than anything else.
4. If you don’t take labor into account it’s way cheaper.
5. Job security, as it does really take a lot of practice/skill  to get really good at it.

Some of the Downsides

1. Making the parts and troubleshooting can get very expensive if you take labor into account
2. Very easy to overload your columns.
3. The autosamplers on Nano-LC systems generally suck. I still know people who do not use them and bomb load everything
4. When it is working, you should not talk about it or stare at it intently. The Nano-LC system will know and break on you.
5. Reproducibility can be poor during long runs of samples as the column and spray tip gradually deteriorates.

I’m generally skeptical when people say they run their nano-spray system for months nonstop on real biological samples. I guess if you are not too concerned with RT and sensitivity reproducibility I guess this is possible,. But if you say run standards every 5-10 runs, you’ll see how much things can go downhill fast. Remember every time you are injecting a sample you are changing your column. That carryover you see is because you are not recovering 100% of your sample from your LC system . And that carry over changes your system in subtle and not so subtle ways.

Here are some tips I have come up with over the years

• Making your own columns, traps and packing them is not that hard. I’ll Put up a tutorial some day. If you buy a nano-column from a company, they are most likely using a Sutter Laser puller and packing using a pressure vessel, just like you can do.
• Most people use a Vented column set up with a Tee or a Cross (for the HV)
• Watch out for those clogging, they are a major point of failure.
• Main points of failure on your LC system (depending on how they work)
• Rotors (rotor seals mainly)
• Piston Seals
• Check Valves
• Tubing getting clogged
• Which is why I do not recommend PeekSIl
• Fittings not connected correctly and introducing dead-volume

Just remember, it gets easier if you just getting started. and don’t be tempted with these all in one nano-solutions you see for sale. They will ultimately fail in some way and you will have a hard time fixing the problem yourself if you do not know how it works. When you have samples piling up, it’s far better if you can fix it yourself. Of course there is always going to be real hardware failures you can’t fix, but most of the routine problems you can