Help Building a Post Column Reaction

[emc]

Our lab is new to HPLC and just recently acquired a Waters Breeze system with fluorescence detector. I need to build a post column reaction setup from scratch using o-phthalaldehyde. I know from the literature that it's a fast reaction and that one can build a reaction coil by knitting PTFE tubing. What I would like advice on is the hands-on practical aspects of stringing the reagent delivery pump/line and reaction coil into the post-column flow.

Since I do not have much experience with HPLC plumbing/connections, I'm afraid of screwing up our existing tubing or purchasing the wrong fittings. Starting from the output end of the column, SS tubing leads from the column and feeds directly into the detector (with no external fitting). I need to tap into that line with a mixing T of some kind (to introduce the reagent) and the reaction coil before the detector. I would like to be able to add and remove the post-column setup easily, which leads me to believe (from what little I know) that some kind of handtight connectors are preferable with a union to close the line when the reactor is removed. Can anyone offer any practical advice on what fittings to purchase, cutting the existing line w/o introducing potential problems down the road (cut the SS tubing near the detector or near the column?), what fittings/connections can be used to join the teflon tubing of the coil to everything else, what kind of mixing T to get, etc?? Any help would be GREATLY appreciated! Thanks!!

[Uwe Neue]

Don't cut anything. You can buy or make straight connectors and purchase the Tee-piece that you need from multiple vendors.

I am glad that you are using the knitted tubes

[emc]

So, if I don't cut anything, how do I tap into the line betweent the column and the detector? Just undo the column fitting? I'm assuming since it's a SS fitting w/ ferrule that I'll at least have cut the end to redo that fitting to avoid a leaky connection, right? When you say buy or make straight connectors, what do you mean exactly? Sorry if I'm asking dumb questions...I'm a newbie.

[Kostas Petritis]

---
Post-column derivatization in general and especially the o-phthalaldehyde reaction can be tricky and you might end up not achieving reproducible results by using a home-made system and not previous experience. You might want to buy a post-column derivatization device from Pickering laboratories or at least consult their on line manuals for useful tips (i.e. look at the library section)...

[emc]

I appreciate the suggestion of purchasing a complete system such as what is available from Pickering, but I'm operating under the request of my advisor (I'm a graduate student) to attempt a homemade reactor first. As a student, my time/effort troubleshooting is (sadly for me) cheaper for him than going straight to a commercial system. I was lucky we even got an HPLC of our own. Had some interim grant money not been about to expire, I would have been left at the mercy of collaborating with another lab whose equipment is already quite busy (and graduation would be much much farther away).

I have already read quite a few commercial manuals/troubleshooting guides for PCRs (including what is available from Pickering). So, I understand what I need to achieve to have a reproducible and efficient reaction. What I need help on is the little practical details (like piecing together the plumbing) that are rarely included in published protocols.

[Uwe Neue]

Here are some suggestions that will accomplish what you need to do:

You use a short piece of tubing between the column and the mixing tee. The mixing tee is on one side connected to the column, on the other side it receives the reagent stream and the outlet is connected to the knitted tube reactor.

Here are suggested part numbers from Waters:

Slip-free connector: PSL618007
Tee (for 1/16 inch compression screw): WAT075215

Additional nuts and ferrules, in case the other connections are not compatible: WAT025604

[Kostas Petritis]

---
If you are a graduate and from the moment you are learning something new (although re-inventing the wheel), it should be fun!

Good luck

PS: the Pickering website also has part number of the exact Tee's and tubing they are using in their systems...

[HW Mueller]

That is what fittings are for: fitting things together. Metal ferrules are notorious for deteriorating, the seat can go as well, but even those can be undone and refitted many times if you don´t mistreat them (don´t tighten them more than needed to stop a leak). There are PEEK or Teflon type ferrules which can´t ruin the seat (the female part of the fitting), nor do they cut into the tubing. As an example, the Upchuch catalog is quite helpful.
Now, if you have special skills you can mess up even with "plastic" connectors. You will learn a lot about fittings if you overtighten a "plastic" version to thje point of converting it to marmelade.