Welcome back to our series on frequently asked questions in HPLC.
Here’s another set of questions that involves the tubing and fittings that we use in the LC.
Stainless Steel Tubing versus PEEK Tubing
PEEK is a type of plastic, polyetheretherketone. It is a very useful material that we use for HPLC tubing, HPLC fittings, in and around the LC world.
Stainless steel is the best. It is incredible. Stainless steel will handle the highest pressures and give you the best fittings. It’s an all-around fantastic material, but it’s very difficult to cut. In fact, I’ll argue you cannot cut it yourself. I have all the fancy tools in my lab. I have the grinder, the wheel cutter, and everything else, but if you cut stainless steel tubing, you’re going to leave a little bit of burrs. These burrs are enough to offset the fitting, and that’s enough to really destroy your efficiency in a modern HPLC instrument. So, for that reason, if you’re going to use stainless steel, you must buy pre-cut lengths. There is no downside to that.
The other approach is to use PEEK. I personally like PEEK because I can cut any length that I want. So, I typically buy a very long roll of PEEK tubing and then cut off the desired length.
Stainless Steel Fittings versus PEEK Fittings for HPLC
The perfect fitting is always going to be the stainless steel fitting. The stainless steel fitting is still very common. It’s great. The problem is, once it’s swaged, in other words, once you tighten it the very first time, that ferrule will seat permanently onto the tubing. That distance beyond the ferrule is critical that it matches the fitting on your column.
Over the years, there have been a bunch of different fittings, which was a horrible idea because all the nuts seem to be interchangeable. They have the same number of threads per inch, but the ferrule depth is very different.
So, with Swagelok-type fitting, by far the most common today, it’s 90 thousandths of an inch. If you look at a classical Waters fitting, it’s 130 thousandths of an inch. If you look at a Radon fitting, it’s 170 thousandths of an inch. You don’t really need to know those numbers; what you need to do is make sure you make the fitting properly. So, either you’re buying stainless steel fittings and you’re making them properly, you’re swaging them properly, or you’re buying them premade, or you can make your own if you get PEEK tubing.
Cautions About PEEK Tubing
PEEK tubing is plastic; it’s a very thick-walled material with a very small diameter. So, they withstand all the typical pressures we would use in HPLC and the typical solvents.
Now, a word of caution: if you’re going to choose PEEK, make sure you look at compatibility with the solvent you’re using. If you’re using a polymer solvent like THF or methylene chloride, I would tread carefully. The pressure rating is not as high, and you’ll get a burst or you’ll get leaking through the tubing. But for most of us running reverse phase – using methanol/water, or acetonitrile/water, or THF/water—PEEK is fantastic. I love it.
What Diameter of PEEK Tubing to Use for HPLC
I like the 0.005-inch inner diameter. That will do great on just about any LC system out there. Here’s the coolest part: in order to cut the tubing, you buy a beautiful little tubing cutter from whoever you’re buying your materials from, and there’s a little hole that you put the tubing through, line it up, and when you press the little switch, it will then break off a piece, and you get a perfectly flat cut.
How Do You Make a Perfect HPLC Fitting with PEEK Tubing?
This is how to make a perfect fitting in your HPLC. I don’t care what kind of column you have; this is how you do it. I’m going to show you using a PEEK fitting. PEEK, again, is Polyetheretherketone. It’s plastic. The key here is that since the ferrule is made out of plastic, there are no steel barbs to stick into the tubing, and it won’t permanently set on the tubing. That means I could use the same fitting to put into an Agilent column, a Phenomenex column, or a Waters column, and I don’t have to worry about if it’s the correct ferrule depth.
Now, let me show you how to make a perfect fitting, and again, I could do this on any column, even on a teeny tiny little five-centimeter Agilent column. The amount of tubing sticking out beyond the ferrule is critical, but it’s really not something we can measure. So, instead of trying to measure it, we know I’m going to need 90,000, 130,000, or 170,000 thousandths.
So, here’s my idea: I’m going to start with an entire inch sticking out, and I’m just going to shove the tubing into the column until it stops, making sure the tubing is bottomed out in the column. Push in on the red tubing while tightening the fitting. You want it to be finger-tight. We use finger-tight fittings for pressures up to 400 bars (6,000 psi).
We call it a universal fitting because this fitting will fit any known column. Since this is the Agilent column, it should be swage-like depth, 90,000 of an inch. Again, we don’t measure it; we just look at it and say it must be 90,000 of an inch because it was set by this column.
Now, the cool thing is, when I’m done using this Agilent column and I decide to use a Waters column, I could take this column off and put this back on another column, like one of my classic old Waters columns, 130,000 inches, and then take it and stick it into the next column. So, the same fitting will fit any column, and not only that, it will give you a perfect fitting every time.
High Pressure HPLC Fittings
We’ll go up to 600 bars, which is 9,000 PSI, using a slightly different style fitting. It’s called the double-winged nut. This nut has a plastic ferrule, but now the nut has a couple of wings on it, and that allows me to really torque on the fitting. To make a perfect fitting, shove it all the way down until it stops. Push the tubing in, and now, as you’re tightening the nut, keep inward pressure on the tubing.
I’m being really paranoid. I want to make sure that tubing doesn’t jump out because if it jumps out a millimeter, you’re going to lose 20% of all the efficiency of your instrument. That $75,000 brand new LC will give away 20% efficiency with one bad fitting. This will give you a perfect fitting every single time, again up to 9,000 PSI, and technically that’s in the UPLC range. We use these on our UPLC columns all the time.
If you are nervous using one of these fittings at high pressure, then you have other options. Some new UPLC manufacturers have come out with a bunch of different fittings. Agilent has one called the InfinityLab Quick Connect. The cool thing is, the ferrule depth has a spring in there. So, put that on the column and finger-tighten it, and once we get finger-tightened, pull down the lever. That tightens it to 20,000 PSI. It’s universal. It will fit any column known to man. Very cool, but they’re expensive.
What’s the takeaway from this?
You can make a perfect fitting every time if you use universal fittings, and it doesn’t really matter who you buy them from. I like the PEEK fittings. There are also some nice high-end fittings like the Agilent Quick Connect. Thermo Fisher has some really nice high-end fittings for UPLC as well. So, you can check those out if you’re worried about running high pressures. You know, I wouldn’t go above 9,000 PSI with my finger-tight fittings. I would want to go to one of the more serious fittings.
Choosing HPLC Tubing Diameter
In terms of tubing diameter, it depends on English versus metric, but I’ll give you a couple of general ideas. In the metric world, 0.17 millimeter ID, the classical green tubing, is good for normal HPLC running 1 mL a minute to 2 mLs a minute. You don’t need anything better than that.
If you go smaller down to 0.12 millimeter, that is the classical red tubing. When we get down to 0.12, now we’re looking at much smaller diameter tubing. You don’t need it if you’re running 1 or 2 mLs a minute. You definitely need it if you’re running 0.2 mLs a minute. So, you know who I’m talking to—the LC mass spec folks. You’ll want to switch to the red tubing, 0.12 millimeter ID. In the English world, it’s 0.005 inch. 0.007 is getting into the sort of the green level, and nowadays, 0.010 of an inch is really too big for modern HPLC work that we’re doing today.
I like PEEK tubing with a 0.005-inch inner diameter or 0.12 millimeter ID. It works great for everything you’re going to do, and it’ll give you skinnier peaks and better efficiency if you use those smaller diameter columns.
Thanks for joining us on our little discussion here on tubing, fittings and tubing diameter. If you want more information or if you would like to see something else demonstrated or a question answered, let us know here.