Getting Started in HPLC

Section 2G. Tubing and Fittings

So far we have not talked about the TUBING and FITTINGS that connect our various LC modules together. For tubing connections between the reservoir and the pump and the injector, the only requirement is that the fittings not leak, and that the tubing between the reservoir and the pump be large enough in diameter so that flow of mobile phase to the pump is not restricted. The tubing connections between the injector and the column, and from the column to the detector, however, require more attention.


Connections between the reservoir and the pump are usually made with Teflon tubing, often of fairly wide bore. Connections downstream of the pump are usually made with stainless steel or PEEK (poly ether ether ketone) tubing. Connections between injector-column and column-detector should be made from narrow-bore tubing (0.010" i.d. or less) in order to minimize dead volume.

This part of the LC system must be carefully designed and constructed in order to minimize the volume of both the fittings and tubing. We say that we have to eliminate dead volume   as much as possible. So we need to keep the tubing length short, and we will usually use tubing of very narrow diameter: 0.010 inch internal diameter (or "ten thousandths") is the usual tubing size for this part of the system (smaller tubing may be required for use with "microbore" columns; larger tubing should only be used with preparative columns). If for any reason we have to replace the tubing or connections in this part of the LC system (between the injector and detector), BE VERY CAREFUL TO SELECT THE RIGHT TUBING SIZE AND THE RIGHT FITTINGS.

Let's talk next about the fittings used in LC. These are called compression fittings and their design is illustrated below.

A piece of tubing is first equipped with a NUT and FERRULE as shown at the right. To connect this end of the tubing with another fitting (the column end, a tee, connector, etc.) for the first time, just screw the nut into the fitting. Be careful not to overtighten the fitting! Usually the nut should be tightened finger-tight and then an additional 3/4 of a turn (270 degrees clockwise) with a wrench.

When reassembling an existing fitting (when changing columns, for example), the nut should be tightened finger-tight and then snugged slightly with a wrench to prevent leaks.

Standard compression fitting used in HPLC.

Unfortunately, not all fittings are interchangeable. This is a major headache for most LC workers, but we have to live with it for the time being. Usually mismatched fittings appear to work, and this makes the situation even worse. That is, the fittings will appear to go together in the usual way. However mismatched fittings will not work properly. The reason is that the main difference in these fittings is the distance between the tubing end and the ferrule, as shown at the right.


Note the difference in the distance from the bottom of the ferrule to the end of the tube in these three tube ends assembled with fittings from different manufacturers.

If this distance is too long for the body of the fitting, the ferrule will not seat properly, and the fitting will leak. If the distance is too short, the ferrule will seat, no leak will occur, but a "dead volume" will be created. This is most insidious, because then our separation will not be as good as it should be, but everything else will look OK. Dead volumes are like the cancer of HPLC - hidden, but a real "killer". Dead volume between the injector and the column permits dilution of the initial sample with mobile phase, causing band broadening. Dead volume between the column and the detector cell undoes the separation already accomplished on the column by allowing re-mixing of separated components.

The fittings for the ends of the column are similar to tubing fittings. A ferrule and nut on the column hold the column into the end fitting - just like tubing is connected together. Because the column is packed with small particles of column packing, it is necessary to have a way to hold these particles in the column. This is usually achieved with a FRIT - a small, coin-shaped piece of sintered metal having narrow pores. Most column frits are nominally 2-micron porosity, although smaller frits (0.5 micron) are used for columns of very small particles (3-micron diameter).


 If we disconnect the column end-fitting, we can see the frit on the top of the column. This frit can be removed and replaced with a new frit if the old frit becomes plugged. However if we do this, WE HAVE TO BE VERY CAREFUL. If the column packing at the inlet is disturbed, it could ruin the column permanently.



2000, LC Resources Inc. All rights reserved.
Last revised: April 06, 2001.