Leaks are a real problem for HPLC systems. It is easy enough to get a leak from a 60psi water main, so it is hardly surprising that at several thousand psi pressure, an HPLC eluent will find an easier way out if it can! Unlike a water main, the flow rate is very low (normally 1ml/min) so nobody is going to get wet, but there are three significant reasons why we really don't want a leak:

Safety. The eluents we use are often toxic (MeOH, MeCN etc). If it just drips out, it will evaporate and we will inhale it, and if it comes out as a fine jet, we may not even see it, and without safety glasses it could easily end up in someone’s eye.

Incorrect Peak Assignment. Peaks elute in a given volume not time. We can only use retention time if the flow is constant. A leak reduces the flow rate, thus increasing the retention times, and can cause a peak to drift out of its retention time window, leading to it being wrongly identified or missed altogether.

Incorrect Quantitation. There are two reasons for this. As a peak’s retention time changes, so does its area. (I had a 200mg/l standard, eluting at 32 minutes. Increasing the eluent strength to make it come out at 27 mins caused the area to change, so that its area was equivalent to 178mg/l). Secondly, if the leak is between the valve and the detector, sample is lost along with eluent, so the results will be low.

Identifying the causes of leaks allows us to adopt measure to prevent them, and hence make the HPLC system more reliable. So here’s a list.

PEEK Tubing.

Love it or hate it, PEEK tubing is here to stay, and there are two things it likes least: Tetrahydrofuran, and being made to go round corners!

If you use THF as part of your eluent, you need to use steel tubing. PEEK is slowly dissolved by THF, and eventually the wall becomes soft enough to allow a leak.

PEEK tubing is quite flexible, but it doesn’t like being bent. This can happen if you cut it too short, so that it is pulled tight at the end of the column and becomes kinked, or if you deliberately bend it to try to make a neat 90o bend for example. It can be done, but you must first heat the tubing with a heat gun till it becomes quite hot. Then bend it while still hot using a 90o edge as a form. If done when cold, the tubing is stretched on the outside of the bend or kink, and the wall becomes thinner, allowing a very fine (almost invisible) jet of eluent to pass through. This can be very dangerous, and safety glasses should always be worn.

Fittings.

Many leaks come from problems making connections. There are four main types of connections used in HPLC:

Stainless Steel Compression Fittings. Stainless steel fitting have the advantage of being strong and able to withstand very high pressures. However they cannot be re-used, without first cutting off and discarding the ferrule. The main reason is that almost all column end-fittings are different, and in particular, have a different ferrule lock distance (the distance from the end of the tubing that the ferrule seats). In spite of this, it is a great temptation to “try and see if it will go”, rather than to cut off the ferrule, clean up the end of the tube, open up the hole in the end, and make a new connection.

Making up a new fitting is always preferable. The ferrule not only locks onto the tubing, but also deforms to fit exactly the internal geometry of the column end-fitting. Not tight enough, and the ferrule will slip off. Too tight, and the ferrule cuts a ring in the tubing wall and will rotate if rolled between your fingers. The problem with re-using a nut and ferrule is that in tightening enough to deform the ferrule to make seal against the end-fitting,  it is almost invariably necessary to over-tighten with respect to the tubing, and hence it will not be long before this union starts to leak.

A couple of other points before we move on. If the ferrule lock distance on the tubing is too long for the new fitting it will buckle whilst being tightened, and may be very difficult indeed to get out again. If the ferrule lock distance is too short, it will introduce a void, causing peak-broadening. Secondly, before making a connection, check that the hole in the fitting is absolutely clear. If you are old enough to remember PTFE ferrules, you will remember the problems they caused when they got stuck inside compression screw fittings! And finally, watch the screw thread. It is possible to cross-thread a fitting. They are only little,  and when you do get a cross-thread, a reasonably strong person will get several turns on the screw before the nut shears off, and they are virtually impossible to drill out. The worst are the Rheodyne long nuts, which are very easy indeed to cross-thread (because of the length of the nut, and the restricted access to the back of a valve.)

If you need nuts, ferrules or tubing, you can find it here, and if you need a tubing cutter, you can find it here

PEEK Fingertight Compression Fittings.

Fingertight fittings have the advantages that they can be re-used, and that they don't require a spanner, and the disadvantage that they don't seal much over 4000psi. So if you are expecting high back pressure, best use steel fittings! There are two main types of PEEK fittings, one-piece and two-piece fittings.

One-piece fittings are the easiest to use and the cheapest to buy the first time around. However, because the ferrule part rotates as you tighten the nut, they wear out much faster, and leak at lower back pressures than the two-piece variety. Two-piece fittings have a separate ferrule, and this does not rotate as you tighten the nut. It also means that you only need to replace the ferrule part when they leak.

Both types need to be done up quite tight if they are not to leak. Also be extremely careful to ensure that the tubing does not slip back through the ferrule as you are tightening it. Once the screw thread or the ferrule gets worn, these MUST be replaced, or a leak will result very soon afterwards.

A word of caution before we move on. If you have a pulse-dampener in the system (sometimes built into the pump) the one thing they really really don’t like is the pressure running up very high and then reducing almost instantly to zero. This is exactly what happens if you use finger tight fittings and they slip off.  So if you do have a pulse dampener in the system, what can you do to protect it?

• Watch the back-pressure, and if it goes much over 4000 psi, find the problem and sort it out
• Set the maximum pressure cut-out to 4000 psi (300Bar)
• Check the pulse dampener membrane if the back pressure drops suddenly. Call me if this happens  and I’ll talk you through it.

If you need new finger-tight fittings you can find them here

Flangeless connections. These are the fittings often used to connect the 1/8” PTFE tubing from the solvent reservoir to the pump. They are similar to the flanged fittings below, but the nuts are different and they require a ferrule, fitted with the flat end towards the end of the tubing. There is a coned shape inside the nut, which compresses the thin end of the ferrule to grip the tubing. They often need to be done up quite tight, and this can cause the ferrule to deform so badly that it cannot be re-used. Because they are used mainly before the pump, a leak can cause air to be sucked in rather than solvent to leak out. You can stop that by mounting the solvent reservoir(s) on top of the pump, so that a slight positive pressure is applied due to gravity and creating a siphon action, thus ensuring that no air can be sucked in. Be very careful if the outer diameter of the tubing is too narrow (the ferrule will be very loose) because then it will be impossible to make a good seal. To buy the 1/8” tubing or replacement fittings, please call us.

Flanged connections. A flanged fitting is made using a tool to flare the end of the tubing outwards. A washer is mounted behind the flange, and the nut presses the washer against the flange, making a seal in the fitting. You need a different nut from the flangeless nuts (it must have a flat end), and we can supply a tool so that you can make the flanged ends yourself. Leaks here usually come from old fittings, or from using the wrong kind of nut, or forgetting the washer!

If you do not feel confident with making HPLC connections, this is covered on our Introductory Course, our Advanced Course, and our Servicing Course, where you will get to make these fittings yourself, and practice with each type of tubing cutter. Should you need help in the meantime, please call us.

Leaks from HPLC Pumps

From the pump to the column, the system is under its highest back pressure, and hence there is the greatest likelihood of a leak manifesting itself. The lower the flow rate, the harder it will be to see, because solvent maybe evaporating as fast as it is leaking out. However the reduction in flowrate thus caused will give rise to longer retention time than normal, and the system back-pressure will be lower than normal. If a buffer salt or acid such as phosphoric acid is included in the eluent, a crusty white deposit will also form as the solvent evaporates, giving a clue as to what is happening.

Perhaps the most obvious source of leaks in a pump is the piston seal. This is a spring-loaded seal which prevents the eluent leaking back into the pump. You have two options regarding maintenance – Prevention: change them every 6 months or so to make sure they never wear out; or Cure: change them when they start to leak! The second option is much cheaper - you won’t get screwed for the price of the seals, you can do it yourself in a couple of minutes, and the seals may last several years if you are nice to them. The downside is that if you use an autosampler, they may let go one night while you are at home watching TV! Instructions on how to change a piston seal are here.

A few notes before we move on.

• Some pumps have a different size secondary piston, and so you may need to keep two sizes of piston seal.
• Pumps with piston back-wash will require backwash seals as well. These are not high pressure seals and hence are less expensive. Be careful not to confuse backwash seals with the seals for a secondary piston (call me or email if you are not sure what you need – 01634 294001 or stuart@laserchrom.co.uk) Some pumps (eg Tosoh Haas, early Kontron) need a tool to compress the leading edge of the seal as it goes in. If you need this, you need it! Nothing you can say or do will make it go in without the tool!
• There is usually a ring behind the seal (it comes out before you get to the seal). This acts as a piston guide to prevent endfloat (ie it allows the piston to go backwards and forwards but not side to side). If this is missing, the seal can slip around, and will wear out quickly.
• Make sure that you use the right seals. Yellow seals may last much longer in highly aqueous eluents. Otherwise use black seals.

Another source of leaks is the purge valve. Many pumps use a cone-shaped piece of Teflon with an O-ring to make the final seal. Closing the valve involves screwing the cone into a seat, so if it leaks either the O-ring has worn out, or the Teflon cone is damaged. The symptoms of a leaking check valve are drips from the waste outlet from the valve when it is closed, and maybe a slightly lower back pressure. Repair/replacement instructions will be in the pump manual, but if you unscrew the valve and keep unscrewing it will usually come out just like that. If you get stuck, call me!

Some pumps have extra filters and one-way valves in the flow path. If these block you will get cavitation (air being sucked out of solution because of the extra reduced pressure as the pump tries to suck in the eluent through the blocked filter). One pump which has several ‘extras’ in the flow path is the Waters M600. Another source of leaks can be fitting certain aftermarket check valve cartridges. They don’t fit the existing housing, and a plastic spacer is used. If this cracks (even a hairline crack) you will get a noticeable leak!

Leaks from the column.

Leaks from the column are relatively easy, because it can only be the compression fittings from the connecting tubing, or the end-fitting itself. We covered the connecting fittings earlier, and the column end-fittings usually operate in essentially the same way. If you have to change a frit, be careful not to over-tighten the fitting when reassembling the column, and be careful not to get any silica powder on the column ferrule surface or it may not seal very well. If you do over-tighten the column end-fitting and it leaks, use one or two turns of PTFE tape (available from B&Q for example!) around the ferrule and try again. Note that if the column is in a column oven, the only clues to a leak will be the back pressure being low, retention times being longer, and sample integration being low ( a leak after the valve loses sample as well as eluent). So to prevent leaks, be careful with finger-tight fittings – do them up quite tight, and don’t let the tube slip back through the ferrule – and if you have to change a frit, keep the column ferrule clean of silica.

Leaks from the Autosampler or injection valve.

Consider the valve first. Almost all autosamplers will use a valve for injection, so the only difference from a manual valve is who (or what!) turns the valve. The only service part that needs replacing is the rotor seal. It wears out with use, so needs replacement after a given number of injections. To access it, you will need to access the rear of the valve (where the connection ports are). 95% of valves will be made by either Rheodyne or Valco. To obtain a replacement rotor seal you can call us, and we will need to know at least the model number of your autosampler, and possibly the model of the valve. This can be found on a sticker on the valve body.

A valve is rated to a specified pressure, normally 5-7,000 psi. From the factory they are usually set to 5,000 psi. To achieve the higher pressures, the tensioning screw(s) need to be tightened. This increases the pressure between the stator and rotor seal in the valve, giving higher pressure resistance, but causing faster wear. In order to use the new high pressure systems operating at over 6000psi, you will need a different valve or autosampler. If in doubt, call us.

Other high pressure leaks can usually be traced to one of the connection fittings. Its can be difficult to keep a ferrule on the connecting tubing when screwing down into a valve port so errors can be made here. Always use a steel nut in steel valve body, and always use a PEEK nut in a PEEK valve body. If you don’t have the appropriate nuts and ferrules for your valve, call us or check here

A couple of other autosampler problems to watch for.

• Needle depth set too high can cause air to be sucked into the syringe unless you use lots of sample. (Needle depth too low and you get a hole in the bottom of each vial!)
• Not topping up the wash solvent reservoir can result in air being sucked in.

Leaks from the detector.

The only likely source of a leak is the flow cell window. In a UV detector, the cell windows can withstand 300-1500 psi depending on the manufacturer. But an RI cell will crack at very low back-pressures, usually under 100psi. So if connecting detectors in series, always connect an RI last. If the cell does crack, there will be horrendous noise (on the baseline, not a splintering crash!) and a puddle will form under the cell. Some detector cases will hold about 20 ml before you see the puddle, so keep an eye on the baseline.

Prevention here is limited to keeping the outlet pipe free from blockage, so that solid samples or buffer cannot dry in the waste line and block it. If the cell breaks, we may be able to help you repair it, but otherwise it means a new cell (cost £500-2000!)