Laserchrom HPLC Laboratories Ltd Technical Bulletin Autosampler problems! Air in the syringe Bent or blocked needle Valve problems Unreproducible injections Sample carryover Wash solution problems Vial, cap and septum problems Run time errors X-Y calibration Units B16-18, Laser Quay, Medway City Estate, Rochester, Kent. ME2 4HU www.laserchrom.co.uk 01634-294001
Introduction An autosampler has more moving parts than the rest of the system put together. Hence it is hardly surprising that it is the source of a number of problems. They tend to fall into three categories; those resulting from lack of care or maintenance, those resulting from human error, and normal wear and tear. Here s a list of the common problems and some suggestions for how to avoid them, and how to fix them once they occur! Air in the syringe The syringe is used to draw sample from the vials, and to pump the wash solution. So we already know where the air must have come from! If the sample level is too low for the amount required for the injection(s) that have been programmed, air will be drawn in once the level goes below the tip of the needle. This can also occur if the needle depth is set too high. Air will also be drawn in if the wash solution runs out, or if it is not degassed prior to use. Run a few wash cycles, and the air should be eliminated automatically into the autosampler waste bottle. Bent Needle A needle has to take considerable force along its axis, as it is forced into through the septum into a vial. Hence in time it becomes weakened, and will eventually bend under the pressure. A clue to this beginning to happen is that the hole in the vial caps starts to move away from the centre. As soon as this is observed, the needle should be checked for straightness, and if necessary, replaced. Needles for autosamplers are inordinately expensive, so the cost must be weighed against the cost of an overnight run being wasted if the needle lets go at 2am! Try to resist the temptation to straighten a bent needle. It sometimes works, but like a car which has been straightened after a crash, the next time it will bend really easily. The other reason for finding a bent needle is that the X-Y calibration is out. As a consequence, the vial positions are not quite where the autosampler thinks they are, and the needle hits the cap instead of the septum. X-Y calibration does go out slowly with time, and is one of the things that should be checked when an autosampler is serviced. If the holes in the vial caps are drifting away from the centre but the needle is straight, this is almost certainly the problem. This is a job only for a service engineer or a very competent user. Replacing a needle is relatively straightforward, although sometimes it is necessary to replace some tubing with the needle (eg Agilent 1050 etc) If there is a needle guide, ensure that this is straight when the new needle is fitted. Autosamplers tend to use reasonable force with the needle, so if the needle is bent, the needle guide could be out of position too. Reconnect the tubing to the needle, and bear in mind that sometimes this forms part of the flow path after injection, so the connection to the needle may need to be tight.
Blocked Needle A needle has a fairly fine bore, and can easily be blocked. The blockage normally comes from a piece of rubber septum which has broken off as the needle passes through. This is known as coring. There are two things which can be done to prevent or minimise this. The first is to use a PTFE septum. Essentially this is just a plastic disc, which splits as the needle passes through. The only drawback is that if the run time is long, or the sample solvent is volatile, the septum does not re-seal, and so evaporation can occur, giving a higher concentration for the duplicate injection. An alternative is to use a rubber septum which has a layer of PTFE on one or both sides. There are two other causes of a blocked needle. One is particles in the sample. This is clearly not an acceptable situation for lots of reasons, but assuming that the sample was correctly made up with no undissolved material, particles can arise because of precipitation when the sample is chilled in the autosampler tray, or because the sample is not sufficiently soluble in the eluent. In this latter case, precipitation occurs at the moment of injection, and subsequent samples are then not drawn up because of the blockage. The second cause is particles in the wash solution. The wash solution should ideally be the same as the eluent at the point of injection, but without the buffer. This ensures that it is totally miscible, does not disturb the dynamic equilibrium in an isocratic system, and has no content which can dry in the needle causing a blockage. Valve problems The injection valve is never made by the autosampler manufacturer, but sourced from a valve specialist such as Rheodyne or Valco. The valve manufacturer supplies the valve, its actuator (the motor which turns it), a control box, and a cable between the control box and the actuator. So if there is a problem and the valve won t turn, assuming the autosampler is sending the control box the signal, the problem must lie with one of these components, and generally it is not a user-serviceable problem. However it is worth removing the valve from the actuator and trying again. If the actuator turns, the problem lies with the valve, which is usually quite easy to remove and replace without paying 100 an hour for a service engineer to queue round the M25! What causes a valve to jam? Either leaving it unused for ages so it dries out, or not flushing it out regularly. If a sample dries to leave a sticky material, then this can easily dry in a valve overnight and cause it to stick. It tends to build up, until the motor just can t make it turn. The danger is that by then the motor itself can burn out, or pull so much current that the cable fails. If it all goes, it will be expensive (possibly 2K) to fix. The secret is to keep the valve flushed out, not to use buffer in the wash solution, and to be very careful when using an autosampler for the first time after a period of prolonged disuse. If in doubt, please call us on the number below for free advice.
Needle Depth too high or too low If the needle depth is set too high, the last part of the sample cannot be drawn up and the needle may draw in air. If the needle depth is set too low, it drives a hole through the bottom of the vials. It is necessary that the needle depth should be adjustable to accommodate different types of vials. So care must be taken when using a new type of vial to establish an appropriate needle depth. This is usually done using the autosampler in service mode, and driving the needle down one step at a time until the vial moves, indicating that the needle has made contact with the bottom. The depth used should be several steps higher than this to allow for variations in the vial base thickness. Wash solution a stronger eluent than the mobile phase In a fixed volume autosampler, at least three times the loop volume of sample is injected, flushing the loop and making sure it contains exactly one loop volume of sample. Fixed volume autosamplers therefore use more sample than variable volume models and are less flexible, but they give much more reproducible injections and this problem does not apply to fixed volume autosamplers. A Variable volume autosampler uses a much larger loop, and meters in a small amount of sample with the syringe. The rest of the loop is filled with wash solution. So if the wash solution is a strong eluent, the same problem occurs as does if the sample solvent is a strong eluent, only much more so, because there is much more wash solution present than sample solvent! The problem is that the peaks become very broad, because the strong eluent disperses the sample into the column before it is itself washed away. The solution is that the wash solution must be no stronger than the eluent, and preferably the eluent itself only without any buffer. If running a gradient, the wash solution should be the eluent at the point of injection. Other wash solution issues 1. The wash solution must not be allowed to run out. Its obvious, but especially if there is a wash cycle after each injection, quite a large volume of wash solvent will be required. 2. The wash solvent must be degassed. Otherwise air can be sucked up, which may be in the syringe for a few injections before finally clearing itself. Hence the injection volume will be suspect. 3. Waste bottle for the wash solution must have enough capacity for all the wash solution used.
Sample carryover Sample carryover occurs if a trace of the previous sample remains on or in the needle when it is inserted into the next sample vial.. Some autosamplers minimise this by leaving the needle in the flow path (making it a part of the loop) so that in the inject position, the needle is flushed with eluent throughout the run. Others have various wash cycles which can be run before or after each sample or each injection. To test for sample carryover, it is necessary to follow an injection with an injection from a vial of clean solvent. Extreme care must therefore be taken to ensure that the vial and solvent is clean! If doing a gradient run, ghost peaks may appear which are nothing to do with the sample, and these will appear if a blank gradient run is performed without making any injection at all. Carryover must also involve peaks which were present in the previous injection. If the current wash routine is proving inadequate to prevent carryover, check the procedure. You can change the wash solvent, perform two or perform more procedures between each run. Also watch out for contamination arising from sample on the outside of the needle. Some autosamplers have the option of washing the needle in a wash port, and then airdrying the outside of the needle. The Sykam model does this and we would be pleased to advise if required. Program Errors - Runtime and Vial Range errors You have to put your hand up for these! If the autosampler is sending the start signals and therefore effectively controlling the system, it must have a longer run time than you give the data system or the gradient controller etc. Otherwise it will make an injection before other devices are ready, and if they are not looking for a start signal, the sample will be injected but nothing will take any notice. Vial 1 will be processed correctly, vial 2 will be injected but ignored, vial 3 will be injected but processed as vial 2 etc. If the system is controlled totally from a PC, which looks for a ready signal from all devices before giving the green light for an injection, then this does not apply. But otherwise, allow a longer run time on the autosampler. Remember for a gradient run, the gradient time will be much longer than the data system run time, because it must allow for returning to the starting conditions and re-equilibrating. The autosampler must wait till this is complete. Injection before the system is re-equilibrated will give variable retention times, and hence dodgy peak identification and quantitation. Vial range errors arise if the samples are not placed in the vial range which is programmed into the autosampler. Its an easy mistake, but it fools the autosampler. If an autosampler reaches a blank space with no vial it will usually stop and report an error. If the cap is not fiited (I know its crazy, but people do!) sometimes the autosampler will notice this and stop anyway, reporting a vial error.
Unreproducible injections If we are asking an autosampler to inject the same sample several times to check reproducibility, we would expect that an automated machine which just runs programs should be able to do this fairly easily. However sometimes this is not the case. Before blaming the autosampler, do check that its not an air bubble in the pump, or integration problems. But assuming the problem lies within the autosampler, here s a few places to look: 1. Not enough sample. The needle barely dips into the sample, so air is drawn in. 2. Needle depth too high. Same as above. 3. Blocked needle. It causes a resistance to flow, and varying amounts of sample get in each time. 4. Sample too viscous, or syringe speed too fast for the sample viscosity. The syringe generates a vacuum and the sample follows more slowly. Slow down the syringe speed, or use a less viscous sample solvent. 5. Worn syringe. If the syringe cannot suck hard enough, and resealing vial septa are being used, there is a reduction in pressure in the vial after each injection, making it progressively harder for the autosampler to withdraw sample. Replace the syringe, or use PTFE septa. 6. Air in the syringe. It can only meter sample accurately in the total absence of air. See the earlier section on dealing with air in the syringe. 7. Wash routine. Sometimes it is found that the first injection gives a lower result than subsequent injections, and the reason is that for the first injection, the syringe starts wet with wash solution, but for subsequent injections it is wet with sample. If this is a problem, the solution is to wash before each injection, not just before each sample. 8. Sample solvent evaporating. This occurs if the sample solvent is volatile, and PTFE septa are used (they do not reseal) allowing solvent evaporation between injections. Hence the concentration of sample is gradually increasing, so the results will increase with each injection (unlike the previous case where only the first injection is low). 9. Sample not down to temperature. If working at (eg) 4 o C, the samples must be allowed to reach temperature before the run is started. Reducing temperature reduces the volume, and hence increases the apparent concentration in a 20ul injection. It can take at least 30 minutes for sample temperature to stabilise. 10. No air gap left between sample and eluent. When a sample is withdrawn from the vial, it is kept separate from the eluent by a minute (5ul) air gap. Failure to do this allows it to diffuse into the eluent that is pushing it around the autosampler, and this causes a variation in peak height from injection to injection.
Vial, cap and septum problems It is important to choose appropriate vials, caps and septa for your autosampler, and for your analytical method. Here s a few check points: 1. Vial Size. Most autosampler use the standard 1.8ml vials. However these come in several flavours. They can be clear or amber, with or without a label, straight sided or with a narrow tip to enable the last drop to be removed, with screw top, snap on top or crimp top etc. Other size vials also offer similar options. The vial size must be big enough to contain enough sample for the injection volume x the number of injections and a little to spare, but also be small enough to allow the needle to withdraw the required amount. If the vial is too big, evaporation causes changes in concentration, and there is a real risk that the needle will suck air. Amber vials are for lightsensitive samples. The label makes sample identification easier, and the top style is to suit you. Crimping is quicker if you have a lot of vials, but the tool costs well over 100! 2. Septa. These can be made of PTFE, rubber or silicone, and the latter two options can come with a layer of PTFE on one or both sides. PTFE is cheaper, but does not reseal after piercing. Hence evaporation can occur between duplicate injections if a volatile sample solvent is used. Be careful to use only one PTFE disc! They are punched out several hundred layers thick and tend to stick together. If you use two at a time, the resistance might be enough to bend a tired needle. Silicone is fine for aqueous samples, but rubber is most commonly used. It reseals, but can leave a slug of rubber in the needle (known as coring). If this happens it can sometimes be cleared, but spoils the run. To prevent this, use a septum with a layer of PTFE on top. Rubber is also soluble in some organic solvents. In this situation it is recommended to use a layer of PTFE on the bottom surface of the septum. Both problems at once? Us a rubber septum with PTFE on both sides! 3. Caps. Caps must clearly be appropriate for the vials. Note that not all screw top vials use the same screw thread, and sometimes the screw will not stay tight. Mixing and matching vials and caps may work, but may not so try a sample before buying. 4. Quality. As with everything this century, there are ultra-cheap versions arriving every day. There are deals to be had, but be careful to test thoroughly before switching. The worst problems are vials which break as you tighten the cap, and vials which have a different thickness of base from batch to batch! The cost saving on the vials might be needed to replace the needle!