Introduction to Modern Liquid Chromatography, Third Edition part 88 ppsx

826 TROUBLESHOOTING f b s needle n c Figure 17.2 Schematic of low-pressure needle-seal. Valve body (b), nut (n), ferrule (f), poly- meric sleeve/seal (s) and distorted portion of sleeve (crimp) that forms a seal around the nee- dle (c). Failure of the injection rotor-seal can result in leaks at the injection valve. The standard 6-port injection valve uses a rotating seal, the rotor, which turns against a stationary stator, to which the tube connections are made. The rotor typically is made of a hard polymer, such as PEEK, Vespel, or one of the fluoropolymers, and contains tiny passages that connect the fluid passages in the stator. The stator may consist of the stainless-steel valve body, or it may be an insert in the valve made of ceramic or another hard, smooth surface. In the schematic of Figure 3.17, the rotor comprises the portion of the valve inside the circle, whereas the stator is the part immediately outside the circle. In the most popular injection valves, the rotor is a flat disk with three kidney-shaped grooves in the surface, similar to that shown in the sketch of Figure 17.3a. These grooves line up with the ends of the tube connections on the stator, shown as the dashed circles in Figure 17.3b. In one position (e.g., load), the flow channels that result connect ports 1–2, 3–4, and 5–6, as seen in Figure 17.3b. When the rotor is rotated 60 ◦ (e.g., to the inject position), a different set of ports is connected, in this example, 2–3, 4–5, and 6–1. If a small piece of hard material, such as a particle of column-packing or a bit of stainless steel from a poorly cut tube, gets caught in one of the passages of the injector, it can scratch the rotor. This can form a connecting passage between two of the grooves, as shown between ports 4 and 5 in Figure 17.3c (arrow). The result is cross-port leakage, where fluid from one hydraulic portion of the system leaks into another. This can show up as fluid leaking out the injection or waste port, as a problem of precision due to liquid leaking into or out of the sample loop, or sometimes as a carryover problem (Section 17.2.5.10). Rotor-seal (and possibly stator) replacement will be required to correct this problem. Also be sure to clean the remainder of the valve thoroughly to remove any particulate matter. More commonly the rotor seal will fail as a result of normal frictional wear. The rotor seals are designed with a service lifetime of > 100,000 cycles. For many laboratories, this will mean several years of operation before failure, so routine replacement of the rotor seal does not make much sense—unless the system has a 17.4 COMMON SYMPTOMS OF HPLC PROBLEMS 827 1 3 4 5 6 2 (a) (c) (d )(e) (b) Figure 17.3 Schematic of injector rotor-seal. (a) View showing kidney-shaped grooves in sur- face of polymeric seal; (b)asin(a), showing connecting ports (numbered, dashed circles) for tubing; (c) scratch between ports 5 and 6 of b, causing cross-port leakage. (d) Cross section of kidney-shaped groove (normal condition); (e) worn seal resulting in burr of seal material at edge of groove (arrow). counter to automatically record the number of injection cycles, so as to allow the remaining rotor seal life to be estimated. When viewed in cross section, the grooves of the rotor shown in Figure 17.3a are U-shaped as in Figure 17.3d. As the surface is worn during normal operation, the rotor will become slightly thinner and often a small burr of rotor material will form at the open edges of the groove, as seen at the arrow in Figure 17.3e. These burrs can break off and shed particles that can block tubing or frits. As the surface or the rotor wears, the contact pressure between the rotor and the stator is reduced, and eventually the seal will leak. Replacement of the rotor should solve this problem; thoroughly clean the valve before reassembly. The pressure limit of the seal between the valve rotor and stator is related to how tightly the two surfaces are held together. Higher pressure-limits require that the surfaces be held together more tightly, but this also increases the friction between the surfaces and the effort to rotate the valve. Normally the injection valves are adjusted to withstand 6000 psi (400 bar) for traditional HPLC applications. For U-HPLC use ( > 6000 psi, Section 3.5.4.3) the two surfaces must be held together more tightly, so rotor lifetimes are expected to be shorter; alternative injector designs may overcome this problem. When an injection valve is disassembled for servicing, 828 TROUBLESHOOTING the rotor-to-stator sealing pressure may need to be adjusted; consult the service manual for additional instructions. A major exception to the general use of rotary injection valves is found in some Waters-brand autosamplers, which typically use a ‘‘seal-pack’’ design that incorporates slider valves and high-pressure seals instead of rotary valves. Some of these parts are user-serviceable, and some require replacement of a subassembly with a new or rebuilt unit. Consult the user manual for more information on troubleshooting and repair. Other points of leakage in autosamplers will vary from one design to another and often are unique to one model. If tightening or replacing a connecting fitting does not correct the problem, consult the autosampler manual for more information. 17.4.1.5 Column Leaks Leaks at the column will be associated with the fittings. Leaks at the tube connections are treated as outlined in Section 17.4.1.3. If the column end-fitting itself is leaking, it may be possible to stop the leak by tightening the nut 1/4-turn. These larger fittings will take more effort to tighten than fittings for the 1/16-in. o.d. connecting tubing. If the fitting continues to leak, it may be best to discard the column, because disassembly of the column end-fitting can result in permanent damage to the column. In the past, it was common to remove the column-inlet fitting to replace the frit if a blockage was suspected, but with today’s column-packing techniques, removing the end-fitting may allow column packing to ooze out and permanently damage the column. For this reason removal of the end-fitting for examination or repair is no longer recommended. Cartridge-type columns comprise a disposable column that is held in a reusable holder. If the end-fitting on a cartridge column leaks, try tightening it to correct the problem. If it still leaks, disassemble the holder, rinse the fitting, and reassemble. In some cases the polymeric seal between the column and the holder may need to be replaced to stop a leak. 17.4.1.6 Detector Leaks The pressure at the column outlet is lower than at the inlet by an order-of-magnitude or more, so detectors are subject to much lower pressures than the preceding high-pressure components. Because leak-related problems correlate strongly with the local system pressure, leaks at the detector are much less common than in other parts of the HPLC system. Fittings at the detector inlet usually are the same type of high-pressure com- pression fittings used in other high-pressure parts of the system; leaks at these tubing connections should be treated as described in Section 17.4.1.3. Some detectors use 1/32-in. o.d. tubing instead of the standard 1/16-in. tubing used elsewhere in the system. This smaller diameter tubing is easily twisted and kinked, so take extra care when working with it. Some detectors operate at sufficiently low pressure on the inlet side that low-pressure plastic fittings can be used; many detectors use low-pressure fittings on the detector outlet because the pressure is quite low. Correction of leaks in low-pressure fittings is described in Section 17.4.1.1. UV detectors (Section 4.4) are the most popular HPLC detectors. A generic version of the detector cell is shown in Figure 17.4. The cell typically comprises a 17.4 COMMON SYMPTOMS OF HPLC PROBLEMS 829 end cap quartz window gasket inlet outlet cell body mobile phase light in light out 10 mm Figure 17.4 Schematic of typical UV-detector flow cell. stainless-steel block (e.g., 10-mm long) with a hole (e.g., 1-mm diameter) drilled through it; quartz windows are held on the ends by an end-cap with O-ring seals. The tube connections to the cell body at the inlet and outlet may be compression-type fittings, which can be adjusted in the normal way (Section 17.4.1.3) if leaks occur. In other cases the tubing is soldered or welded to the flow-cell body, in which case factory repair or replacement will be required. The inlet tubing connected to the cell usually is thin-walled, narrow-diameter tubing (e.g., 1/32-in. o.d.) that also functions as a heat-exchanger to stabilize the temperature in the flow cell. Be careful not to twist or kink this thin tubing when tightening fittings. Because air bubbles create noise in the cell, the UV detector often is operated with an after-market back-pressure restrictor on the cell outlet (Section 4.2.1). This creates enough pressure (e.g., 50–100 psi) to keep the bubbles in solution, but not so much pressure as to cause the window seals to leak. Spring-loaded back-pressure restrictors work well to accomplish this. An alternative is to use a narrow-bore waste tube (e.g., ≤0.010-in. i.d. =≤0.25 mm i.d.), but as the flow rate is increased, the pressure also is increased. With this type of restrictor, a high flow rate may create sufficient back-pressure to exceed the upper pressure-limit of the detector cell (e.g., 150 psi), causing leakage at the window seals. Once a detector cell leaks, it may continue to leak if the window seals become distorted. Some detector cells are user-serviceable, whereas others will require service by a factory-trained technician, or complete replacement—consult the detector manual for more details on your model. Other types of detectors have different flow-path and detector-cell designs. For any flow-through detector (fluorescence, electrochemical, conductivity, etc.), leaks can occur at the detector flow cell. Evaporative detectors, such as the evaporative light scattering, corona discharge, or mass spectrometric detector will have fluid leaks only at the interface, so leak isolation may be a little easier. Consult the appropriate section of Chapter 4 for a general discussion and generic design of 830 TROUBLESHOOTING specific detectors. This information, plus details from the detector manual, will help you locate and correct leaks in specific detector models. 17.4.2 Abnormal Pressure Abnormal pressure is always a symptom of some other problem in the HPLC system. Normal system pressure will be different for different HPLC systems and applications, so it is a good idea to record the system pressure on a regular basis. For example, if you use a batch record or run sheet, record the pressure as part of the system suitability records; then you will be able to compare a questionable high- or low-pressure symptom to the historical record (Section 17.2.4). Conventional HPLC systems are capable of operation up to 6000 psi (400 bar), but most applications operate at pressures of 1500 to 3000 psi (100–200 bar). U-HPLC systems are designed to operate at pressures > 6000 psi—as much as 15,000 psi (1000 bar) or more. However, as of this writing, these systems typically are operated in the 8000 to 10,000 psi (550–650 bar) range. Ideally the pressure will be constant during an isocratic separation; however, small pressure fluctuations in the range of 1–2% of the operating pressure (e.g., 10–20 psi, 1–2 bar) are normal for many applications. Pressure also will vary with mobile-phase composition, as illustrated in Table 17.1, so pressure changes during gradient elution are normal. Methanol (MeOH) is more viscous than acetonitrile (ACN), and blends of MeOH and water are considerably more viscous than either MeOH or water alone. On the other hand, the pressure generated by ACN/water mixtures decreases in a fairly linear fashion as the mixture is changed from 100% water to 80% ACN. More detailed information on solvent viscosity can be found in Table I.3 of Appendix I. Table 17.1 Example Pressures for Various Solvent Blends B-Solvent a % B-Solvent a Acetonitrile Methanol 0 1650 psi b 1650 psi b 20 1840 2520 40 1700 3265 60 1390 2920 80 1010 2150 100 710 1080 a A-solvent is water. b Approximate (calculated) pressure for 250 × 4.6-mm, 5-μm particle column operated at 35 ◦ Cand 2mL/min(Eq.2.13a). Most HPLC pumps have pressure-limit settings that serve to shut off the pump if the limits are exceeded. The upper pressure-limit protects the system against damage or leaks if a blockage occurs causing excessive pressure. Most workers will set the upper limit well above the normal maximum operating pressure for 17.4 COMMON SYMPTOMS OF HPLC PROBLEMS 831 all methods. For example, if normal method pressures are 2000 to 3000 psi, the upper limit might be set in the 4000 to 5000 psi region. This will provide some protection, yet is not low enough to result in frequent pump shutoff. The lower pressure-limit shuts the pump off if the pressure drops below the set point, such as when a mobile-phase reservoir runs dry. There is not much danger of pump damage if the solvent supply is exhausted, and the pump will not pump air, so the column will not dry out. Nevertheless, it is a good idea to set a lower pressure-limit in the 20 to 50 psi region so that the pump stops if the solvent supply is interrupted. Pressure problems are discussed below and summarized in Table 17.4. 17.4.2.1 Pressure Too High Higher-than-normal pressure is a symptom of a blockage (assuming that the system settings are correct). Most commonly the pressure will rise gradually with successive batches of samples as debris collects on the in-line filter, guard column, or frit at the head of the column. This is normal with methods for the analysis of samples that may not be completely free of particulate matter. Remember, the pressure can increase by as much as 60% over the starting pressure during a gradient (Section 17.4.2, Table 17.1), so set the upper pressure-limit to accommodate this normal pressure fluctuation. It is also important to remember that column pressure is related to particle size, column length, and column diameter (Eq. 2.13a). Nominally equivalent columns can also differ in their pressure drop (e.g., two different brands of 150 × 4.6-mm i.d., 5-μmparticleC 18 columns). Finally, HPLC systems designed for higher pressure operation (e.g., U-HPLC) often have very small orifices and reduced tubing diameters (e.g., ≤0.005-in. or ≤0.12-mm i.d.), resulting in > 1000 psi of system pressure without a column installed, so allowances for normal background pressure must be taken into account during troubleshooting. Occasionally a sudden pressure increase will occur, and likely trigger the upper pressure-limit with a resulting pump shutdown. This can happen upon injection of a very dirty sample, such as untreated plasma, if sufficient particulate matter is present to completely block a frit or a piece of connecting tubing. Blockage can also take place when buffer and organic solvent are mixed on-line under conditions where the buffer solubility is poor and precipitation occurs, such as blending phosphate buffer and acetonitrile in some HPLC equipment. Problem isolation for excessive-pressure problems is quite simple. Just work your way upstream from the column outlet, loosening the tubing connections as you go, with the pump operating. (If you strongly suspect a blockage at a particular point in the system, such as the in-line filter, start there and save time.) When a fitting is loosened and a sudden pressure drop results, the blockage is immediately downstream from that fitting. Remember, there normally is > 1000 psi pressure drop across the column (depending on the flow rate), so much of the pressure drop observed when loosening the column-inlet connection is normal. With conventional HPLC systems designed for operation ≤6000 psi, once the column is removed, the pressure should be very low (e.g., ≤100 psi). As noted above, systems designed for higher pressures (Section 3.5.4.3) and sub−3-μm particles may have a system pressure of 1000 psi or more with no column attached. For reference purposes, it may be useful to go through the blockage isolation procedure with a normally operating system, to see what the normal system pressure is at each connection (for some fixed flow rate). 832 TROUBLESHOOTING Once the problem location is isolated, appropriate corrective action should be taken. For example, if a piece of tubing is blocked, the tubing should be replaced. If the in-line filter is blocked, replace its frit. If the column is blocked, column reversal may help (the procedure is included in the discussion of split and distorted peaks in Section 17.4.5.3); otherwise, it may be necessary to replace the column. Before putting the system back into service, consider if steps need to be taken to prevent the problem from recurring—or reduce its impact the next time. For example, if the problem is frit blockage from sample particulates, you may want to institute a filtration or centrifugation step during sample preparation, and use an in-line filter (Section 3.4.2.3). If buffer precipitation blocked a tube in the pump or mixer, consider reducing the buffer concentration or pre-mixing the buffer and organic solvent. 17.4.2.2 Pressure Too Low System pressure that is too low is a sign of a leak or a pump problem (assuming all the system settings are correct). If the pressure is cycling, the problem is more likely at the pump (see the further discussion in Section 17.4.2.3), whereas if the pressure is steady and low, a leak is more likely. Because low-pressure and cycling-pressure problems are closely related and sometimes hard to tell apart, consult Section 17.4.2.3 for additional information. If the pump has shut off due to the lower pressure-limit, and the cause is not obvious (e.g., an empty mobile-phase reservoir), restart the system and see what happens. Sometimes the lower pressure-limit sensor is too sensitive and will stop the pump if a momentary drop in pressure occurs, such as with the passage of a bubble through the pump. If this is a regular occurrence, it may be best to disable the lower pressure-limit. Mobile-phase leaks can also cause low pressure; identify and correct the source of the leak using the instructions of Section 17.4.1 and Table 17.3. Although mobile-phase leaks are the most common type of leak, it is possible for air to leak into the system through a loose low-pressure fitting (Section 17.4.1.1). If efforts to find a liquid leak are fruitless, tighten each of the low-pressure fittings to see if this corrects the problem. If a proportioning valve is not sealed properly, it may be possible to pull air into the system through an unused solvent supply tube. A sticking inlet check valve can prevent the pump from building sufficient pressure. Check-valve sticking is particularly problematic with ball-type check valves (Fig. 3.12a) when used with acetonitrile (ACN). When ACN is used as a solvent, the machined surface of the sapphire valve seat can catalyze the polymerization of minor contaminants in the ACN (aliphatic amines) [15, 16]. This polymer then results in a smoothing of the contact surface, so the ruby ball sticks to the seat via increased surface tension. Sonicating the check valve in methanol seems to correct this problem, at least temporarily. There is also speculation that sonication in dilute nitric acid might serve to remove this polymeric buildup [17], but this had not been confirmed at the time this book was completed. HPLC pumps that use active check valves (Fig. 3.12b,c) are not subject to this sticking problem; unfortunately, pumps with ball-type check valves cannot be retrofitted with active check valves. A pump that is starved for mobile phase will not be able to generate the expected pressure. Check that sufficient mobile phase is available at the pump, by 17.4 COMMON SYMPTOMS OF HPLC PROBLEMS 833 carrying out the siphon test described in Section 3.2.1. Impediments to free-flowing mobile phase include blocked inlet-line frits, faulty proportioning valves, and pinched or blocked tubing. Insufficient mobile-phase degassing or insufficient pump purging can leave enough air in the mobile phase that the pump loses prime, thereby lowering the pressure. Ensure that the mobile phase is properly degassed (Section 3.3), and purge the pump by opening the purge valve, then pumping 10 to 20 mL of degassed mobile phase to waste (a high flow rate will sometimes displace bubbles in the pump). Frequent bubble problems can point to a faulty degassing module. If this is suspected, try an alternate degassing method (Section 3.3) to isolate the problem. A worn pump seal can cause leaks (Section 17.4.1.2), but before leaks are apparent, the seal problem may prevent the pump from being able to provide the expected pressure. Carefully check for seal leaks at the drain hole on the bottom of the pump head (between the inlet check valve and the pump body), and check the system logbook (Section 17.2.4) to see if the system is due for a scheduled seal replacement. Seal replacement (see Section 17.4.1.2) should correct any problems. When the pump head is removed, check to see if there is any piston damage—a scratched or broken piston also can cause the pump to underperform. 17.4.2.3 Pressure Too Variable As mentioned above, the HPLC system pressure in the normally fluctuates. Typically this fluctuation is 1–2% (e.g., 10–20 psi, 1–2 bar) of the operating pressure, but this will vary between systems and applications. It is therefore a good idea to make a note of the normal pressure variation as part of the records kept for each batch of samples (Section 17.2.4). Also keep in mind the normal pressure cycle during each run for gradient elution. A summary of variable-pressure symptoms and solutions is given in Table 17.4. The most common sources of these problems are bubbles in the pump, sticky check valves, worn pump seals, broken pump pistons, and an inadequate mobile-phase sup- ply to the pump. The identification and correction of these problems is almost exactly the same as for low-pressure problems (Section 17.4.2.2), so further instructions are not needed. The main difference between low-pressure and variable-pressure problems is that the latter may be limited to one pump head of a dual-piston pump, or one pump of a two-pump system. Thus part of the system may be working normally (higher pressure) while part of the system is not delivering enough mobile phase (lower pressure). The two most likely problem sources are a bubble in a pump head or a sticky inlet check valve. The simplest initial approach to correcting the problem is first to purge the pump to see if this fixes the problem. If it does not, sonicate the check valves (Section 17.4.2.2) in methanol. Additional information is found in Table 17.4. 17.4.3 Variation in Retention Time Retention times for analytes should be constant within a sample batch (e.g., same day, same batch of mobile phase) if all chromatographic conditions are held constant. If a change in retention is observed, it indicates that at least one condition has changed. Because it is impossible to hold all variables exactly constant, there is a normal variation in retention time for every method. Typically this is in the range of 834 TROUBLESHOOTING ±0.5% of the retention time or ±0.0.02 to 0.05 minute; this normal variation can be determined from historic sample-batch records. When the retention-time variation exceeds the normal variation, steps should be taken to assess the cause and take corrective action. A tool to implement the initial divide-and-conquer approach (Section 17.3.1) is to calculate the retention factor k (Eq. 2.6), and then compare changes in k and retention time t R with the help of Table 17.5. Table 17.5 will guide you to one of the following sections for more information about possible changes in the mobile phase, column, or column temperature. As discussed in more detail below, mobile-phase changes tend to occur in a stepwise fashion when some intentional change is made, column changes usually take place over a period of weeks or months, and temperature changes tend to cycle during the day. These general patterns can greatly aid the identification of the problem source. An alternate way to classify retention-time problems is by the observed change in t R —increased, decreased, or variable. If you want to approach the problem in this manner, consult Table 17.6 and Section 17.4.3.6 first. The following sections (17.4.3.1–17.4.3.5) cover each of these symptoms. It may be appropriate at this point to run the system reference test (Sections 3.10.1.3, 17.2.1) to determine if the problem is related primarily to the equipment or the method. Finally, there is much overlap among the various causes of variable retention, so it is a good idea to read all of Sections 17.4.3.1 through 17.4.3.5 in order to gather as many ideas as possible, if the solution to the problem is not quickly reached. 17.4.3.1 Flow-Rate Problems A change in the flow rate will change t R but not k, because k is independent of flow rate, while t R varies in inverse proportion to flow rate (an exception can occur for pressures > 5000 psi, because of a slight dependence of k on pressure). Never underestimate the power of operator error—it is a good idea to verify that the proper flow rate is selected. Once a setting error has been eliminated, the only possible cause of a higher than normal flow rate is a problem with the system controller, which will require the skills of a trained technician to fix. Flow rates that are lower than normal cause retention times to be too long, a problem that can be caused by bubbles in the pump, pump starvation, faulty check valves or pump seals, or leaks. Sections 17.4.1 and 17.4.2.2 describe corrective actions for these problems. As discussed in Section 17.4.2.3 for pressure, the causes of variable pressures or flow rates often are the same as low-pressure or low-flow problems 17.4.3.2 Column-Size Problems A rare, but possible cause of a change in t R but not k is installation of the wrong column size, but correct type (e.g., Symmetry C18). Of course, the column size cannot change without operator intervention. The obvious fix is to look at the column label and then install the proper column. 17.4.3.3 Mobile-Phase Problems A change in the mobile phase (e.g., %B) can result in changes in both t R and k. When a new mobile-phase batch is prepared incorrectly, any changes in retention 17.4 COMMON SYMPTOMS OF HPLC PROBLEMS 835 will be noticed at that time—whether the mobile phase is pre-mixed (‘‘off-line’’) or prepared by on-line mixing. When on-line mixing systems are used, small variations in mobile-phase composition may occur due to problems with the proportioning system. Alternatively, small, continuous changes in the mobile phase (and sample retention) can occur over time, although this is much less common. For example, a volatile buffer, such as ammonium carbonate, may evaporate so as to change the pH of the mobile phase. If continuous helium sparging is used for degassing, a volatile organic component of the mobile phase could be selectively evaporated. Errors in formulating the mobile phase are a likely cause of shifts in retention (with increased retention for a reduction in %B, and vice versa). The rule of 2.5 (Section 2.5.1) indicates that a 10% change in %B will change the retention factor by approximately a factor of 2.5 times; a 1% error in mobile phase %B can account for ≈10% change in k. An error in mobile-phase pH can have a much larger effect on the retention of acidic or basic solutes (Section 7.3.4.1) than neutral analytes. The concentration of mobile-phase additives, such as ion-pairing reagents (Section 7.4.1.2), also can affect retention. During method development the robustness of the separation to small changes in mobile-phase composition should have been examined (Section 12.2.6). The results from robustness testing can be useful in determining the specific mobile-phase error that was made. From a practical standpoint, however, the most direct solution is to make up a new batch of mobile phase and determine whether the problem has been corrected. 17.4.3.4 Stationary-Phase Problems With continued use of the column, changes in retention and selectivity are common, but t 0 is unaffected; consequently values of both k and t R will change. Retention shifts due to a change in the stationary phase rarely are the only symptom observed. Usually the plate number N will also drop, peak tailing will increase, and the column pressure will rise. Past records of method use (Section 17.2.4) in combination with recent data on the performance of a column (values of N, pressure, etc.) can be used to avoid its (highly undesirable!) failure during the assay of a series of samples. As a rule, a column lifetime of 500 to 2000 sample injections should be expected for most applications (and will account for <1% of the total cost of analysis). Some methods can degrade columns more quickly, while other methods may allow a longer use of the column. Expect shorter column lifetimes when the column is operated outside the 2< pH<8 region or at temperatures > 50 ◦ C. The use of in-line filters (Section 3.4.2.3) and guard columns generally will extend column life. In any event, the column should be considered a consumable item that will wear out (hopefully gradually) over time. The easiest way to check for stationary-phase related problems is to replace the column (module substitution, Section 17.3.5). If a guard column is in use, first replace or remove the guard column to see if the problem is resolved. If the column repeatedly fails prematurely, check to be sure that it is operated within its recommended limits (consult the column care-and-use instructions for specific guidelines). In some cases it may be appropriate to find and use an equivalent column (Section 5.4.2) that is more stable. If the failure is due to the injection of dirty samples, additional sample-preparation steps (Chapter 16) may be necessary. . (Section 17.2.5.10). Rotor-seal (and possibly stator) replacement will be required to correct this problem. Also be sure to clean the remainder of the valve thoroughly to remove any particulate matter. More. (arrow). counter to automatically record the number of injection cycles, so as to allow the remaining rotor seal life to be estimated. When viewed in cross section, the grooves of the rotor shown in. the seal between the valve rotor and stator is related to how tightly the two surfaces are held together. Higher pressure-limits require that the surfaces be held together more tightly, but this