Leaks and Obstructions: Troubleshooting Common Problems Close to the Point of Sample Injection

The “autosampler” is an indispensable component of modern high-performance liquid chromatography (HPLC) systems. Contemporary technology enables automated, rapid, and precise injections of samples with very little carryover. However, the rotor/stator assembly at the heart of injection valves used in these samplers is also a weak point. Routine wear of the rotor can lead to leaks and release debris into the mobile phase, leading to obstructions downstream from the injection valve. In this installment of “LC Troubleshooting,” we take a close look at how the commonly used “flow-through needle” sampler works, discuss how and where leaks can develop, and solutions to the problems when they do occur.

In the past few months, we have encountered a handful of problems related to autosamplers in my laboratory, so I thought it would be a good time to discuss in some detail where, why, and how these problems occur, along with solutions and suggestions for prevention. Previously in this “LC Troubleshooting” column, John Dolan addressed problems with autosamplers in about a dozen different installments, including a series of four articles more than 20 years ago. This series addressed a variety of problems not covered in this installment, including reproducibility and carryover (1–4), and readers are referred to these rich resources if they are interested in learning more about these topics. There are many different approaches and technologies used for sample introduction (that is, injection) in modern liquid chromatography (LC) systems. In this installment of “LC Troubleshooting,” I am focusing on the “flow-through needle” (FTN) design, because it is the one we use most frequently in my own laboratory, and it is the design found most often in the field. Readers interested in learning about other designs and approaches are referred to the article on LC sampler technologies by Steiner, Carsten, and Dong (5), as well as Michel Dong’s book (6).

The Flow-Through Needle Sampler: How It Works

Steps Required for Sample Injection

Figure 1 shows an illustration of the fluid paths through a FTN sampler. Specific details of the design vary from one instrument manufacturer to the next, but this illustration captures the ideas essential to the discussion here. At the heart of the device is a six-port, two-position valve. The illustration shows the fluid path through the valve and the connected components in the two positions: 1) “mainpass” on the left; and 2) “bypass” on the right. The valve spends most of the time associated with an analysis cycle (that is, sample injection, separation, and column re-equilibration) in the mainpass position. Here, mobile phase produced by the pump flows into the valve, through a “metering device” (a high-performance syringe designed to draw microliters of sample from a vial at ambient pressure), through a sample loop and associated needle, back to the valve through the “needle seat,” and on to the LC column. Then, after one analysis is complete and before the next separation begins, the valve moves to the bypass position in preparation for drawing the next sample from a vial. When the valve is in the bypass position, most of the sampler components are isolated from the mobile phase flow path. The needle is pulled out of the needle seat, and either a robotic arm takes the needle to a sample vial, or the sample vial is brought to the needle by a robotic arm. The vial septum is punctured by the needle, and a specified volume of sample is drawn up into the needle as the metering device plunger retreats, pulling liquid through the needle and sample loop capillary into the barrel of the metering device syringe. Once the sample has been drawn into the needle, it is returned to the needle seat, completing the flow path between ports 2 and 5 on the valve. At this point the system is prepared to introduce the sample into the mobile phase stream by switching the valve back to the mainpass position, effectively “injecting” the sample into the column. This series of steps is then repeated as many times as there are samples to be analyzed.

The Valve Rotor is the Star of the Show

All the action associated with the injection valve is localized to two main parts: 1) a stator, which, as the name suggests, is stationary, and does not move; and 2) a rotor (often referred to as the rotor seal), the part that rotates when the valve switches between the mainpass and bypass positions. The primary functions of the stator are to connect the valve body to the other component parts of the sampler, and the rest of the instrument (the pump, column, needle seat, and metering device), and to guide the fluid to the rotor in specific locations. The stator is commonly constructed mainly of stainless steel, and the surface that contacts the rotor is commonly coated with a material that makes it smooth and wear resistant. The primary function of the rotor is to provide a fluidic path between two ports of the stator, through a groove cut in the rotor surface. Most rotors are made of polymeric materials filled with a solid to give them the structural integrity needed to resist deformation when they are pressed against the stator with the force need to seal well at ultrahigh-pressure liquid chromatography (UHPLC) pressures. The rotor shown in Figure 1 has two grooves. In the mainpass position, one of the grooves connects ports 1 and 2, while the other groove connects ports 5 and 6. When the valve is switched to the bypass position one of the grooves connects ports 1 and 6, while the other groove connects ports 4 and 5. The image shown in Figure 2 was captured using a light microscope and a brand new rotor for a six-port, two-position valve like that illustrated in Figure 1. The focus of the image is on just one of the grooves. Here we see that the channel cross-section has a roughly rectangular geometry. To further illustrate that the mobile phase flows through the stator, into the rotor, and then back out through a different port in the stator, a simplified, but semi-transparent view of the stator-rotor assembly is shown in Figure 3.

The Rotor Seal Can Also Be the Cause of Many Problems

Given that most rotors are made from polymeric materials, they are the literal weak point in the rotor-stator assembly. Frequent rotation of the rotor against the metal stator surface under the high force needed to seal at UHPLC pressures inevitably leads to distortion or loss of the polymeric material. The flow channels through the grooves can become distorted if the polymer itself flows and changes its shape, and in a severe case can block liquid flow through the groove altogether. On the other hand, the polymeric material can be eroded by the mobile phase, particularly under harsh mobile phase conditions (for example, very low or very high pH, organic solvents such as tetrahydrofuran). Finally, particulates from injected samples or the mobile phase that get stuck between the rotor and stator can cause scratches in the rotor and/or stator surfaces.

Internal Leaks

Two examples of many possible outcomes of these effects are shown in Figure 4. On the right side of the illustration, we see that a small channel has developed as a result of the erosion of rotor material that allows a small flow between ports 1 and 2, even though the intended flow path connects ports 1 and 6. In the bypass position of the valve, this means that some mobile phase flow intended to go from the pump directly to the column will actually leak into the flow path connecting the metering device to the needle. In my laboratory we recently observed exactly this scenario. This type of failure is difficult to diagnose because the leak is internal to the valve. In our case, we suspected there was a problem when we observed small puddles of liquid on top of our sample vials after the sampler had drawn sample from a vial. We concluded that mobile phase leaking from port 1 to port 2 moved through the metering device and out through the needle after the needle was pulled out of the vial septum, leaving a small puddle of mobile phase there. After changing the rotor in this sampler, this problem was not observed any longer.

External Leaks

Figure 4 further shows that an external leak (left side of the illustration) from the valve body can develop if rotor material is lost to the point where a channel develops between one of the grooves and the edge of the rotor body. This type of problem is easier to diagnose because either liquid will be visible on the outer surface of the valve body, or some precipitated mobile phase salts may be observed as white residue on the valve body if the leak rate is very small. In either case, simply replacing the rotor should resolve this problem as well.

Solutions and Prevention

In most cases where a scratched or eroded rotor is indeed the cause of a leak, simply replacing the rotor will resolve the problem. However, when we have the valve disassembled, we look carefully at the surface of the stator that contacts the rotor surface and inspect it for scratches or other defects that affect the smoothness of the surface. Such defects also occur with routine use of the valve, albeit usually at a slower rate than rotor wear, and will dramatically increase the wear rate of a new rotor. Thus, if a defect is observed on the stator surface, it too should be replaced.

There is no good way to prevent normal wear of the rotor material, thus it is only a matter of time before the rotor has worn to the point where a leak develops and it must be replaced. Of course, simply waiting for a failure of this type to occur may not be the best strategy, particularly in a laboratory environment where instrument up-time is particularly valuable. In these situations, it is wise to replace the rotor regularly as part of a routine preventative maintenance program. What constitutes the “right” frequency of rotor changes depends nearly entirely on the number of injections made with the sampler each day but is also influenced by the operating conditions (for example, mobile phase pH, sample “dirtiness,” and operating pressure). Users interested in establishing a schedule for routine replacement of rotors are encouraged to consult with their instrument manufacturer for a recommendation.

The Other Big Problem: An Obstructed Needle Seat

Although most of this installment has focused on problems related to the injection valve, the other problem we encounter relatively frequently in my laboratory is partially occluded needle seat capillaries. This capillary is the one that connects the needle seat to the injection valve (lower left corners of the illustrations in Figure 1). In the context of normal operation, flow only ever goes one way through this capillary—from the needle seat to the valve. In most cases, the diameter of the needle seat capillary is smaller than the capillaries upstream of the needle seat all the way back to the injection valve. As the rotor wears, small pieces of rotor material are released into the mobile phase and pushed through the sampling loop and needle. When these pieces of rotor material encounter the smaller diameter needle seat capillary they can become stuck and occlude the mobile phase path, leading to much higher than expected pressure drop across the sampler unit. Diagnosing this particular cause of increased pressure drop can be tricky, but the systematic approach I have recommended for diagnosing pressure problems will work in this situation as well (7,8). We have found that backflushing the needle seat capillary can sometimes push out the obstructing material, and return the pressure drop to normal. However, we generally find that this is only a temporary fix, and some short time later (typically days) the capillary becomes obstructed again. Thus, generally our solution is to simply replace a needle seat capillary as soon as we are convinced that it is indeed obstructed.

Summary

In this installment of “LC Troubleshooting,” I have discussed problems we face most often with the “flow-through needle” design of modern LC samplers, which is the most common type of autosampler in use today. Most of these problems originate with routine wear of the rotor part of the injection valve, which is typically made of a polymeric material, that then leads to leaks that can be either internal or external to the valve itself. Simply replacing a worn seal resolves most of these problems, but they can also largely be prevented by establishing a regular preventative maintenance schedule that involves replacing the rotor before problems arise.

Acknowledgments

I’d like to thank Molly Stein and Micah Stoll for their work on the illustrations shown in Figures 1 and 3, respectively.

References

(1) Dolan, J. W. Reproducibility and Carryover — A Case Study. LCGC North Am. 2001, 19, 290–296.

(2) Dolan, J. W. Autosamplers, Part II — Problems and Solutions. LCGC North Am. 2001, 19, 478–482.

(3) Dolan, J. W. Autosamplers, Part I — Design Features. LCGC North Am. 2001, 19, 386–391.

(4) Dolan, J. W. Autosampler Carryover. LCGC North Am. 2001, 19, 164–168.

(5) Steiner, F.; Dong, M.; Paul, C. HPLC Autosamplers: Perspectives, Principles, and Practices. LCGC North Am. 2019, 37, 514–529.

(6) Dong, M. W. HPLC and UHPLC for Practicing Scientists,2^ndEd. Wiley, 2019.

(7) Stoll, D. R.; Grinias, J. P. Treat It Like A Circuit, Part I: Comparison of Concepts from Electronics to Flow in LC Systems. LCGC Int. 2024, 1 (4), 6–10. DOI: 10.56530/lcgc.int.ol2372p5

(8) Stoll, D. R.; Grinias, J. P. Treat It Like a Circuit, Part II: Applications and Troubleshooting. LCGC Int. 2024, 1 (5),6–12. DOI: 10.56530/lcgc.int.wy6073a3