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Down-proofing Your HPLC & UHPLC Systems

Maintaining laboratory instrumentation often involves balancing in-house expertise, downtime, and the cost of outside services

Angelo DePalma, PhD

Angelo DePalma is a freelance writer living in Newton, New Jersey. You can reach him at

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downproofing your HPLC and UHPLC systems

Keeping the Repair Staff Away—Up to a Point

Those factors take on special significance for high-performance liquid chromatography (HPLC), where successful execution—particularly for ultra-high-performance systems (UHPLC) and sub-two-micron separations—relies on the precisely tuned interplay of complex electrical, mechanical, and chemical operations.

As Tracie Brombos, VP of operations at GenTech Scientific (Arcade, NY) observes, HPLC systems have more moving parts than most instruments. “More chemicals come into contact with those parts, so maintenance takes on a higher priority.”

Best practices

Viet Pham, senior product marketing manager at Waters (Billerica, MA), notes that proper storage of HPLC columns is essential for optimizing longevity and performance. Pham recommends storing used columns in 100 percent acetonitrile and avoiding long-term storage in buffered eluents. If the column was used with buffered mobile phase, he suggests flushing it with HPLC-grade water followed by 100 percent acetonitrile. “Failure to flush with water may cause the mobile phase buffers to precipitate within the sorbent bed and potentially harm the column. Completely seal the column with the supplied end plugs to prevent the chromatographic bed from drying.”

LC system setup and storage are next on Pham’s list. “When preparing a system, you should prime all solvent lines—not just the ones you plan to use—to ensure you have fresh solvent everywhere.” In addition to replacing old solvent in all the lines, this practice ensures efficient degasser performance by evenly distributing the vacuum pressure across the entire manifold. Priming all lines greatly reduces the risk that algae or bacteria will grow in the old solvent and subsequently affect system robustness.

“Similarly, users should make sure that the pump seal wash is primed and running properly,” Pham adds. Users should also consider the mobile phase they are using for seal wash and their washing frequency, especially when using corrosive or high-concentration mobile phases.

Users should prime the needle wash and purge solvents in the autosampler as well. Solvents should be miscible with the sample to minimize the amount of carryover between injections. “These solvents should be primed at the same time that the mobile phase lines are primed to ensure fresh solvent is used throughout the system.”

Waters recommends a minimum of 30 minutes’ warmup for optical detectors to ensure a stable baseline. Users should regularly check and be aware of the detector’s lamp energy, since a precipitous drop in energy indicates potential issues with the lamp, flow cell, or internal optics. Pham suggests calling a field engineer to fix such problems. When the analysis is complete, operators should flush the detector unit with water (for reverse phase chromatography) and store the system in a suitable organic-aqueous mobile solvent mixture to mitigate bacterial growth when the system is sitting idle.


The sensitivity of modern HPLC has advanced as samples become more precious, and autosampler consumables have kept pace with these changes. To conserve samples, needles must enter deep into sample vials to access the limited sample volume. Sampling systems that are out of dimensional tolerance may result in needle damage and system downtime.

Cleanliness of vials and septa has also become critical with the increasing sensitivity of HPLC and UHPLC systems. Ten to 15 years ago, contaminants, usually residue from the manufacturing process, began appearing in vials and septa. The contaminants were always present but were noticed only when systems became more sensitive.

“As system sensitivity continues improving, the chemistry limits of materials being used in autosampler consumables will become increasingly challenged,” Pham notes. “Vendors will need to innovate processes or materials to ensure the consumables keep up.”

Qualification checks

Ade Kujore, marketing specialist at Cecil Instruments (Cambridge, UK), notes that modular HPLC systems offer analytical scientists straightforward troubleshooting, repair, and maintenance. In modular HPLC systems, the main components (pumps, detectors, autosamplers, etc.) are supplied as separate parts.

“The advantages of this type of system are that the users may select the types and brands of components they prefer,” Kujore says.

Instruments are shipped to meet specifications, but users should check periodically that this happy situation continues. In addition to daily care, users should routinely perform operating qualification checks.

“Should qualification checks highlight an instrumentation issue, then it is prudent to consult the manufacturer,” Kujore advises. “Experienced users can check things themselves or refer them to in-house metrologists who can perform routine cleaning and check pumps, seals, and detector flow cells; align detector lamps; or change injection valve rotors.” Depending on company policy, a trained manufacturer’s representative or approved contractor should be consulted for serious repair and certification issues.

Kujore’s “Golden Rules”

  • Ensure that injected liquids and mobile phases are free of particulate materials. “If in doubt, pass each sample through a 0.45 μm filter, and use a filter at the liquid outlet of the mobile phase reservoir.”
  • “Visible air bubbles are the enemy!” Degas all mobile phases entering the pump. Many users now use on-line degassing, but in many cases, old-fashioned off-line ultrasonication and/or helium sparging are also acceptable.
  • Similarly, before a mobile phase is first used in an HPLC system, purge each pump to ensure that no air is inadvertently introduced.
  • Acetonitrile is unkind to pump check valves, as it tends to makes them stick. “So after acetonitrile mobile phases have been used for a project, prime and purge the pump(s) with water or methanol.”
  • Some solvents should simply never be used as HPLC mobile phases because they can corrode and degrade pump components. Refer to the manufacturer’s pump user manual for details.
  • During times a system is not being injected, circulate the buffered mobile phase through the pump(s) at low flow, such as 0.2 ml/minute. After using the buffered mobile phase, flush the system with 15/85 percent methanol/water. “It is also useful to back-flush the pump’s pistons manually or automatically.”
  • Use tubing of appropriate materials, lengths, and internal diameters. “This may sometimes require installing new sealing unions and screw fittings.”
  • Mobile phase leaks and unusually high back pressures are causes for immediate concern and must be investigated.
  • Use only spare parts and consumables that your manufacturer has approved. “It is not worth using inferior parts to save a few dollars.”

Following the suggestions in this article will not guarantee your HPLC system will never fail. It will, however, reduce occurrences, both inconvenient and severe, that can cut into your lab’s throughput and productivity.

For additional resources, including useful articles and a list of manufacturers, visit