Daily and Routine LC Maintenance

To minimize downtime, be sure your High-Performance Liquid Chromatography (HPLC) is properly maintained. If not, the pressure of the system can build up, the lines can get blocked, and the columns don’t last forever. 

Daily and Routine LC Maintenance will help you in

1. Improve instrument performance.
2. Prevent extended, unscheduled downtime.
3. Increase data quality and consistency.

Here are some suggestions to help you maintain your HPLC and increase your instrument's reliability.

Daily LC Maintenance

Before daily analysis, everyone has to check Liquid chromatography for better performance and also avoid the problem

1. Check the mobile phase level.

• The mobile phase level plays a vital role for running an LCMS (Liquid chromatography–mass spectrometry). An empty mobile phase creates air bubble in flow path thus pressure fluctuation and many problems. It is better to check the mobile phase level first if level is low fill up.

2. Mechanical pump oil level and oil color checking.

• The oil level in the rough pump of mass spectrometry should not be empty or lower than the warning level. The color of oil should be colorless or light yellow color. Dark yellow or close to the orange color of oil means, it should be changed.

3. Check the nitrogen generator flow level, outlet level and operation pressure

• Without proper nitrogen gas pressure, mass spectrometry will be unstable in vacuum conditions. It will create problems with the analysis. Checking nitrogen generator flow level and pressure is always a wise decision.

4. Check column pressure

• Column pressure should not exceed its operating condition. It will hamper the column's lifetime. The log should be maintained for pressure records. So that it can be easily notified that column pressure is higher than usual.

5. Check solvent waste, dispose of when full

• Solvent waste should check because of the overflow of waste from the disposal chemical bin.

6. Surface cleanup

• Dust or other unwanted waste can create lot of problems for analysis and also for the flow path in the capillary. Surface cleanup mandatory action for daily maintenance.

ALSO READ: SOP for Preventive Maintenance of HPLC

• By developing a program of regularly scheduled events that ensure your instrument is running well, you will minimize unplanned downtime for unexpected repairs, improve component lifetime, and ensure more consistent performance. All of this adds up to a cost-effective strategy for protecting data quality and increasing overall lab productivity.

• Routine LC maintenance programs should establish specific inspection, cleaning, and replacement schedules for the serviceable parts of the instrument. 
• This includes 

1. Mobile phases
2. Pumps
3. Autosamplers
4. Columns/guards
5. Detectors

• Specific protocols and recommendations will vary by manufacturer and model, so always consult your instrument manual when developing your procedures. In addition, consider the types of methods and samples you are running-more frequent service will be required for high throughput testing of dirty samples than would be needed when fewer samples or cleaner matrices are being analyzed. 

Routine LC Maintenance

Mobile Phases

• Many performance and troubleshooting issues can be traced back to mobile phase problems, primarily related to contaminants and leaks. Contaminants can be microbial or particulate and can ultimately cause incorrect and variable mobile phase ratios, system blockages, and high back pressure. 
• To avoid this, use only fresh, filtered mobile phases that are made with HPLC-grade solvents, and clean or replace your solvent inlet filters regularly. Glass filters are inexpensive and environmentally friendly, so it is best practice to simply replace them regularly. Stainless steel filters can either be replaced or cleaned by sonication in isopropanol (note that glass filters should not be sonicated because they can fracture and/or shed fragments). 

• Always store prepared mobile phases in clean bottles and keep them capped to prevent contamination. Be sure to use proper bottles with secure caps; storing mobile phases in old solvent bottles topped with lab film or foil is not recommended because this allows the evaporation of volatile solvents and additives, which can change the concentration of the solutions.
• Because particulates can also contaminate the degasser, it is important to flush the degasser lines with isopropanol (use water first and then isopropanol if using a buffered mobile phase to prevent precipitation). The degasser tubing can become brittle over time and crack; this causes the unit to become very noisy, indicating that the tubing needs to be trimmed or replaced. 

• Maintaining a detailed instrument logbook that tracks symptoms such as this will help you determine appropriate time intervals when setting up a routine LC maintenance schedule.
• Leaks are another common mobile phase issue that can be minimized with a good routine maintenance plan. Small leaks can cause loss of sample, increased retention, and decreased signal while large leaks can cause the instrument to automatically shut down, which stops data collection and could mean you need to rerun the batch. 
• To prevent leaks, include regular inspection of tubing and fittings for moisture and residue in your routine maintenance plan. Verify that the end fittings between the mobile phase tubing and the column are tightened and secured, but take care not to overtighten because this can damage the fitting. You may need to remove the fitting and inspect it for any signs of damage, or simply replace it at regular intervals. 
• High-pressure fittings are more durable than standard fittings and are a useful alternative that can allow for longer intervals between maintenance events.
• Your routine LC maintenance schedule should be supplemented by daily checks of mobile phase components and system plumbing. Before starting your analyses, check mobile phase clarity and preparation date, be sure the levels in the mobile phase and waste containers are adequate, and trace tubing lines and fittings with a dry lab tissue; this will help you spot small leaks that can be missed by visual inspection alone.

Pumps

• Binary and quaternary pumps are another part of the system that should be included in any routine LC maintenance plan. Serviceable parts include the pistons, piston seals, inlet and outlet check valves, and the purge valve frit. 
• Pistons and valves are the more durable parts of the pump and may only require replacement only once or twice a year. Simply wiping them with water : methanol (50:50) and inspecting them for wear when changing the piston seals is adequate, but be sure to keep replacement parts on hand so they are available when you need them.

• In contrast, the piston seals and purge valve frits need more frequent attention. Piston seals will wear rapidly, especially if using high buffer concentration mobile phases, and often need to be changed every three to six months. If your system has a seal rinse option, check to ensure that there is an adequate supply of fresh seal rinse solution (typically 90:10 water : isopropyl alcohol) as part of the routine maintenance plan. For closed-loop seal wash kits, inspect the level of seal rinse solution often and replace it regularly.
• Following these steps and making sure the seal rinse lines are properly primed will help maximize the lifetimes of in-pump consumables.
• Purge valve frits can become dirty from collecting debris from pump seal wear and usually need to be replaced at the same time. Leaks at the pump head, inconsistent retention times, and unstable pressures can indicate the piston seals are breaking down and need to be changed while high back pressure or irregular peak shapes can point toward a clogged purge valve frit.

• A careful review of the instrument logbook can help you decide what service interval is best to set for your instrument. 
• In addition to following a good routine LC maintenance plan, you can further extend pump lifetime by storing it under favorable conditions. When the pump will not be in use, even if just overnight, first prepare it by flushing it with 90% water for about 15 minutes to remove any buffer salts, followed by 100% organic. Then, either turn the pump off or reduce the flow to 0.1 mL/min.

ALSO READ: HPLC Column Care and Maintenance

Autosampler

• To prevent leaks, clogs, and carryover, injector valve rotor seals, stators, stator faces, needles, and needle seats should be inspected frequently and replaced as needed.
• When inspecting these parts, take particular care to examine the autosampler valve components (stator, stator face, etc.) for blockages or scratches. Generally speaking, rotor seals and needle seats will need to be replaced more frequently than stators and needles.

• As with other parts of the LC system, mobile phases with high concentrations of buffer salts can accelerate wear and their use may need to be offset by more frequent LC maintenance. One of the best ways to prevent autosampler problems is to filter or centrifuge samples prior to injection to remove particulates. Syringe filters and filter vials make this a simple task that is well worth the time because it helps prevent injection valve damage and back pressure increases due to blockages.

Columns and Guard Columns

• As a regular part of LC maintenance, inspect the column connections (as well as all other system connections) for leaks, pinched tubing, and loose fittings. When replacing tubing, use short lengths of narrow tubing to minimize system volume, thereby reducing peak dispersion and loss of resolution. Always check the tubing ends to ensure a clean, square cut because irregular cuts can cause void volumes to form. 

• Also, it is best practice to replace a fitting if it is leaking because it is likely damaged and further tightening can exacerbate the problem.
• There are no serviceable parts in the column compartment itself, but turning the oven off when not in use will help lengthen the lifetime of both the column and the oven. 
• Filtration is another way to extend both column lifetime and service intervals. Using a guard column or UltraShield UHPLC precolumn filter offers excellent protection from both particulates and contaminants. Be sure to keep a log of baseline pressure as increasing pressure is a good indication of a blockage; set the guard column replacement schedule based on the pressure trends you observe in the logbook.
• When it is time to replace the guard column, check that your system pressure returns to normal. If it does not, it may be time to regenerate or replace the column. To regenerate an HPLC or UHPLC column, flush it in the direction shown on the column with a series of solvents. Use a minimum of 20 column volumes of each solvent and flush in the order shown in Table I.

Table I: Solvent flushing sequence for LC column regeneration.

• Once a new or regenerated LC column has been installed, be sure to follow all usage guidelines, including pH limits, temperature limits, and storage solvent. Maintaining a column log in addition to an instrument log is an excellent way to prevent problems when returning a column to service or preparing it for use in a different method.

Detector

• Detectors are complex and have relatively few customer-serviceable parts. Regular inspection and cleaning protocols should carefully follow the instrument manual. Spikes or an increase in baseline noise may indicate a failing lamp, so keeping an extra on hand is recommended. Always consult your instrument manual before attempting installation.

Effective Schedule

• The most fundamental element of an effective routine LC maintenance program is that it is performed regularly following a planned schedule. To maximize effectiveness, minimize disruption, and avoid downtime for unexpected instrument failures, the schedule should be set considering instrument logbook history, sample matrices, number of samples run, and method details (e.g., use of buffers, additives, or derivatization reagents). Instruments used heavily for analyzing dirty samples under harsher method conditions will require more frequent maintenance than those used lightly to analyze relatively clean samples. 

ALSO READ: Maintenance of Flow Cell