Reactivating Your GPC/SEC Instrument After Shutdown

Gel permeation chromatography/size-exclusion chromatography (GPC/SEC) has developed in recent years into a widely used and established analytical technique used to separate synthetic and natural macromolecules based on their hydrodynamic radius. GPC/SEC is a liquid chromatographic separation technique whose areas of application include organic solvents, sometimes with additives such as salts or acids, or aqueous applications with salt content in a pH range of 2 to 12. This article will discuss the steps that should be taken when reactivating your GPC/SEC instrument after an extended shutdown period.

Gel permeation chromatography/size-exclusion chromatography (GPC/SEC) systems are often shut down during extended measurement breaks or vacation periods. In such cases, separation columns are removed and stored to preserve performance and reduce solvent, energy, and consumables usage. Reactivating the system requires careful attention to ensure operational accuracy and efficiency (1).

The recommended procedure should begin by connecting a fresh bottle of the same solvent used before shutdown. All system modules should be powered on during activation. Initially, the flow rate should be set to a low value (for example, 0.1 mL/min), and system pressure must be monitored closely. At this stage, columns should remain disconnected. If pressure increases unexpectedly at a constant flow rate, the manufacturer’s customer service should be contacted. If pressure remains stable, the flow rate can be gradually increased to accelerate flushing.

Certain components of the system are not continuously flushed with eluent and may be overlooked during solvent exchange. These include the injection valve, which typically has two positions: the main pass (used during analysis) and the bypass (used during sample loading). Both positions must be flushed, either via the operating software or manually, depending on the system configuration. All other switching valves, such as column switching valves, refractive index (RI) recycling valve, and two-dimensional (2D) valves, should be treated similarly to ensure complete solvent exchange.

Proper flushing of the reference cell in the RI detector is essential to achieve a stable baseline with minimal drift and fluctuation. If a viscosity detector is present, the capillary bridge and retention vessels must also be flushed thoroughly with the new solvent. These steps are critical for restoring optimal detector performance and avoiding measurement artifacts.

Finally, all waste and recycling lines must be rinsed to prevent contamination and ensure smooth operation. Neglecting these components can lead to baseline instability, carryover effects in subsequent analyses, or partial clogging.

Column Installation in GPC/SEC Systems

GPC/SEC columns are the core components of the chromatographic system and must be handled with the utmost care to ensure reliable performance and extended operational lifetime. Proper storage during periods of non-use and thorough conditioning before reuse are essential to maintain column integrity and reproducibility of results.

Storage conditions vary depending on the column type. These specifications are detailed in the respective column user manuals. In general:

• Aqueous-phase columns should be stored in water containing sodium azide (as a biocide) or in a water–methanol mixture.
• Organic-phase columns are typically stored in pure, salt-free, non-corrosive organic solvents such as tetrahydrofuran (THF) or toluene.

Once the GPC/SEC system has been properly prepared, column installation can proceed. Start by verifying the compatibility of the fittings between the column, injector, and detector. Although most columns use standardized 1/16-inch stainless-steel fittings, slight variations in design exist. Improper connections can lead to leaks, compromised chromatographic performance, or even damage to system components. For optimal results, use standard stainless-steel nuts with one-time compression ferrules. Plastic fittings (PEEK or polypropylene) should be used cautiously, ensuring chemical compatibility with the mobile phase and any planned solvent changes.

If the column has been stored at low temperatures (for example, in a refrigerator), allow it to equilibrate to room temperature before installation to prevent any thermally caused damage.

During installation:

• Stop the flow or briefly open the pump purge valve to avoid solvent spillage.
• Remove the column end stoppers from both ends of the column.
• Connect the inlet capillary (from the autosampler) to the inlet of the first column or precolumn.
• Direct the column outlet to a waste container initially to monitor flow and ensure proper flushing before connecting to the detector.

After thorough rinsing, the column outlet can be connected to the detector via the capillary tube.

It is important to note that flushing system components located downstream of the column should only be performed after the entire column system has been thoroughly rinsed with fresh solvent. A complete solvent exchange typically requires flushing with approximately five column volumes to ensure that the previous mobile phase is fully displaced.

Spending additional time on careful system setup, including proper column installation and solvent equilibration, has a consistently positive impact on the quality and reproducibility of GPC/SEC analyses. Attention to these preparatory steps minimizes baseline disturbances, improves peak resolution, and helps prevent damage to sensitive system components.

Changing Solvent in GPC/SEC Columns and the Entire System

After flushing the GPC/SEC column(s) and system, it is important to recognize that the solvent used for column storage or system shutdown is often not the same as the solvent intended for analytical measurements. Regardless of the column or instrument manufacturer, it is strongly recommended to switch between mutually miscible eluents only. If the solvents are not miscible, an intermediate solvent must be used to prevent phase separation and damage to the stationary phase.

• If solvents A and B are fully miscible and additive-free: Direct exchange is acceptable.
• If A and B are miscible, but B contains additives (for example, salts): First flush with additive-free B, then proceed with B.
• If A and B are immiscible: Use an intermediate solvent C that is miscible with both A and B and compatible with the column.

Solvent changes should always be performed slowly, ideally at a flow rate of 0.1 mL/min, to minimize mechanical stress and allow the stationary phase to adapt. Columns may be used for different applications, provided that all solvents involved are chemically compatible with the stationary phase. Compatibility should be verified using the column's operating manual. Even the addition of less than 1 mL of an incompatible solvent can cause irreversible damage to the column packing material.

A complete solvent exchange typically requires at least five column volumes. For standard analytical GPC/SEC columns, this equates to approximately 50–75 mL. For systems with multiple columns, the required volume increases proportionally.

Additional Considerations

• Frequent solvent changes may accelerate column degradation. Regular checks of sample or plate count measurements are recommended to assess column performance over time.
• If multiple solvents are used regularly, it may be advisable to dedicate separate columns to each solvent system.
• When switching to viscous eluents, increasing the oven temperature can reduce solvent viscosity and lower system pressure.
• Organic stationary phases may swell or shrink depending on the solvent. These swelling/deswelling processes require time and should be managed with low flow rates during solvent exchange. For example, transitioning from THF to chloroform should be done slowly, as the differing swelling behavior can cause mechanical damage to the column gel material.

Always consult the column manual for solvent compatibility, recommended flow rates, and temperature settings (2) to ensure safe and effective operation.

Reminder: Refer to the flushing procedure outlined earlier. Ensure all valve positions, including injection and switching valves, are flushed appropriately. Don’t forget the RI detector’s reference cell and, if present, the viscosity detector components. Proper flushing is key to stable baselines and accurate measurements.

Starting the First GPC/SEC Measurement

The instrument, including the columns, has been fully flushed and is now ready for initial measurements. In accordance with good scientific practice, chromatography should first be evaluated using solvent injections (blank injections) to assess baseline stability and reproducibility before analyzing sensitive samples. To simulate later operating conditions, all modules and software settings should already match those intended for actual sample measurements.

In addition to blank injections, determining the current plate count number of the column system is an important performance metric. This is typically done using monodisperse, low-molecular-weight compounds. For THF applications, suitable substances include butylated hydroxytoluene or toluene; aqueous systems are often tested with ethylene glycol. The plate count number should be compared to values obtained before system shutdown. Some users also employ checkout samples or polymeric internal standards to perform an initial system validation.

For example, if three blank injections are performed, their chromatograms should be superimposable at identical injection volumes, indicating that the system has reached a stable state. When using checkout samples, their chromatographic behavior can also be compared to measurements taken before system shutdown, aiming for close similarity.

Following a shutdown and column storage, recalibration of the entire system is strongly recommended. If solvent injections, plate count number determinations, and/or checkout sample analyses show no irregularities, the system is considered ready for calibration and subsequent sample measurements.

Summary (Figure 1)

• The system should be restarted with the same solvent, but using a fresh bottle, at a low flow rate, and carefully monitoring the system pressure.
• Careful installation and thorough solvent equilibration of GPC/SEC columns are essential to preserve column integrity, ensure reproducible results, and prevent damage to system components during reactivation.
• Solvent exchange in GPC/SEC systems must be performed slowly and carefully using only chemically compatible, miscible solvents, potentially with intermediate solvents, while adhering to column-specific guidelines to prevent damage and ensure reliable chromatographic performance.
• Before initiating GPC/SEC measurements after system reactivation, it is essential to validate baseline stability and column performance through blank injections, plate count determination, and check sample comparison to ensure analytical reliability and reproducibility.

References

(1) Radke, W. Tips & Tricks: Treating Your GPC/SEC System Properly The Column 2022, 18 (11), 10–13.

(2) Agilent, GPC/SEC eBook Series – GPC/SEC Troubleshooting and Good Practice, 2024.