Maintaining and Cleaning the HPLC Flow Cell

If you want the best LC data, you need a properly working UV flow cell that is matched to the application. The primary goal is to choose the flow cell length that provides the best sensitivity while maintaining chromatographic resolution.

Unfortunately, Analysts often think about flow cells only when they troubleshoot problems with the HPLC system. Maintenance of the UV flow cell is simple and every user of the HPLC system can do it. To start, keep in mind four general preventive measures:

1. Many flow cells have quartz windows or fused silica components, so avoid using alkaline solutions with a pH greater than 9.5, which can attack these components and impair optical performance.
2. To prevent crystallization inside the flow cell, always flush it with water after running buffers or salt solutions, especially at high pHs.
3. When your LC is unused overnight, ensure your flow cell contains at least 10% organic mobile phase to prevent algae growth.
4. Some modern flow cells use coated or uncoated fused silica capillaries. You can adversely affect coated capillaries by turning on the UV lamp without solvent flow, so read the manufacturer’s recommendations carefully.

Flow Cell Selection

• It is essential to carefully select a proper flow cell for your HPLC system. You may need a high-pressure flow cell when several detectors – or a detector and a fraction collector – are connected in series. In addition, check that the flow cell has the proper volume and optical path length to ensure the optimal resolution and sensitivity.
• Many analysts assume that small-diameter columns always require very low-volume flow cells, but the column length, particle size, and gradient slope all play a role. For example, a 4.6 mm column running steep gradients produces a smaller peak volume than a long 2.1 mm column operated under high-resolution conditions.
• To minimize any unnecessary dispersion that could reduce the apparent chromatographic resolution, the flow cell should be no larger than about 10% of the peak volume.
• Note also that absolute cell volume is not always the best parameter to consider; the critical parameter is effective cell volume.

Flushing the Flow Cell

• If the system was shut down unexpectedly during an unattended sequence run, the sample may be still trapped in your flow cell. 
• If the system was stopped overnight with salt buffer, it is important to start the system flush with a lower flow rate and watch the system pressure. 
• A warm-water flush using the 60°C setting on your column compartment is an effective way to clean your UV flow cell.

ALSO READ: Troubleshooting for UV Detector of HPLC

Flow Cell Cleaning Solvents

• The flow cell can be cleaned by pumping a solvent through the system. Generally, the recommended solvent is the highest polarity solvent you have recently used. Therefore, ethyl acetate (often solvent B) would be used. See the below table for alternative solvents.

Suggested Flow Cell Cleaning Solvent

Solvent	Description
Methanol	Miscible with most LC solvents. It will force other solvents through the system so that they will not interfere with the next sample, Break down the tertiary structure of the stick and allow it to flush through.
Acetone	Common organic solvent with high dissolving properties.
Dichloromethane (DCM)	Effective at solubilizing organic molecules
Dimethylsulfoxide DMSO)	The strong solvent will clear the organic sample.
Dimethylformamide (DMF)	Strong solvent if other solvents do not produce the desired results.
Tetrahydrofuran (THF)	Strong solvent.
Mildly acidic aqueous solution	0.5M acetic acid in distilled water can be used to clear samples that are not removed by organic solvents.
Mildly basic aqueous solution	A dish-type detergent solution can wash through particulates not picked up by the acidic solution. The emulsification effect of detergent is an effective tool as well to help wash through oily samples. Follow the detergent with sex- eral minutes of clean distilled water

Quick Flow Cell Cleaning

• Quick cleaning is advantageous because very little solvent is required and repriming is not necessary. To quickly clean the system:

1. Remove the column from the system.
2. With a syringe, inject the selected solvent into the lower column mount.
3. Insert a bypass tube in place of the column.
4. From the Peak Trak menu select the Tools > Manual Control… command and pump 100 mL of Solvent A at 100 mL/min.
5. Replace the bypass tube with a column and start a run. The Companion will attempt to calibrate the flow cell.

If the calibration is successful, the system is ready for use.

If the alert message persists, refer to the thorough flowcell cleaning solvents and techniques.

ALSO READ: Types of HPLC Detectors

Thorough Flow Cell Cleaning

To thoroughly clean the flow cell:

1. Remove the column from the system and insert a bypass tube.
2. If using an alternative solvent, place the solvent inlet line into a container of the selected solvent.
3. Pump 100 ml of solvent through the system at 100 mL/min.
4. If an alternative solvent was used, return the solvent inlet line to the appropriate solvent container.
5. Select the Tools > Auto Prime menu command to prime the system.
6. Replace the bypass tube with a column and start a run. The Companion will attempt to calibrate the flow cell.

If the calibration is successful, the system is ready for use.

If the alert message persists, refer to the alternative flow cell cleaning solvents and techniques.

• The solvent strength and high flow rate will typically clear the obstruction and return the flow cell to optimum performance.

Alternative Cleaning Techniques

The following alternative techniques should be considered if the thorough flow cell cleaning instructions do not clear the obstruction.

1. Pump water/DMF/methanol (50/25/25) for 3–4 minutes followed by pumping straight methanol (or ethanol) for 2–3 minutes.
2. Pump 5–10 mL DMSO followed by methanol, ethyl acetate, then hexane.
3. After the lines have been filled with solvent, reduce the flow rate or pause for up to 18 hours. Doing so will allow the solvent to remain longer to dissolve the obstruction.

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