Liquid chromatographs play an important role in the separation and analysis of biomedical macromolecules, ionic compounds, natural products, and other high molecular weight compounds. However, during use, the fluidic system is often troubled by bubble problems, which affect the accuracy of experimental results. This article provides a detailed analysis of where bubbles occur in a liquid chromatography, their causes, and solutions.
Two Common Locations Where Bubbles Occur
Pump head suction tubing (white tubing) – an area where the system pressure changes dramatically.
Detector flow cell – an area where bubbles easily accumulate and affect baseline stability.
Root Causes of Bubble Formation
The core principle of bubble formation can be summarised in two points: temperature rise and pressure drop.
Temperature factor: When the ambient temperature or column temperature approaches the boiling point of the solvent, the solvent may vaporise and form bubbles; small bubbles originally present in the solvent may expand due to heating.
Pressure factor: Even after sonication or filtration, tiny bubbles may still remain in the mobile phase. When system pressure drops, these tiny bubbles can grow and coalesce, interfering with normal experiments.
Bubble Problems in the Pump Head and Suction Tubing
Pump head bubbles
The pump head experiences the most dramatic pressure changes in the system, making it a prime location for bubble formation. The pump relies on negative pressure to draw liquid, and this negative pressure environment causes small bubbles in the mobile phase to grow. When the pump chamber switches to positive pressure, the enlarged bubbles may not completely shrink and can become trapped in the pump chamber, affecting the accuracy of liquid delivery.
Solutions:
Use the specially designed purge valve on the pump head to expel bubbles.
Observe column pressure stability; stable column pressure indicates successful bubble removal from the pump head.
Bubbles in the white suction tubing
Case A: Air leakage at the connection point of the white tubing
Characteristic: Visible bubbles appear. Because the tubing experiences alternating atmospheric and negative pressure, leakage manifests as air entering the tubing rather than liquid leaking out.
Action: Replace the tubing directly.
Case B: Clogged filter
How to diagnose: Remove the filter; if bubbles stop appearing, the filter is clogged.
Cleaning steps:
Sonicate the filter in 30% nitric acid solution for about 20 minutes.
Sonicate the filter in water, changing the water several times during the process.
Case C: Vaporisation of the mobile phase
Common situation: When room temperature is too high in summer, low‑boiling solvents such as acetic acid, ammonia water, or petroleum ether may easily vaporise.
Action: Sonicate the mobile phase and lower the room temperature.
Bubble Problems in the Detector Flow Cell
The flow cell is another location where bubbles easily accumulate, significantly affecting the baseline. After the mobile phase enters the column, the pressure gradually decreases, allowing tiny bubbles to grow. Because the cross‑sectional area of the flow cell is much larger than that of the steel tubing, bubbles are more likely to expand inside the flow cell and may become too large to shrink and exit through the tubing.
Handling bubbles in a refractive index detector flow cell
A refractive index detector cell can withstand only a few kilograms of pressure. Treatment methods include:
Repeatedly flushing the flow cell.
Raising the waste bottle to apply a small back‑pressure using the liquid column in the waste tube. However, this method may make bubble removal more difficult; currently no better alternative is available.
Removing bubbles from a UV detector flow cell
When the baseline is chaotic and irregular, bubbles in the flow cell should be suspected.
Method 1: Adjust the back‑pressure regulator
Repeatedly adjust the back‑pressure regulator until the baseline stabilises.
Method 2: Manual bubble removal steps
Remove the back‑pressure regulator and connect a spare steel tube.
Start the pump to aspirate liquid and adjust the detector to zero.
Increase the pressure upper limit by 50 kg above the current column pressure (e.g., if the column pressure is 60 kg, set the maximum pressure limit to 110 kg).
Manually block the outlet end of the waste steel tube while observing the pump pressure rise and the change in the detector’s A/V value.
How to judge successful bubble removal:
If bubbles are present in the flow cell, manually blocking the waste tube will cause the A/V value to change dramatically. When the tube is unblocked, the A/V value may rise sharply again – this indicates that bubbles are present but not completely removed.
When the A/V value remains essentially unchanged both when blocking and unblocking the waste tube, bubble removal has been successful.
After successful removal, the baseline will become stable.
Principle of How Bubbles Affect Detection Values
When the flow cell is filled with either air or liquid, light transmittance is high. However, when bubbles are present, light transmittance decreases significantly. Manually blocking the waste tube compresses the bubbles, increasing light transmittance and causing the A/V value to drop sharply (become more negative). Understanding this principle helps in more accurately diagnosing and solving bubble problems.
By systematically identifying the location of bubbles, understanding their causes, and taking targeted corrective measures, the operational stability of the liquid chromatography and the reliability of experimental data can be greatly improved. Regular inspection and maintenance of the fluidic system, especially the areas prone to bubble formation, are key to ensuring proper instrument operation.
