Bubble formation on mixing of solvents can lead to a number of problems in HPLC analysis which can be prevented by degassing of mobile phase.
Unstable and noisy baselines
Lower flow rate precision of pump due to cavitations in piston chamber or effect on check valve performance. Excessive pressure can develop which can lead to eventual pump failure.
Air bubbles passing through detectors lead to spurious peaks
Air bubbles can contribute to flow transfer of mobile phase through the HPLC column due to creation of dead volumes.
Before a discussion on the features of degassing techniques let us understand how the problem arises. Solvents equilibriate with atomspheric gases on exposure to laboratory environment. On mixing the solvents the solubility of air is less than it is in same proportion of pure solvents and excess air will tend to bubble out.
Outgassing can occur on mixing or anywhere in the LC system where rough surfaces produce nucleation sites for bubble formation. Practically it is not necessary to remove the entire dissolved air but only a fraction can be removed to bring it below the supersaturation level in the mobile phase.
Low pressure mixing systems are more prone to bubble formation. In low-pressure mixing the solvents are mixed at atmospheric pressure and outgassing can take place anywhere down the flow path.
On the other hand in high-pressure mixing solvent are blended after passing through the pumps and mixing takes place under high pressure in the mixing chamber. The mixture may be saturated but under elevated pressure outgassing is prevented. Bubble formation, if any, can take place when the mobile phase exits the column and returns to atmospheric pressure.
Commonly used degassing practices for HPLC mobile phase are:
- Helium purging
- Vacuum degassing
Boiling is the most effective technique to get rid of dissolved air completely but it is never advised because of loss of volatile components along with the gases and also it takes a long time to equilibrate the mobile phase to the required ambient temperature conditions.
Helium purging removes up to 80% of dissolved air. For organic – aqueous mobile phase an equal volume of helium for purging is adequate. The rate of supply of helium can be reduced after some time as excessive purging can lead to loss of more volatile mobile phase components.
Vacuum degassing removes more than 60% of dissolved air. One option is to apply vacuum during filtration of mobile phase through 0.45 or 0.22μm porosity membrane filter. On – line vacuum degassing is available on most commercial available systems. Mobile phase is passed through porous polymer tubing placed in a vacuum chamber inside the HPLC. The porosity of the tubing allows expulsion of gases through the walls but the liquid is retained in the tubing
Sonication using ultrasonic baths is common in most laboratories but as a stand-alone technique it removes only up to 30% dissolved air so sonication in combination with any other technique is recommended..
For practical purposes it is advisable to degas mobile phase in both low and high pressure mixing systems and combination of different techniques can eliminate most of the problems associated with bubble formation.
As on-line vaccum degassing is offered on most commercially available systems sonication in combination with on-line degassing gives satisfactory results.
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