I have an old HPLC machine. Every time when I put my filter into 50%:50% water and acetonitrile mixture, my filter always generate bubbles. However, once I change the solvent to 100% acetonitrile, no bubble comes out. Does anyone know the reason? Why it only happens in the mixture bottle？
ps：I placed the water and acetonitrile mixture overnight to make sure no bubbles before I use it.
So many reasons... Your filters could be dirty. The water is filled with gas (air, because it has not been degassed). Methanol/Water mixtures (1:1) results in an exothermic reaction full of gas, which is completely different than just using a bottle with 100% pure liquid. Mixtures of liquids start off with gas dissolved in them. Pure solvents like ACN have lower viscosity than water when being drawn through the solvent pickup filter (so fewer bubbles are seen), but will compress and out-gas more when compressed by the HPLC pump (after, on the high pressure pump outlet side, which you can not see).
Leaving the solution to sit overnight will not prevent bubbles (leaving water out overnight will just allow bacteria to grow and contaminate the solution and HPLC). The mobile phase solution(s) should be filtered & degassed before and during use.
USE an HPLC degasser (i.e. An inline vacuum degasser or continuously sparge all liquids used with ultra-high purity helium gas). This is standard practice with HPLC to reduce pump cavitation and improve pressure stability over time. Placing the mobile phase bottles containing the solution in an ultrasonic bath (or using a vacuum filter) may help reduce the volume of gas dissolved in the solution, but once you remove the bottles from the ultrasonic bath (or remove the vacuum), the gas will begin to dissolve right back into the solutions.
- Here is a related article link that may be of interest to you. "Air Bubbles Exiting the HPLC PUMP, Reason Why"; https://hplctips.blogspot.com/2020/02/air-bubbles-exiting-hplc-pump-reason-why.html
PS : Always check the fit and condition of the solvent pickup filter(s) to the low pressure tubing to make sure they are secure. Always use fresh clean solvent pickup filters on all lines. Any filters which are kept in pure organic solvents that are filtered often may last for several months before needing replacement (i.e. 6 months). Filters which are used with aqueous solutions or buffers, due to bacterial, algal or even mold growth, often require regular cleaning or replacement, ~ one or two months. Solvent filters are consumable items. Replace them. Failure to use fresh clean pickup filters in your HPLC may lead to total contamination of the flow path (column, pump, A/S and detector(s)).
BTW: I find it useful to tip the flat bottomed solvent pickup filters 45 degrees inside the bottles. This seems to reduce the formation of bubbles rising up through the tube (instead, they are offset, captured).
So many reasons... Your filters could be dirty. The water is filled with gas (air, because it has not been degassed). Methanol/Water mixtures (1:1) results in an exothermic reaction full of gas, which is completely different than just using a bottle with 100% pure liquid. Mixtures of liquids start off with gas dissolved in them. Pure solvents like ACN have lower viscosity than water when being drawn through the solvent pickup filter (so fewer bubbles are seen), but will compress and out-gas more when compressed by the HPLC pump (after, on the high pressure pump outlet side, which you can not see).
Leaving the solution to sit overnight will not prevent bubbles (leaving water out overnight will just allow bacteria to grow and contaminate the solution and HPLC). The mobile phase solution(s) should be filtered & degassed before and during use.
USE an HPLC degasser (i.e. An inline vacuum degasser or continuously sparge all liquids used with ultra-high purity helium gas). This is standard practice with HPLC to reduce pump cavitation and improve pressure stability over time. Placing the mobile phase bottles containing the solution in an ultrasonic bath (or using a vacuum filter) may help reduce the volume of gas dissolved in the solution, but once you remove the bottles from the ultrasonic bath (or remove the vacuum), the gas will begin to dissolve right back into the solutions.
- Here is a related article link that may be of interest to you. "Air Bubbles Exiting the HPLC PUMP, Reason Why"; https://hplctips.blogspot.com/2020/02/air-bubbles-exiting-hplc-pump-reason-why.html
PS : Always check the fit and condition of the solvent pickup filter(s) to the low pressure tubing to make sure they are secure. Always use fresh clean solvent pickup filters on all lines. Any filters which are kept in pure organic solvents that are filtered often may last for several months before needing replacement (i.e. 6 months). Filters which are used with aqueous solutions or buffers, due to bacterial, algal or even mold growth, often require regular cleaning or replacement, ~ one or two months. Solvent filters are consumable items. Replace them. Failure to use fresh clean pickup filters in your HPLC may lead to total contamination of the flow path (column, pump, A/S and detector(s)).
BTW: I find it useful to tip the flat bottomed solvent pickup filters 45 degrees inside the bottles. This seems to reduce the formation of bubbles rising up through the tube (instead, they are offset, captured).
I think it's because of the water, because every time I use the water alone or with a percentage with an organic solvent when I filter I get bubbles, so after the filtration I put my sample in the ultrasound for a few minutes and I inject it in HPLC or LCMS.
I fully agree with William Letter.
In addition to using a degasser you could try a brief ultrasonic treatment, just before you start up the HPLC.
- Badges
- Science method