Chromatography Forum

Hello,

I'm having issues with air bubbles in a gold system HPLC and the resulting oscillation on the UV reading.
I have looked at previous postings here and I have troubleshot the list below:

1) Not a degassing or mixing cell problem: Solvents were fully degassed and the problem does not happen when bypassing the column.

2) I have tried multiple columns: All of them show the presence of equally spaced air bubbles after the column. Even when the column has very low back pressure.

3) I have tried adding a narrow capillary right after the column and right before the UV detector entrance: The problem persists.

The UV oscillation is so bad it is killing my UV reading and resolution. I have tried changing the UV reading frequency and had no luck.

Any advice would be helpful.

bests

J. Paulino

You have not provided ANY basic HPLC method conditions so how can we make suggestions? Please provide all of your method details. Brand and model of HPLC, Flow rate, % composition, mobile phase, detector settings, flow cell type, all column dimensions and type and so on. Statements such as "1) Not a degassing or mixing cell problem: Solvents were fully degassed and the problem does not happen when bypassing the column." does not tell us what we need to know. What did you replace the column with? Was it just a zero dead volume union or short section of tubing? What was the back-pressure before and after this change? What is the period of oscillation (if it changes that way) ? What is magnitude of it in real units (scale)? Help us help u.

Try adding a capillary after the detector. Note: make sure the back pressure of the capillary does not exceed the the maximum pressure of the detector.

Hello,

Thank you replying. I apologize for the lack of information previously.
I’m using an HPLC sytem gold model 125 from Beckman. The detector is a model 166 with a optical flow cells of 4uL/5mm/ss;2.8ul/0.5mm/ss.
I have tested 2 columns:
1) an old C4 (300 Å, 5 μm, 4.6 mm i.d. x 250 mm), which shows the UV/bubble behavior at flow rates as low as 0.1 mL/min all the way to 0.4 mL/min
2) a newer POROS™ R2 10 µm (4.6 x 100 mm), which only shows UV/bubble at flow rates of 1ml/min (which is the minimum flow rate recommended for that column).

The run condition:
flow rates tested 0.1 to 1 mL/min
mobile phases: H2O 0.1%FA and MeOH 0.1% FA
mixture: 50/50 A/B

I have bypassed the column with a dead union connector that gives a lower back pressure than with the column.

The UV oscillations are on the +/- 0.5 AU.

I assumed the problem was due to a difference in pressure before and after the column, but since the addition of a capillary did not help I'm at a lost.
I have not tried the capillary after the detector, that will be done soon.

bests

Do you have a dynamic or static mixer, what volume?

Since air bubbles are continuously present , you may try to replace the tubing going to the detector inlet , there may be a crack somewhere.