Chromatography Forum

Dear Members,
We have a method using methanol – 0.1M ammonium acetate (70:30) as the mobile phase. High salt concentration is needed to avoid tailing peak.
Column: 150 x 4.6 mm, C18, 5 um
Temperature: 25 C
Flow rate: 0.8 – 1.0 mL per minute (depend on retention time of the main substance).
The problem is gradually increment of the pressure during run (approx. 1 – 2 kgf/cm2 per 15 minutes) with or without sample injection. We have tried both premixing and injection port flushing, but the increment was still occurred. Could anyone give me suggestion to prevent this pressure increment?

Note: The pressure would be back to normal after the column was washed with water – methanol (30:70).

Best regards,
Siswanto Tanuatmojo

Syx,

The pressure increase comes probably from the high concentration of ammonium acetate in your mobile phase. Maybe pressure increase comes from partial precipitation of the salt in your chromatographic column. Maybe, if it does not affect your chromatography, and assuming you have a column heater, you could increase a little bit your column temperature 5-10 C to increase the solubility of the salt...

Actually maybe there are better ways to keep your compound from tailing than 0.1 M ammonium acetate but I assume that changing your method is not negotiatiable...

I am observing samething with my mobile phase ACN:Buffer (sodium acetate 20 mM, pH 4.0) and using premixed. However For my case some air bubble get entrapped from place unknown, tried potentially every tubings etc. when I open the purge valve a bubble comes out and after just moving this buuble out pressure again comes to same but start rising very gradually. In my case everytime there is a buuble. But Dont know where from it enetring using vaccum filtered sonicated mobile phase.

teflon tubing is somewhat permeable to air. It's possible that you are getting air diffusing in via the line from the reservoir to the pump.

What if changing the method is not an option? The method gives satisfied result… but should be done not more than 6 hours to stay within acceptable pressure area.

Buy a column switching valve, the cheapest binary pump you can find, and two new columns from the same lot. While one is washing (cheap pump), the other is running (good system). The validation and calibration is a bit trickier, but your software should be able to handle it. See, there is no problem you can't solve with new toys. Of course, if your volume of work is not big enough, don't do it.

Dear
Why do u go for higher conc of buffer u can go with lower pH, it will give u better symmetry, think so it can solve u r problem. Otherwise u can increase temperature 35 to 40 ° C


Raj B

Raj-Can you please detail me whats the relationship between using higher pH with gradually increasing pressure.


I look for your perspective in this regard.