Chromatography Forum

Hi,everybody
i repeat an experiment of BP(azithromycin). in the "related substances",it use 1.8g/L Na2HPO4 adjusted pH to 8.9 as mobile phase A ,CH3OH/CH3CN(1:3)as mobile phase B. Column:WATERS Xterra.
The method uses gradient elution.0min(50%B)-25min(55%B)-30min(60%B)-80min(75%B)-81min(50%)-93min(50%B). And my column pressure is drifting all the time !!
PS:The HPLC i used is SHIMADZU 2010,its channel ID is relatively micro, can it be possible sulphate educed in the channel??

Your A and B phases have different viscosities, so it is normal for pressure to vary as your mobile phase composition varies. As long as it holds pretty steady when conditions are not changing, and your peak retention times do not move around much, you're OK.

Your A and B phases have different viscosities, so it is normal for pressure to vary as your mobile phase composition varies. As long as it holds pretty steady when conditions are not changing, and your peak retention times do not move around much, you're OK.

yes, the column pressure's dirfting is usual in the gradient elution,but my column pressure drifted very greatly,sometimes is 14MPa, and then turn to 0.3MPa!! Is this related to the instrument?

I think it's due to the column bed. It's kind of polymer column and it swells at high content of organic mobile phase,, thus the pressure increases.

Influence from the column is possible, but not from 14MPa to 0,3MPa, an XTerra column is still a silica based column. Please check flow rate from pump A and pump B, and if the magnetic valves are working properly. Second step is to check for leackage. What is your flow rate and column dimension?

Influence from the column is possible, but not from 14MPa to 0,3MPa, an XTerra column is still a silica based column. Please check flow rate from pump A and pump B, and if the magnetic valves are working properly. Second step is to check for leackage. What is your flow rate and column dimension?

Thankyou, my flow rate is 1mL/min, column is 4.6*250mm. I think it's not the problem with the column.I suspect the nonreturn valve has problem.

Hello!
I have the same problem with the column pressure when working on Azithromycin related substances. What can i do to solve the problem? We worked on 4 different HPLC's, and we noticed the same problem. We changed the column and there was no change. The problem is still there. Please give me a hint!
Thanks.

Are you seeing pressure drift (steady pressure change throughout run) or pressure fluctuations (sudden random changes in pressure)?

I'm seeing pressure fluctuations (sudden random changes in pressure: from 110 bars to 80 bars, then suddenly pressure drops at 30 bar, then it rise back to 80, and that's the way it goes). The pressure graphic looks like a heart electrocardiogram, with up and downs.
The method is from EP 6.8 "Azithromycin". In my opinion is a lethal weapon for any lc chromatograph. The solvent mixture is 1.73 g/L solution of ammonium dihydrogen phosphate adjusted to pH 10.0, the mobile phase A is : 1.80 g/L solution of anhydrous disodium hydrogen phosphate adjusted to pH 8.9 and the mobile phase B: methanol R1, acetonitrile R1 (250:750 V/V);
Time (min) Mobile phase A Mobile phase B
0 -25 50 → 45 50 → 55
25 - 30 45 → 40 55 → 60
30 - 80 40 → 25 60 → 75
80 - 81 25 → 50 75 → 50
81 - 93 50 50

Flow rate: 1.0 mL/min.
Detection: spectrophotometer at 210 nm.
Injection: 50 µL
temperature: 60 °C
column XTerra MS C18; 250x4.6; 5 microns
Our HPLC's are from Agilent and have quaternary pump.

I have validated that method and had same column pressure problems , even my column was dead after working a few days from begining of validation. I have been using same column that is refered by EP (that Xterra ) but it did not last long enough to finish validation. I lost 2 columns . I do not know if it was my luck or another unpredictable variable. I thınk column temperature was too harsh ( 60°C ) for constantly working for days and customer support admitted that.
We use an MS column which can resist 80°C with a guard column and have same choromatogram RRT etc publised by EP. I do not remember exact column name now .
Method works for over 1 year in product stability period without any problem now.
Actually I just realized you are talking about a BP method but probably they are same . I hope it helps.

Best Regards.

Can you make an effort to remember the name of the MS column you are using?

Thanks.

I really wish to remember that column but I left from company where I have validated that methot long time ago.
All I remember , column has same dimensions , chemisty ( C18 ) but and MS quality column which can resist temperature up to 80°C . Probably Xterra MS C18 but I really do not want to give you wrong information.
I also remember column condition progress was taking a bit long time before to get identical gradient profiles and static starting and ending pressures . I recommend you change your pump frit before analyses. This has been giving me less pressure problems and less unwanted peaks due to gradient profile .

Best regards.

We use a guard column with a XBridge column (that can resist up to 80 degrees C) and so far we dont have problems. Thank you very much for helping me out.

Thanks again,
Daniela