Chromatography Forum

Im having a high pressure issue withour UPLC for 6 weeks now. I removed the guard column thinking that was the issue but it made little difference. When i used the union i was getting a pressure of 1200psi for 2ml/min flow rate which is fine. Where as with my column i was getting over 15000psi at 0.5ml/min with the column. So i tryed a new column (same spec) and the same issue occured i.e high pressure. When i have nothing on the line ie no column or union the pressure is 70psi.....so this again suggests no blockage of any line.... the method was developed before and im doing a method transfer from one site to another so the method worked previously and it it is infact a waters method for explosives. I got a full PM done on the system yesterday but the problem is still there!!It cant be the moblie phase cause i used hplc grade and i filter through 0.2um. The engineer said that the seal wash housing and the seal ring were worn and heavily oxidised and need to be replaced....could this be the issue???...he said it could be introducing air into the sytem


Column: C18 Column: Acquity UPLC BEH C18, 2.1 x 100mm, 1.7µm or equivalent.

Column temperature: 50°C

Autosampler temperature: 5°C

Detector: Photodiode Array (PDA) detector.

Wavelength: 254nm and 210nm

Injection Volume: 5µL

Eluent: Mobile Phase A – 100% Organic free water

Mobile Phase B – 100% Methanol

Gradient:

Time
(min) Flow
(mL/min) %A %B Curve

Initial 0.500 69 31 -
4.00
0.500 69 31 6
9.00
0.500 40 60 8
10.00
0.500 40 60 8
10.10
0.500 69 31 8
12.00
0.500 69 31 8

What pressure do you normally see with this analysis? Have you confirmed that the column heater is in fact working and that you are actually getting correct flows from your pump?

i have run the analysis and when it has "behaved" i had pressure of about 9000psi but its so erratic!. After the PM on friday it looked all good. the enginner put the flow up to 0.5ml within one minute and the pressure was 9200psi...which looked great....it stayed like this for 20 minutes then it gradually starting increasing and increasing till eventually it was going to max out at 15000psi and crash the system. I think what your suggesting that it is the method is the problem but what method seems fine at the begining and starts going crazy after a few minutes! and then when i started the sytem again i couldnt even get a 0.2ml flow rate without the system going over 15000psi. I beleive the system is getting air in it from some where.....but even when i do all the priming and purging the problem still persists so it must be being introduced constantly from somewhere....although i could be wrong. i will again state what the engineer said to me about the seal housing being worn and therefore this is where the air is being introduced but i got to wait for a week and a half before that part comes in. So what else can i be doing......i have done alot of trouble shooting and really have run out of ideas. remember it just had a full PM ( except the seals) so that eliminates alot of things.

When you replaced the column, was it from the same lot as the previous column? Maybe you have gotten a bad lot from Waters?

Do you have 0.2-micron filters inline between the pump and autosampler and the autosampler and the column? I know you pressure checked the system without a column and with a restrictor, but if you have small particulate buildup over time, maybe the capillaries won't plug but a column will.

Also, for the method, if you have the ability to control the temperature over time, make sure you don't have a wrong setpoint for the temperature compartment somewhere in the method after t0. 9000 psi at 50 C may become 10000+ at 25 C. And verify that it will in fact hold a setpoint using an external method (like an IR temperature gun).

Columns are from a different lot. In fact i just used some other columns for different methods and the same pressure issue occurs. I have 0.2um guard colunm filter on and i have Stainless Steel Slip-On Inlet Filter in the mobile phase. I also filter all my mobile phase,strong wash seal wash etc with 0.2um filter and my mobile phase is just solvent and obviously hplc grade. I see what your saying about the particulate build up and would explain why it is gradually increase in pressure. but i beleive with the filters im using that it would eliminate that possible cause. my method is set up properly too .

also i would like to add when i bought a brand new column the pressure issue happened immedialtey so that would suggest it is not the column. The system it self was not used for 6 months before i came to it...i really hope something hasnt grown inside some pipe....but again i really doubt it.....i cleaned it extensively with solvent when i came to it and then before i got a new column i washed it with IPA,methanol, water for about 8 hours....there is not much more i can do than that

If I understand correctly:
You had a flow of 0.5ml/min on your system, isocratic and after 20 minutes your pressure is ramping up?
Maybe you should repeat this with only pumping A, and see if the same happens, if it does, check everything in where the mobile phase flows: your bottles, your solvent filters, your lines, ...
If not, try the same with B, if not, try the combination, if not, start using gradients, ...
Just try to narrow down if possible.

Ace

Im using a gradient with a 0.5ml/min flow.
4.00 69 31
9.00 40 60
10.00 40 60
10.10 69 31
12.00 69 31
I have tryed all of those you said but found it acted the same way with all the pumps.

You may want to make sure the system is clean by flushing with isopropanol, and maybe some dilute nitric (check with Waters to see if this is recommended). Or, maybe some mix of polar/non-polar solvents - Agilent sells an HPLC Flushing solvent that is (IIRC) 50% IPA, 25% MeCl2, 15% MeCN, and 10% cyclohexane. I have had service engineers tell me to replumb the LC to pump solvent directly to a hot column compartment and then back to the autosampler and then to waste, using that mixture. It would seem that you might be seeing chemical contamination of the column, and if all things are equal between the instrument upon which you developed the method and this instrument that has been sitting for 6 months, you may want to suspect some sort of fouling has occurred.

Perhaps before you go through all the trouble of solvent flushing, you could instead bypass the degasser and re-run the test you performed (isocratic over 20 minutes)? That would eliminate the degasser as the contamination point. Then you could bypass the injection valve and go straight to the column compartment, etc, to see if it is one particular component that is fouling the column.

ive already done the clean down of the syetm with IPA etc...did that about 3 weeks ago....as you can see im running out of ideas.I ran the column vs union on the system again and this is some of the results i got:
Flow psi Mobile Phase Condition
0.5ml/min 250 50/50 Union outlet disconnected
1.0ml/min 500 50/50 Union outlet disconnected
0.5ml/min 3151 50/50 Union/All connections connected




when I put the column on and disconnect the column out let I still get extremely high pressure. For example 6000psi for 0.100ml/min. Also if I put the union on and try to put the flow rate up to 1ml/min the pda outlet and inlet start leaking. I will state again it cant be the column that is at fault. This a new column with no injections run on it . So it doesn’t make any sense that the column can change so dramatically.

did you already replaced the tubing from the injection port to the column?
Maybe it is partially blocked?

Ace

Ok i believe i have the issue solved...fingers crossed.....turns out it was caused by multiple issues. The outlet connection from the PDA when disconnected reduced the pressure on the system by about 1000-2000 psi. Our older column ran at a psi of 13500-14000 psi when the pda outlet was connected and when the outlet was removed it ran at about 11000psi. So when we bought a new column, the new column has a higher running pressure than the old column by about 1000-2000psi. So when you add these up the new column with the pda outlet cable attached added a considerable amount of pressure to the already exsisting run causing the pressure to go over the limit of 15000psi and causing the system to crash. Is it normal for a column of the same model and make to differ while running at the same flow rate by 1000-2000psi?

Thanks everybody for your help...confirmed....bad new column from waters and a faulty connection causing all the issues.Have gone back to the old column and it is working fine. Bad luck getting bad column!