UPLC issue...high pressure
Posted: Fri Aug 10, 2012 1:35 am
Im having a high pressure issue with this method below. 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. My conclusion is that its the column!!....correct....but its very strange that both columns arent working i.e
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....so it must be the column! the method was developed before and im doing a method transfer from one site to another so the method worked previously.....the only thing i can think of is that the columns werent maintained properly (they are not new columns im using). I have the system on now and if i slowly increase the flow and keep its constant the pressure is dropping gradually. what do you guys suggest? oh yes as well the mobile phase is just solvent and im even filtering that with a 0.2um filter.
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
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....so it must be the column! the method was developed before and im doing a method transfer from one site to another so the method worked previously.....the only thing i can think of is that the columns werent maintained properly (they are not new columns im using). I have the system on now and if i slowly increase the flow and keep its constant the pressure is dropping gradually. what do you guys suggest? oh yes as well the mobile phase is just solvent and im even filtering that with a 0.2um filter.
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