Dear Samir,

"all my peaks appear right after the the solvent Peak" - this sounds like incorrect eluent composition (too high % organic) which would also explain the lower pressure than anticipated. I would clean all frits (in the eluant bottles) and valves. You may also want to check the gaskets of the pumps.

Hope this helps!

Best regards

Holger

Dear Samir,

"all my peaks appear right after the the solvent Peak" - this sounds like incorrect eluent composition (too high % organic) which would also explain the lower pressure than anticipated. I would clean all frits (in the eluant bottles) and valves. You may also want to check the gaskets of the pumps.

Hope this helps!

Best regards

Holger

Do you monitor the pressure profile during the run? Is the pressure profile smooth or is wavy with regular pulsations? If this is the case, a part called check-valve on the pump needs cleaning. Make sure that you de-gas the mobile phase under vacuum ultrasonication. When ACN and water mix, the solubility of dissolved air decreases which leads to bubble formation. You did not mention the mobile phase composition. Inorganic salts precipitate in high ACN concentration and it may take a long time to wash off the precipitated salts esp. if it is a phosphate buffer. 

You will not see an instantaneous change in pressure when there is there is sudden change in composition of the mobile phase. There is significant volume, the so called dwell volume, after the pump and before the column. 

What is your pressure when the column is off line?  What is the pressure when you make null and blank injections?  Is your sample clear and filtered through a 0.10 um filter?

Simply I will suggest if possible go for calibration/ validation the HPLC, it may help.

As mentioned above salts dissolved in the mobile phase could be an issue. These crash out of solution at high concentrations of ACN. If this does happen one way to dissolve them is to run very hot water through the system without the column. This will clean your HPLC to clean the column I use a column heater and run water through it. If it is not salts. You can use the program lab adviser and run a pressure test, leak test, and valve test. You need a capillary to and blank nut to do these tests.  To get into lab advisor shut down the engines. Go to all programs Agilent Technologies then lab advisor. Open lab advisor and connect to the instrument by clicking connect. On the left I believe you will see service and diagnostics click that and then click the pump. This will allow you to choose what test to pick. I would do the valve test first. I think your active inlet valve might be bad. If it is be sure you order the valve and the cartridge that goes inside it. Also be sure not to over tighten and crush the cartridge. If the valves are good then a leak test would be next. If will walk you through this in the software.  Best of luck to you.

Dear samir,

As mentioned above salts dissolved in the mobile phase could be an issue. Do you use any additive in the aqueous phase as a buffer? I would recommend an acid or an base (depending on their compounds and their column). Moreover, the solvent of the sample should be similar to the original terms of chromatographic method, so I'd advise against injecting pure acetonitrile.

Kind regards,

Beatriz

it seems you need to sonicate guard column, if its there in methanol. 

second you can try to give high end organic solvent wash to column.

check this may it work

best of luck

KK

Dear Raju, There have been many studies that columns (and guard columns) should not be sonicated. Sonication causes the chromatographic bed to re-arrange and eventually settle. Sonication usually creates a void at the column head.

Hi Samir,

If a hardware issue is causing the problem, the problem isolation and diagnostics depend of the hardware configuration.  If you could let me know the individual module models, ie G13XX,  in the stack, the eluents, any gradient profile, basically your method conditions, and I will try to help you out.  Thanks.  Doug

Good afternoon everyone.

First of all, thank you very much for your input.

I am using the following conditions:

• Column : Kinetec C18 column, 150 mm × 4.6 mm, 2.6 µm particle size (Phenomenex, U.S.A.);
• UV/Vis detector: 280 nm; Injection volume: 20 µL; Flow rate: 0.6 mL/min
• Mobile phase: H2O (containing 2.5% Acetic acid) – Solvent A; ACN – Solvent B. Gradiente: 0 min, Solvent A/B, 80/20; 24 min, Solvent A/B, 20/80; 30 min, Solvent A/B, 20/80; 30 min, Solvent A/B, 20/80; 33 min, Solvent A/B, 80/20; 40 min, Solvent A/B, 80/20

The Agilent modules are G1322A (degasser), G1311A (pump), G1313A (autosampler) and G1316A (column).

We did a pressure test and leak test. We found that the pump was droping pressure faster than it was supposed to.

So we opened the pump and cleaned it.

We performed again the tests and it passed.

However, the pressure is very unstable, varying almost 10 bar.

The chromatograph is much better though.

Any thoughts now? 

Once again, thank you very much.

Pressure increase due to the compositions of the mobile phase (as listed) should be minor.  Pressure will increase as more water is pumped.  However, it would make sense to reverse the column wash it with 95% water, 95% ACN, then mobile phase.  I would assume the column has been previously used and abused by not thoroughly washing out the buffers.

Hi Samir,

Thanks for the additional information.  The G1311A uses low pressure mixing thru a multi channel gradient valve to create the correct solvent composition.  A tube carries the mixed solvent from the MCGV to the active inlet valve on the pump head where it enters the first of 2 pump chambers.  the AIV closes and a sapphire piston pushes the eluent thru a sapphire ball and seat out let check valve to the pressure sensor/pulse damper.  A stainless steel tube brings the solvent back to second pump chamber on the pump head where a second sapphire piston operates 180 degrees out of phase with the first piston, so one piston fills, while the other piston delivers.  the eluent is then pushed thru the bypass valve to either the column or waste.  The bypass valve has a particulate filter in its base.

I  would recommend some easy, no tool, checks.  first, open the bypass valve about 1/4 turn. set the flow rate at about 1 ml/min with water or ACN. check the pressure.  if the bypass valve filter is not plugged, the pressure usually runs 0-1 bar.  if its higher than that, the filter may be plugged causing backpressure on the pump head.  this can cause backpressure so air bubbles don't flush out of the pump with a purge.  this can cause the pressure variation your seeing.  the procedure to change the filter should be in the user manual.

Second, check the MCGV for leakage.  make sure all the solvent lines, A-D are full of eluent. power the pump off and remove the tube from the MCGV to the AIV.  There should be a finger tight nut on the tube going into the AIV.  Remove the tube from the AIV.  If the MCGV is sealing properly, no solvent should come out of the tube.  If solvent comes out with the power off, the MCGV is not sealing and probably needs to be replaced.  This can cause the solvent composition to be incorrectly proportioned and cause poor peak resolution and retention time shifts.

Next, check the solvent filters in the eluent bottles for plugging.  with the eluent lines full of solvent to the MCGV, remove the lines from the MCGV and check for adequate flow.  If flow seems restricted, remove the solvent filter and repeat.  If the filters are restricted, replace them.  I've never had any positive results trying to clean them.

Check if you can see any air bubbles in the lines from the degasser to the MCGV,  MCGV to the AIV when the pump is running. could be a bad ferrule, leaky line, bad MCGV, or the degasser isn't working.

I would try this and if still having issues, let us know.  There are several other more involved things to try.

Good luck. 

Simple ,Wash with hot water Before starting any analysis.By this all the settled buffer particles can be dissolved and removed .