Hello all,
I am working in a biochemistry lab using HPLC 1260 Agilent model for several years now.
For the past couple of months we were struggling with the high pressure of the instrument. We cannot get a new pump or replace it (it costs more than the budget we have!. So far we managed to decrease the pressure with running IPA, hot water and sometimes Acetonitrile for several hours followed by washing with water and it always helped us.
The problem is, for the past week, we again have this high pressure problem (more than 200 bar at 0.5 mL/min and none of these washing steps has helped us.
I cannot think of any other solutions right now and would appreciate any comments that you might have.
Hi, does the high pressure refer to the entire system including the column or without column? If the high pressure occurs without the column, partially clogged capillaries, injection valve bores or inline filters (at least the 1200 systems had a PTFE frit located in the purge valve) may be the cause. Try removing the capillaries from the detector further to the injector to localize the problem. If the problem only occurs with the column installed, replace it.
Niloofar Gopal Pour: Troubleshooting must be performed in a logical manner by someone with several years of professional chromatography training. Why do you think you have a "high pressure" issue? Many HPLC columns and/or mobile phase solutions would result in pressures over 200-bars at 0.5 mL/min and be considered normal. What column dimensions and particle size are you using? What is the specific mobile phase used???? Please Specify the exact conditions you are using so we can put into context what you are doing. Maybe nothing is wrong? Get help from someone familiar with the instrument (they will know which parameters you need to share with us). Note: NOT someone inexperienced, such as "Sasha Nealand" at your school.
Start troubleshooting at the beginning. What is the system backpressure of the system when the prime-purge valve is opened and only 100% ACN is flowing at 1.000 mL/min ? This is a very important basic level test to start with. If the back-pressure with ACN is more than 12 bars, with the PRIME PURGE VALVE OPEN, then your PTFE frit is clogged (replace it every month. It is a maintenance item and when clogged can generate high pressure during your analysis). If it is less than 12-bars, then the frit is not clogged and you can close the valve and proceed with troubleshooting by replacing the column with a calibrated restriction capillary (this is what we use for testing). *After replacing the frit, evaluate the pressure again and report back.
- This article is very important to read. We find so many clients ignoring the basic maintenance of their HPLC system, esp at schools (most never had any training) and observe increasing back-pressure over time. They do not understand why. The solution is often very simple. Maybe it is the reason for your issue? "Troubleshooting a GRADUAL HPLC PUMP PRESSURE INCREASE OVER TIME (When PURGING, DURING an ANALYSIS or when FLOW is DIRECTED TO WASTE)"; https://hplctips.blogspot.com/2024/04/troubleshooting-hplc-pump-high-pressure.html
"The HPLC Restriction Capillary; Troubleshooting, Qualification and Running Without A Column"; https://hplctips.blogspot.com/2018/04/the-hplc-restriction-capillary.html
"Pressure Drop Across an HPLC / UHPLC Column"; https://hplctips.blogspot.com/2011/08/pressure-drop-across-hplc-uhplc-column.html
Andre Stratmann Thank you for your advice Andre. This is the pressure without column and yes, we have resolved clogs in the tubes before. However this time it is not resolved with all the solvents I tried. I will check the filter. That is something I never checked before.
Thank you very much!
William Letter Thank you for your detailed answer and the valuable links William.
I consider the current condition high pressure based on my previous experience with the same system. I have been using this instrument for 6 years in the same condition, and the pressure has never been this high (for us, 30-40 bar is normal for 0.5 mL/min no column, open purge ACN: below 10)
My mobile phase is water.
I have not attached the column and this problem is for the instrument with no column attached.
Based on your explanation, I realized it is highly possible that our PTFE filter is clogged. Back pressure with ACN is about 200 bars with an open valve which explains the issue. I will try to change the filter because we did not need to do this before.
Thank you for sharing your information. It was very useful!
Niloofar Gopal Pour wrote: "Back pressure with ACN is about 200 bars with an open valve which explains the issue."
If your system shows more than 12 bars with ACN, at 1 mL/min, and 200 bars is certainly far too high, then extreme abuse has occurred and you may have far more damage then just frit replacement (but replacing the frit is still the first place to start and see what happens). How could anyone ignore such a finding for so long???
The HPLC system is designed to have the pump's PTFE frit replaced each month as part of maintenance (a ~ $6 disposable part). We teach this in our introductory HPLC training classes to new students. Learning proper maintenance procedures are critical to using the instrument.
Additionally, please check and replace the solvent pickup frits used with your pump. Depending on filter type (glass or stainless steel) and the mobile phases used with, these important filters should be replaced every few months (esp if you use the glass ones, as they can not be cleaned) or if you use 316 SS frits, they can be cleaned in solvent (ultrasonic) and often re-used. Clogs or obstructions in these pickup frits may lead to increased system back-pressure. Both the pickup filters and THE PTFE are critical filters, requiring attention to maintain a clean flow path for the HPLC. It sounds like your HPLC system has not been properly maintained for many years. It may be wise to bring in a professional to have the system fully cleaned and serviced.
You must budget for parts, supplies and training to use these complex tools. Additional money should be put aside for repairs and consumable items too (i.e. columns, solvents etc). This general article may also be of interest to your school's lab group.
"HPLC Maintenance & Repair Parts To Have on Hand for HPLC Systems"; https://hplctips.blogspot.com/2013/11/maintenance-parts-to-have-on-hand-for.html
Also, if anyone tells you, or ever suggests to you that one way to solve a high-pressure problem (esp one where no column is being used and the pressure is high when the system is directed to waste!) to "running IPA, hot water and sometimes Acetonitrile for several hours followed by washing with water", then please IGNORE that person. They have no understanding of HPLC systems at all and following their suggestions may waste time and/or cause serious damage to your HPLC system.
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