I'm currently using an HPLC system (Agilent 1100) with VWD and RID detector. My system has been down for about a month for RID repairs (optical unit failure, repaired from Agilent itself), andsince the RID is back, I can't seem to stabilize it.
Corrective and preventive actions:
- Pump pressure unstable: Changed the Outlet and Inlet check valves - Temperature well controlled in the RID (35°C) and the column compartment (60°C)
- Flushed system with MeOH and IPA to remove potential microbial growth during stop time
- Flushed RID with water, with a capillary after the detector to have backpressure and remove bubbles
- Mobile phases (all aqueous) degassed (sonic bath) and inline degasser
Most importantly, the system was stabilizing normally before the optical unit repair, with the same mobile phases and same conditions.
Thank you!!
Proper troubleshooting is needed to solve the problem. You say the system was down for one month. That is a long time so you should assume nothing and start at the beginning to make sure all systems are working as designed. This must start by first establishing a smooth pump cycle. Basic fundamentals of liquid chromatography must be followed to achieve this. Let us take a look at just a few of the basics:
(1) Degassing. You must have a mobile phase degassing in place. This could be from continuous helium sparging or the use of an inline electronic vacuum degasser (e.g. G1322A or G1379A/B). *Sonication is nearly worthless. If using a vacuum degasser, then the module must be operating perfectly, and not due for service (vac degasser modules require service every 3-5 years, or earlier if a problem develops (esp the G1379-series which are prone to early failure). Degasser modules must be fully flushed before use and must not have a steady YELLOW or RED status light showing after ten minutes of ON time). Broken degassers contribute to mobile phase contamination. They are critical to achieve a reliable baseline with an RID!). We see hundreds of clients running broken vacuum degassers and wondering why they are having baseline problems (the answer is right in front of them). LINK: "Common Degasser Errors"; https://www.hplctools.com/Troubleshooting%20HPLC%20Degasser%20Repair.htm
(2) Properly prime, flush and evaluate the system: Remove the HPLC column. Prime ALL the pump channels with fresh, clean liquid. Next, install a capillary restrictor (in place of the column) and use it to achieve a back pressure of about 50 bars at the desired flow rate you plan on using. With the restrictor in place, set up the pump composition needed (%) and run the mobile phase through it. Monitor the pump back-pressure "stability" (known as "ripple"). It should be less than 1% (often, ~ 0.1% with an Agilent 1100 Quat or Binary Pump). Fouled solvent pickup frits, MCGV channels, sticking valves and/or a fouled AIV will result in cyclic (up/down) operation with air bubbles in the lines. If you can not achieve a nice steady flow under these basic conditions, then there is no reason to proceed with your method. You must first establish and demonstrate that the pumping system has been maintained and is in good working condition. *Establish a clean flow rate with stable baseline, low pump "ripple" before doing anything else.
You may need to perform preventative maintenance on your pump (replace the PTFE frit, replace the piston seals, replace the pistons, replace the AIV cartridge). Normally, the AIV and MCGV lasts for ten plus years so do not require service unless abused. The outlet valve should also last a decade or more and generally only needs to be run in an ultrasonic bath for 20 minutes (placed in a beaker with some pure water and HPLC grade IPA). Run a Pressure Leak Test (Max pressure tightness test) and also a Pump Leak test after servicing the pump. It should pass with ease.
Two other comments:
(1) You mentioned your column is maintained at 60C, but your RID is held at 35C. That makes no sense. You need to establish the same temperature (or close to it) for the mobile phase as in the detector's flow cell. Any difference in temperature will establish a temperature gradient (baseline shift that goes on forever!). *Place all SS lines inside Teflon tubing to insulate them. Set all thermostats to the SAME temperature. Keep the connection lines as short as possible and out of the ambient air. Maintain ROOM temperature within 1C, if possible, to minimize drift.
(2) Throw out the HPLC column if there are any concerns about its suitability. Columns are inexpensive consumable items. Using a fouled column will waste thousands of dollars worth of solvent, chemicals and TIME. Always have a new one available, as a spare, to save time/money.
Some links to articles which may help you.
- "HPLC Baseline Stabilization Tips for Refractive Index Detectors (RI or RID)": https://hplctips.blogspot.com/2018/02/hplc-baseline-stabilization-tips-for.html
- "Common Causes of Baseline Noise in HPLC, UHPLC"' https://hplctips.blogspot.com/2014/09/common-causes-of-baseline-noise.html
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