Hi Ramkrishna,

Looks like your washing method was not that helpful.

To regenerate your C18 column, I'd recommend the following steps:

1- Start with 95% deionized water:5% Acetonitrile and let it go for at least 30 min.

2- Wash the column with 95% Acetonitrile: 5% deionized water for another 30 min.

If the pressure is still high, run the above but reverse the column to get rid of any trapped compound in your column.

To prevent this happens again, always filter your sample with syringe filter.

Hope it works.

Regards,

Hello, the addition of TFA 0.5-1,0%w/w can help to wash out column.

Dear Ramkrishna,

Attache column to HPLC and run re-generation procedure as follows.

You can give column flushing with the solvent in the ratio 90:10 (Water: Acetonitrile) for 30 minutes thereafter to re-generation procedure is to be done as follows,

1) Keep column oven temperature 35°C

2) Run Plain Water (H2O) for 30 minutes

3) 0.1% H3po4(Orthophosphoric Acid) for 30 min

4) Run Plain Water (H2O) for 30 min and check pH at outlet with pH paper to get the idea that orthophosphoric acid completely washed. pH of outlet should be toward alkaline more than 5.5pH.

5) Run Acetonitrile for 30 minutes

6)Run Methanol for 30 Minutes

Then you can use for further analysis.

Regards,

Vijay Bagul

Normally, if the column used has been properly equilibrated and an observed gradual increase of pressure "over-normal" continues run-to-run, this indicates one of two things: (1) That Column fouling is taking place OR (2) you have a clogged HPLC pump OUTLET FRIT. Both of these issues will result in what you observed. More info follows...

Let us take a look at these two problems in a bit more detail.

(1) Column Fouling: Columns ger fouled with sample over time. This may be from overloading the column with sample (use less sample!) or from improper column washing. Make sure you wash the column down with a solution that is stronger than the mobile phase used for the analysis. In your example using MeOH and water ("5 mM Ammonium acetate in a 1% acetic acid solution is basically water) you will need to use a different solvent for this purpose. The wash solvent should be fully soluable with your samples and stronger than MeOH. Perhaps ACN would be best? Remember to separate your Analysis method from your column wash methods. When the analysis method is complete, the solvent composition should NOT be directed back to the starting conditions within the same method (a common newbie mistake). *We used to do this back in the early 1980's, before computers. Professional training today is different and a second 'new method' (known as "The wash method") is programmed to run after the analysis method. Create and save a "Column Wash" method where you change from MeOH in your example to ACN (which is "stronger" and will elute off different material). In this example; Flush the column with acidified water plus a max of 95% ACN. Create and save another method, a third method, that equilibrates the column in your initial analysis mobile phase (this is how the pros do it, using separate methods for each task. Makes it easy to automate the tasks). Now you should have three dedicated methods that can be called on or run sequentially to: equilibrate the column, run the analysis, wash the column, equilibrate the column .... This helps to insure all runs are washed, equilibrated and analyzed the SAME (machines are really good at this).

(2) Obstructed Pump Outlet Filter: As a professional consultant I am asked to troubleshoot many HPLC system issues where the system back-pressure slowly increases over time (days, weeks, months) and while #1 above is often to blame, there is another reason. Inside every HPLC pump is a small filter designed to catch piston seal debris and other larger particulate matter from making its way into the very narrow passages of the HPLC system, especially the injector. These very inexpensive filters are consumable parts which are designed to be replaced on a regular basis to maintain proper function. If they are not replaced, then they fill with debris resulting in a PARTIAL OBSTRUCTION of the flow path. This is observed as a slow increase in system pressure over time. It also may appear as an increase in back-pressure when directing solvent to waste (pump to waste, not to column). When directed to waste only, the expected back-pressure should be very, very low. In fact you should know what the normal pressure should be given a specific solvent and flow rate for your system. If the pressure is higher than normal, then the frit is probably overdue for replacement. Change it. *PS. In a week, an article on this exact topic will appear on my free "HPLC CHROMATOGRAPHY HINTS and TIPS" training blog [https://hplctips.blogspot.com/].

• BTW: HPLC Columns are consumable items and do not last forever. With some applications, 200-injections would be perfectly acceptable before disposal (i.e. amino-acid analysis, Lipids etc). We do not have enough info to know if this is the case here.

"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

For that column I suggest always filter your mobile phase buffer, and samples with 0.2µm filter.

You can also use 0.2um precolumn filter or guard column and change this once the pressure goes up.

what is the ratio of methanol to buffer? is it gradient of Isocratic?

Generally I would not ever run a column 100% water. a post run flush with 95% water for 30 min and graduate to methanol for 30 minutes should do.

Column care instruction for that specific column is available and will provide a procedure for rejuvenating the column, - flush from high aqueous, through to MeOH, then THF and back to aqueous or something like that.