Chromatography Forum

Someone used an empty 4 liter bottle of HPLC grade water for some sort of waste, the bottle was not labeled in any way to indicate that it was not HPLC water. I used the bottle to make a 0.1%TFA mobile phase. I noticed that my baseline looked horrible, and when running a gradient the pressure was different then what it usually is. I went to make new mobile phase and noticed the 4L bottle of "HPLC Water" had an off smell. I took the pH and it was 4.3, and has a TDS of ~100, its not flammable. My best guess is that it is discarded drinking water samples for ICP-MS analysis, which would have been acidified with 0.1% nitric acid

I attempted to flush the system with IPA at 0.5ml/min over night but still have high pressure, and a nasty baseline. Any suggestions on the best way to flush out the system? I replaced the frit in purge valve, replaced my guard column, and am about to back flush my column.

To make matters worse I was initially making new mobile has due to baseline noise which I suspect may be due to a lamp going out, so now i'm trying to troubleshoot contaminated mobile phase, a column that is dirty from the contaminated mobile phase in addition to a possible dying lamp(6718 hours).

My plan
replace lamp
run lamp diagnostics
backflush column (on different instrument)
flush lines/system of contaminated water

I think that change of the lamp not corrected pressure problem. Better tray new column with new buffer.

It would be really helpful to know EXACTLY which make, model and type of HPLC system you are using, esp the model # of the pump. Constructive suggestions depend on it. *Be sure to find and work with an experienced chromatographer when following the below generic suggestions.

FIRST: Dispose of the column used. *Columns are inexpensive consumable items just like filters and solvent. Trying to flush unknown contaminates from a column is a waste of time and money. Any experienced chromatographer will know this. Any inexperienced chromatpgrapher will not and continue to wastet time and money. All of the data collected on the system using the old column afterwards may be considered invalid so dispose of it and replace with a known clean fresh column AFTER flushing down the flow path and servicing the instrument as described below.

Using contaminated mobile phase in your system also implies that the solvent pickup filter(s) (connected to the bottle line) should be: If glass, then disposed of; If stainless steel, then properly cleaned before re-use.

Next, place a restriction line in the HPLC (no column) to create enough back pressure to run the pump(s) and flush, in this order, Pure Water, Pure Methanol, Pure IPA as "flush" solutions through the flow path to waste. Repeat with acidified water, then acidified methanol next, back to pure water, then pure MeOH. This may remove some of the unknown contaminates. Replace the pump's inlet filter again (they are cheap).

Your HPLC inline degasser membranes are probably contaminated from exposure to the dirty solution used. Depending on model, degassers require professional cleaning and service every 3 to 5 years so if due, this may be a good time to have it serviced. The membranes may be fine, they may be damaged or contaminated too and the only way to know if to have the system professionally serviced. The membranes are easily damaged from contact with a long list of chemicals (which may have been in the solution you used). In any case, for now, do not run fresh mobile phase through the degasser to the column. At the very least, make sure you flush out the degasser separately or better yet, have the degasser professionally serviced first. *A contaminated degasser will continue to contaminate the entire flow path of your HPLC system ["An Often Ignored HPLC & LC/MS Contamination Source. Did you check your Vacuum Degasser?"; https://hplctips.blogspot.com/2015/08/a ... -lcms.html ]

As you also note that your UV/VIS lamp may or may not be bad (if it really has over 60K HOURS, then it has been bad for at least 58K hours so probably no one is maintaining this instrument or resetting the counter when they install a new bulb), test it. The detector that you are using will have one or more formal SOP's which describe how to test the lamp's output energy using a specified flow cell, mobile phase and settings. Review the manual. Perform the tests to determine the lamp's condition. Be sure to evaluate the flow cell used for window contamination too as using a contaminated flow cell will result in misleading lamp energy test results.

Also, run a wavelength calibration too, after verifying the lamp and flow cell are ok.