Chromatography Forum

Hey,

I am working on the Analysis of a quite "sticky" compound (bile acid phospholipid conjugate with lipophilic rings and chains as well as integrated hydrophilic parts) which somehow contaminated our Waters System.

Blank run with only MeOH shows our analyte as contaminant http://i.imgur.com/ul95SnN.png
Now this is going on for 3 weeks now and I have flushed the System with many solvants:

3times with 15 column volumes of each (normal and reversed column direction): H2O:AcN 95:5, THF, H2O:AcN 5:95
1time with 20-30 column volumes of each (normal column direction): 100% methanol, 100% acetonitrile, 75% acetonitrile–25% isopropanol, 100% isopropanol, 100% methylene chloride, und 100% hexane.

Now we tested a brand new column + pre column and the contamination as seen above persists. What parts of the machine can I clean or have to replace that might provide this nice and comfortable home for my analyte?

Looking Forward to your help

Thanks!

Assuming this is a carryover issue (you dont really say) there is a frit in the injector which you can change, which can help. Also, a change in your needle wash might be called for (7:2:1 ACN/IPA/water + 0.1% formic).

as AA said, you could replace the needle wash frit
then use a needle wash solvent that dissolves your compounds well

beside of this:
Waters has a document online, dealing about contamination in LC/MS:
"CONTROLLING CONTAMINATION IN LC/MS SYSTEMS"; #715001307

there are some suggestions about cleaning solutions in it, like the "magic mix"
somewhat ACN/MeOH/IPA/H2O, 25%each + 1% formic acid.
- disconnect the column (replace it with a restriction capillary), flush all lines with the cleaning liquid. let it flow through the system e.g. over-night at low flow (e.g. 0.2 ml/min).

if not succesfull, you could try a cleaning with nitric acid 6 mol/L (LC only)
As always: be very careful when using HNO3. Dangerous reactions may occur when in contact with organic solvents.
- collect all effluent separately
- flush the whole system (all lines and injector etc) with sufficient amount of water first
- then do the nitric acid cleaning (about 30-50ml)
- flush the whole system with water again until the effluent's pH isn't acidic anymore

Thanks very much! I´m quite new to this and appreciate your help, I will try it.