Matrix / Ghost peaks on 2420 ELSD

[TMC]

I am having a new problem with an ELSD lipid method that I have been developing. The problem is that recently matrix blanks and samples are producing ghost peaks while diluent blanks are not. The retention times and areas of the ghost peaks are not reproducible from one injection to another. Here is some background information. The diluent is a mixture of hexanes/IPA/water. The matrix blank is 10 % of 400 mM sucrose/20mM phosphate pH 7 in water and 90 % diluent. Samples are centrufuged at 16000 rcf for 30 minutes. HPLC method is 0.7 mL/min 0.1 % TFA in 80 % organic (isocratic at start followed by gradient). Ghost peaks are occurring in both isocratic and gradient parts of the method and have a height of about 1-2 LSU units. ELSD conditions are 25 psi nitrogen, 35 C nebulizer, 50 C drift tube. Injection volume is 30 uL. I have tried to isolate the problem by changing each one of the following variables one at a time:

Cleaned nebulizer with methanol then water, cleaned the drift tube with heated water, removed guard column, tried an older column, new mobile phases, extend the high organic washout, extend the re-equilbration time at the end of the method.

This is a relatively new column (<100 injections) and the method I am using has not changed from when I had no ghost peaks.

Any ideas out there?

[Kostas Petritis]

Are the ghost peaks sharp or potato like?

[HPLCCONSULT]

ELSD's can exhibit "ghost" peaks for many reasons. Too many to list here, but one common one that I see is caused by drift tube sample build up. Over time, most of your samples end up being cooked onto the inside wall of the drift tube. As temperature, humidity and solvents change, bits of sample will break off the wall and fly by the detector causing a peak to be seen. Drift tubes should be cleaned on a regular basis (consult the manufacturer for instructions) to prevent this. Another reason for "extra" peaks is that you may be sending through some non-volatile material (i.e. phosphate) that is also seen by the detector. The ELSD type detectors are great at picking up anything and everything in your mobile phase (and in the tube) so it is important to make sure that everything is soluble and volatile.

*ELSD Method Development 101 Note: Try raising the drift tube temperature and/or increasing the nebulizer gas flow rate to see if your problem goes away. Do this cautiously as you do not want to vaporize "real" things (what you really want to do is set up a series of runs where you inject a known standard (low concentration) using various gas flow rates and temperatures. Vary one at a time. Record the actual S/N ratio of the peak in each run. Study the results and you will be able to determine the best gas flow rate and drift tube temperature for that sample using that method (Only).

Also: With gradient runs, the atomization is changing on the fly (as the flow rate and temperature are not changing in real time to keep up with the system) so some materials can appear and disappear during the run.

[TMC]

Thank-you for your responses.

The peaks are sharp.

Cleaning the drift tube did not help. If the drift tube was dirty, I would think that all injections would have ghost peaks? Your suggestions are good, however the frustrating part is that the method worked fine before under the same conditions.

I am leaning toward particles in the samples/matrix blanks and not blanks as injections from the same vial are not reproducible with respect to the ghost peaks. However I have not changed the sample preparation procedure.