ELSD and Phospholipids

[FPF]

I am using a Waters ELSD model 2420 with normal phase conditions (hexane, isopropanol, water) to analyize phospholipids. I am experiencing poor signal-to-noise and excess noise during analysis.

I think that the phospholipids are being retained inside the drift tube. Does anyone have a cleaning procedure for the drift tube other than water?

[AA]

It is highly unlikly that these compound are being "retained" inside the drift tube. What evidence do you have to make you think this?

[shaun78]

excessive noise when using an ELSD is generally caused by either too low of a nebulizer temperature, or not enough difference between the temperature of the drift tube/nebulizer (generally, 20C is recomended).

[juddc]

Backing off a bit even from your temperature settings, are you sure that your mobile phase is clean? Dissolved solids or (heaven forbid) particulates can make for high background noise as well.

Are you sure your column isn't bleeding or shedding?

Is your gas flow rate sufficient?

Is the quality of your gas sufficient?

I also run a 2420 and have done some development toward assaying phospholipds with it with pretty good results, albeit under NARP conditions. I had no background noise issues and I find the instrument to be quite robust.

[Kostas Petritis]

Sometimes when you are you working with normal phase solvents, during evaporation, the temperature of the drift tube/nebulizer can decrease at such a point, that your mobile phase is not misible any more, creating a lot of background noise.

I would suggest to increase both drift tube and nebulization gas to maximum and wash the system with isopropanol. Then try to repeat the experiment with high temperature settings. Phospholipids are not volatile at all so you shouldn't have any problems working at higher temperatures...

PS: Column bleeding can be another cause...

[Mark Tracy]

Check your gas supply and change the filter and make sure the tubing is clean. Also check your drain and vent lines and make sure they are unobstructed and flowing freely.

[FPF]

Thank you for your replies. Maybe some more information on column type and other settings might prove useful. For the column, I am using a Phenomenex Luna CN 3µm particle size. On the ELSD, I have the tube temperature at 40°C, the nebulizer heater off and the gas flow at 15 psi. As for solvents, they are all HPLC grade from Fisher.

I have not worked with the particular method that utilizes this detector, as it was inherited through an acquisition. I've used this particular model of ELSD to analyze the phospholipids using reverse phase conditions and have had great results with good reproducibility. This is my first time working with normal phase solvents and haven't used normal phase since college.

[shaun78]

A 40C temperature in the ELSD is not going to evaporate your solvents. Because you have water in your mobile phase, the tube temperature must be at least 120C.

With the nebulizer temperature off (or colder than the tube) you are creating a case where solvent can condense on the nebulizer.

This is most likely the cause of the noise (both factors combined).

[Schmitty]

A 40C temperature in the ELSD is not going to evaporate your solvents. Because you have water in your mobile phase, the tube temperature must be at least 120C.

With the nebulizer temperature off (or colder than the tube) you are creating a case where solvent can condense on the nebulizer.

This is most likely the cause of the noise (both factors combined).

All of the newer ELSDs can achieve excellent nebulisation with little temperature input from the nebulizer block or drift tube.

[shaun78]

All of the newer ELSDs can achieve excellent nebulisation with little temperature input from the nebulizer block or drift tube.

Wow. They have changed that much over the past three to four years?

Curses to technology!

[AA]

For a 2420, I think you will need a slightly higher drift tube temp (45 or 50 C) and definiatly a higher gas pressure (35 or 40 PSI). Higher gas pressures will give you smaller droplet that are easier to evaporate.

[Kostas Petritis]

As I said before, when you operate with normal phase mobile phases things are different and you might want to operate in higher temperatures...