Phospholipids using ELSD

[Kostas Petritis]

OK Robotjock here are some suggestions,

I do not really know which ELSD you use, and there maybe differences due to the different nebulizers but:

When I worked with normal phase solvents (i.e. mixtures of hexane with other solvents/additives), I noticed things precipitating and immicibility problems in the nebulizer. Then I touched the nebulizer and saw that it was extremely cold. Actually what was happening (I think) is that evaporation of hexane is pretty fast and endothermous, which was decreasing the temperature of the whole nebulizer at such a point that you start seeing immicible phases. These immicibility issues, depending on your mobile phase, can increase your background noise or even the peak area of your analyte as during the analysis you will have less and less of your analyte reaching your detector.

In orded to test it, I heated the nitrogen lines that were used for the nebulization to about 50 C (that was the temperature of the nitrogen at the exit of the nebulizer) and no problems what so ever from that point.

I suggest that you follow this strategy (heating your nebulizer), first pass through 100% isopropanol (you do not have to use a column for this) for about 30 minutes, then you can use your normal phase solvent and your separations conditions. In addition, it might be a good idea to increase your drift tube temperature as well.

Once you do this, let us know if it worked (or not).

[Robotjock]

Mr. Petritis;

Thanks for your suggestion.

I've also wondered about differences between ELSDs from the various vendors. Will have to investigate at PittCon. However, don't have $$ allotted for a new ELSD. [May be a demo from vendor would provide convincing data to support purchase. ]

The N2 heating results are interesting. Won't this just faciliate the evaporative process and faciliating the deposition of the large, waxy phospholipids within the instrument?

However I've nothing to lose and all to gain. How did you heat the N2 lines?

Happy Thanksgiving to all...

[Kostas Petritis]

Robotjock,

I have my doubts when you say that these waxy materials you observe are phospholipids and I have my reasons to believe so.

For the case of ELSD, I took a large metal tube, put it in coil and in a water (or oil, I can not remember anymore) bath and attach the end directly to the nebulizer with an adaptor. If you can not do that, just have a longer plastic tube in coil and connect it normally to your ELSD (becareful because it may melt).

I didn't have to make more normal phase work, so I didn't make any permanent modifications. However, I worked on the construction of a capillary column control system where we had an electrical heater connecting in line to a plastic tube and the whole temperature was controlled in a feedback based mechanism (one temperature sensor and a temperature controller). So there are more elegant solutions if ever you want to have a permenant solution...

[Kostas Petritis]

I just remember something more (it has been more than 6 years so I started forgeting the details). In addition with what I said, I worked also with a prototype dual wall nebulizer where you could actually run a liquid through it to warm (or cool) the nebulizer. In this case, I just warmed it.

Now that would be more difficult to do... I suggest that you start with what I suggested and we'll take it from there (depending on the success).

[Schmitty]

I have an ELSD loaned to a group doing phospholipid quantification in my building. I also help them with instrument problems (cleaning, troubleshooting, etc.). I have only had to break-down the MKIII (Alltech ELSD) once. It was white around the nebulizer, but they were not experienceing anything that was inhibiting their ability to complete an analysis run.

The method is from the International Lecithin and Phopholipid Society, method AM101.

Mobile phase A is: Hexane/IPA/AA/TEA (81.42/17/1.5/0.08%)
Mobile phase B is: IPA/H2O/AA/TEA (84.42/14/1.5/0.08%)

Gradient:
Time %B
0 - 5
5 - 20
8.5 - 40
15 - 100
17.5- 100
17.6- 5
29 - 5

Flow is 1mL/min, except from 22 min to 27 min where it is increased to 2.

Let me know if I can't post this much detail about a method, and I can edit it out. Good luck

[Robotjock]

Schmitty,
Thanks for the detailed information - the more the better. Limits the assumptions one needs to make.

The conditions are similar, but not exact, to what I was using. I was using an Alltech model 2000 ELSD. However, as previous post states the nebulizer was being blocked. Will add Method AM101 to action queue.

Mr Petritis,
May be a current instrument has incorporated such a dual-walled nebulizer. Will check on it.

I'll post results when obtained.

"to work, to work I go,
the more I play the more I know."

[Waleed]

am glad that am here with you and your partners desussing such ntrested articls... i want t ask you something please.. am trying to use HPLC instrument with UV detector to separate phospholipids. and according to the material present in my lab. i have Si column, spherosorb C8 and nucleosil C18 columns.. and i found an article that use this method but i dunno if it will work out to me becasue the specific column used in that method is microparticulate Si.. the mobile phase used is n-hexan with acetate buffer.... please if you think that it will work to separate these phospholipids tell me abou it... and if you have any avilabe procedure uses the mentioned parameters wish that u will send it over....
this method i need must be compatable with the instruments i have , i need this procedure so urgently...