Chromatography Forum

Dear Chromatographers,

Could anyone share the information about RP-HPLC determination of phosphatidyl serine? We only get some method using ELSD and TLC. We do not have ELSD.

Best regards,
SYX

The said compound has very "weak" chromophors,

what is the concentration range of it in your samples ?

For low concentrations UV detection will be useless

Which other types of HPLC detectors do you have?

CLND or MS would work for this...

Bad news for me ...

I just have DAD and RID.
Is it possible to use derivatization mode?

RID should also work although the method won't be sensitive...

There's a new detector out called the Corona CAD which may be a solution for you too? It is also a evaporative detector but involves charging.

hmm... i think i should try RID first as Mr. Kostas said. But I will consider the other option if RID won't be run.
Otherway, I will try TLC-Densitometric method with iodine vapor derivatization.

Let me be cocky. I'll eat my hat (or any suggested) if this compound does not have retention on the ZIC®-HILIC column.

However, as pointed out above the detection constitutes a problem and you need to find a suitable detector like ELSD, ECD or MS (or an old fashioned colorimetric procedure for post-column detection)

However, as pointed out above the detection constitutes a problem and you need to find a suitable detector like ELSD, ECD or MS (or an old fashioned colorimetric procedure for post-column detection)

... that's the problem. Have any idea for last option (colorimetric post-column detection)?
I need any reference about this technique. I never use it before and feel unfamiliar.

For options on derivatization reactions, look at the book by Lunn and Helwig. For options how to do it best, look for "knitted tubes". I believe that SeQuant can help you with the hardware. For the principles of the technique, you can ask me.

Dear Mr. Neue,
I will search and read the book first, then ask you anything that I do not understand to you. Thank you.

If you are in the mood for something experimental...

Phosphatidyl serine and phosphatidyl ethanolamine both have primary amino groups. These primary amines will react with ortho-phthalaldehyde and a mercaptan under mildly alkaline (pH 9-10) conditions. The derivative has absorbtion maxima at 330 and 230 nm and fluorescence emission at 465 nm. Please don't use mercaptoethanol; there are many superior alternatives. You could do this either by automated pre-column derivatization or by post-column reaction.

On the other hand, if you are going to be doing this frequently, maybe you should just write a strong case to your management for an ELSD.

Since we are taking about phospholipids:
What kind of phosphatidylserine are you trying to measure? Native or synthetic? (i.e. does it contain unsaturated fatty acids?)
What is the goal of your method?

I agree with Mark. If you need an inexpensive, sensitive and quite robust detection then post-column reaction with OPA is an interesting alternative.

However, as pointed out above if you intend to run numerous of samples then an investment in a new detector will pay off.

Mr. Klaus, we bought phosphatidyl serine derived from soy bean. We want to determine phosphatidyl serine in tablets.

I will do the experiment with OPA and mercaptan. What kind of mercaptan that is most recommended to try?