analysis of phospholipids using HPLC with UV detectors

[Waleed]

dear all:
i wish that i can find suitable procedure for separating phospholipids using HPLC with UV deterctors and Si 5 columns... mobile phase monitoring is well established with n-hexan and phosphoric acid buffer.. but the problem is that Si columns are defferent and have many applications and i ddnt find a particular method using this procedure..

[tom jupille]

Phospholipids generally do not have much in the way of UV chromophores until you get down to very short wavelengths. Unfortunately, while hexane is transparent in the low-UV region, silica gel columns usually require the addition of a more polar solvent to hexane in order to elute phospholipids, and these polar solvents often have a higher UV cutoff.

In essence the combination of adsorption chromatography and UV detection for phospholipids is not a good one.

[Waleed]

thank you very much for this explanation.. but the only thing i can say is that the HPLC instrument we have in ur laboratory is provided only with UV detector. and there s alternate detector which is flouro spectrophotometri detector.. and i ddnt find suitable procedure for both. the only procuder i found and am trying to apply it is the separation of phospholipids using UV detection and micro particulate Si 5microgram column.. and am wondering if i can use this procedure? and if i cant can you pleaze provide me any person who can help me to find alternate procedure? by the way other columns i have are: C8 water sphesorb 5micro,C18 nucleosil 100 5 micro and C8 nucleosil 5 micro....
i wish if u can help me because i need it urgently....

[Waleed]

one final thing... the mobile phase thats described in this procedure is composed of n-hexan and acetate buffer...

[tom jupille]

I'm sorry, but it is difficult to use a hammer to assemble a bicycle, and it is difficult to use a UV detector to analyze phospholipids. It is simply the wrong tool for the job.

That said, I'm assuming that your mobile phase is composed of n-hexane and acetic acid ("acetate buffer" would require an aqueous solution, which is immiscible with hexane). The problem is that acetic acid absorbs in the low-UV and interferes with UV detection. You could try substituting isopropanol for the acetic acid, and then using phosphoric or nitric acids as necessary as counter-ions.

Here's a link to a review which may help:
http://www.cyberlipid.org/phlipt/pl40004.htm

As you will notice, most of these references use ELSD.

[Waleed]

thank you very much... i know what o you mean... i have forget to tell you that the mobile pahse used in that procedure is composed of n-hexan, acetate buffer and 2-propanol... please if you have time to visit this link
www.iupac.org/publications/pac/1992/pdf/6403x0447.pdf
and then you can tell meif i can use this procedure or not. by the way am trying to analyse blood plasma phospholipids ....
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[Waleed]

one more thing what do you think about using flouro spectrophotmetric detection ? do you think it will work out? if you have any refrence or website that can help me just send it over.. i will be thankfull