Chromatography Forum

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..

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.

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....

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

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.

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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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