Chemical Forums
Chemistry Forums for Students => Analytical Chemistry Forum => Topic started by: sdekivit on October 22, 2005, 01:01:56 PM
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hey,
i have a question about GLC cause i have to analyse the fatty acid composition of phospholipids. The way i want to analyse this is to compare with an external standard, a mixture with fatty acids ranging from a chain of 12 C to 18 C . To do this' i'll make methylesters from the fatty acids.
Now i have to chose a type of column but i'm kinda lost. They said to me i should chose a polar stationary phase. But i have apolar compounds with a different chainlength. All are methylesters so the type of the polar group is unchanged, so i should separate on the basis of a different chainlength. That's why i thought of a apolar stationary phase.
Why do i need a polar stationary phase ? is it because the compund would be too much in the statiuonary apolar phase ?
(my reasoning is based on the fact that apolar compounds are attracted to apolar phases)
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Fatty acids tend to elute as broad tailing peaks on non-polar columns (ie DB-1 or siloxane type columns) so they are typically esterified (and hence made more non-polar) to make the peaks sharper. A broad tailing peak is difficult to integrate (poor quantification). You can buy an FFAP (I believe it stands for free fatty acid phase) column from Agilent and do the analysis directly (no esterification required) up to about C22 or so.
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What is GLC?
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What is GLC?
Gas-liquid chromatography. Shame on you Mitch.
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Fatty acids tend to elute as broad tailing peaks on non-polar columns (ie DB-1 or siloxane type columns) so they are typically esterified (and hence made more non-polar) to make the peaks sharper. A broad tailing peak is difficult to integrate (poor quantification). You can buy an FFAP (I believe it stands for free fatty acid phase) column from Agilent and do the analysis directly (no esterification required) up to about C22 or so.
ty :) and than in this polar stationary phase unsaturated fatty acids stay longer in the column because they can interact with the stationary phase (pi-electrons) right ?
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That sounds reasonaible. Empirically, I have noticed that oleic acid (C18 with one site of unsaturation) elutes later than stearic acid (C18 saturated) on this column. I have not tested highly unsaturated samples. On the less polar columns with polyethylene glycol stationary phases, the esterified acids elute with the more unsaturated materials coming out later. This would be due to a greater amount of miscibility of the unsaturated acid derviatives (alkene is more polar than alkane) in the somewhat polar stationary phase.
As for the peak tailing, hydrogen bonding is likely the most dominant factor responsible for distorted fatty acid peaks on other columns (see McNair, HM; Miller, JM Basic Gas Chromatography, Wiley, 1998, p 60).
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Gas-liquid chromatography. Shame on you Mitch.
Heh, you don't see it called GLC too much anymore. It's usually just GC now.
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true, but i prefer saying GLC and not GC because now i can see a liquid is used as a stationairy phase (althoug a solid stationairy phase, GSC, won't be used anymore these days)
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i have tried to seprate the fatty acid methyl esters using GCMS technique with a polar stantionary phase column" ODB" ,,, and it provide good results... so maybe i think u have to try this,,, there u will find many procedures how to do this... about the external standerd whats the nomber of C atoms u used?
and as u know FAME is not high polar compounds .. so it can be easly separated in the Si phenyl alkyl stationary phase..
am wondering if u have a suitable procedure how to separte these phospholipids using HPLC with UV detection.. because i found many articles but non provided me good resluts,,,
thnx
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Hi. just want to give some suggestions regarding FAME analysis.
I have been running this test for quite sometime and found that column OMEGAWAX from Supelco is a good column for this.
does your analysis include determining trans fatty acid? if yes, this column will not be able to separate it out though.
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What is GLC?
Gas-liquid chromatography. Shame on you Mitch.
I use GSC so yeah I have no real excuse. ;P