Chemical Forums
Specialty Chemistry Forums => Biochemistry and Chemical Biology Forum => Topic started by: keetner on November 22, 2012, 05:12:35 AM
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Hi guys,
So I had spotted this TLC plate with different lipid standards (their identities are known). The thing I'm wondering about though is, when I spotted oleic acid, I got a huge expansive blob at the top of the solvent front. Also, with phosphatidylethanolamine, it created faint streaking above and below the point. I'm wondering though, what could cause this? Method = iodine staining.
Phosphatidylethanolamine
I tried looking up some information about possible bands/streaks, but the best I could come up with (besides bad spotting) is something about the difference in hydrocarbon compositions of the streaks. The article said if I were to take samples of the streak above and below, I would find more polyunsaturated fatty acids above and more saturated below. However, it was talking about phosphatidylcholine in bovine retina cells -- I was working with egg yolk.
They gave some reason, which was this: When phospholipids from rod outer segments are subjected to TLC on silica, partial resolution into groups of molecular species occurs (Roque and Giusto, 1995).
I'm not really quite sure what it's saying here, however.
I'm certain there must be something to it, because I wasn't the only one who observed this streak. And it doesn't seem like your conventional 'bad spotting' streak, either.
Oleic Acid
I'm not sure what to think of this one. Most of the stuff I come across attributes it to bad spotting but again, I feel like there has to be a reason why the spot is so expansive (other people in my lab got it as well). Could it also have to do with the large hydrocarbon tail? I would have assumed the points would have been slightly 'neater' (especially since I am dealing with standards here).
Any way, any help would be greatly appreciated -- thanks!
Oh, I've attached my TLC plate photo in case you wanted a better view. Photo isn't the best, but I assure you, the mentioned points were there. Oleic was spotted 2nd from the right, phosphatidylethanolamine was spotted 4th from the right. Looking at it now, I wonder if that large spot at the top might be from the triglyceride sample (spotted at the 1st point from the right).
(https://www.chemicalforums.com/proxy.php?request=http%3A%2F%2Fi.imgur.com%2FLZTfD.jpg&hash=0f6fbbfc7156febcb9b935d5f31ac4afc1a0959c)
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I'll try to touch base with someone in a few days, but one thing I would expect is that phosphatidylethanolamine even from a single cell would be a heterogeneous mixture. The fatty acids with which glycerol is esterified are heterogeneous with respect to the number of double bonds and the number of carbons in the chain. Lipid experts would probably know me than I would, but perhaps you could stain with something specific for double bonds, and only the unsaturated fatty acylesters would show up. Just a thought.
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What solvent(s) are you using for the TLC?
My guess with the oleic acid is that it's not really binding to the stationary phase very strongly and is basically staying only in the mobile phase (which is why it's all the way up at the solvent front). Maybe trying a more basic TLC plate (such as basic alumina) or using an apolar TLC plate (such as C-18 silica), you would be able to get a cleaner spot with oleic acid.
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The solvent was: hexane, chloroform, methanol, water (30:30:30:1).
Unfortunately, this experiment was not meant to be anything particularly extravagant, so I am unable to try any of the things you guys recommended!
I'd still be interested in any information though.
Thanks for the responses!