I'm intending to measure some carotenoids from fish samples, but the carotenoids are in very low concentration. Therefore I have to increase the size of the fish sample, which contains a lot of lipids. I can't inject in the HPLC a such an oily sample, because I can clog the column. I thought in YMC-Pack PVA-Sil, intended to separate lipids, but The HPLC only has a UV detector, I don't have a ELSD. Should be enough the UV detection to locate the lipids in the chromatogram and collect my carotenoid (hopefully free of lipids) and quantify it in a different column? Any other HPLC column suitable for lipid separation?
Thanks!
Carotenoids are great. They have visible-range absorbance maxima. Make use of that.
It's usually possible to find a wavelength where only the carotenoid is visible. If your detector really doesn't go into the visible range, you can collect the entire colored range by eye and quantify it with a regular visible spectrophotometer.
Just make sure you don't leave them sitting in room light. They do get broken down with time.
You can try derivatization of carotenoids so you can separate them from other matrix interferences so as to avoid chromatographic separation.
John has the idea. Run carotenoids under several wavelengths until you find out where they elute from your column. Or check column eluates/ fractions with a regular visible spectrophotometer. Then collect only that fraction when you have lipids in the sample also.
In -20/-80 C the lipids would settle/freeze, then the supernatant can be safely injected into a C18 or C30 (preferably) reverse phase "semi-preparative" HPLC column to monitor/ elute the carotenoid fractions. I wonder if C30 matrix are available to prepare in house packed small-scale chromatography columns to elute with acetone: water mixes to enrich those carotenoids. Sigma has a carotenoids kit as well (with individual components as standards for use as HPLC standard runs). Thanks, Biswa
Consider sample clean up prior to the extraction steps. When dealing with high fat samples or samples with low levels of carotenoids, a saponification step of samples can be helpful. Several sample preparation methods are available in earlier literature, but it will take additional time.
Thanks all for your answers. I have a DAD, so I can measure the carotenoids. My fear is that UV is not enough to detect the lipids, because I have seen that an ELSD is used for this purpose. I want to be sure that I can relay on UV detection to locate phospholipids, triglycerydes, cholesterol etc in my column, so I can collect the carotenoid of interest safely and measure them in a different column.
I knew about saponification, but this can produce false positives of the carotenoid that I want to measure (meso-zeaxanthin).
Biswra, could you please tell me the conditions to freeze the lipids? I tried before but I couldn't freeze the them.
Thanks all again.
Increase concentration of lipids, or change the solvent to precipitate lipids. Alcohol + increasing amounts of water?
RP C30 column is the better option. It is able to separate carotenoids and their E Z isomers and you will not have trouble with lipids if you are using PDA detector.
Finally the YMC-Pack PVA-Sil works great. It separates the lipids from the carotenoids that I want to collect.
- Badges
- Science topic