I developed an HPLC method on a C8 column for the separation of the constituents of a green plant extract.
During the purification of these extracts by preparative HPLC, I noticed that the resolution decreases with the number of injections. During the regeneration of the column in the backflush mode, I eluted a green compound with IPA or CH2Cl2 as mobile phase. This is consequently a very hydrophobic compound.
Since colored in green, I supposed that it was chlorophyll, but after a short research in the literature, I'm not convinced by my assumption, since chlorophyll doesn't seem to be so strongly retained on apolar phase.
What is your advice?
What could be this green compound if it is not chlorophyll?
I cannot see what else it could be. Chlorophyll is strongly retained on reverse phase. One needs at least 90-95% MeOH or MeCN to elute it from C18 columns and I would not be surprised if part of it builds up in the column after several injections. Chlorophyll may be less but still quite strongly retained on C8 columns.
I cannot see what else it could be. Chlorophyll is strongly retained on reverse phase. One needs at least 90-95% MeOH or MeCN to elute it from C18 columns and I would not be surprised if part of it builds up in the column after several injections. Chlorophyll may be less but still quite strongly retained on C8 columns.
Just by looking at the chlorophyll a, b and c structures (see attached link) all of them seem pretty hydrophobic to me.
http://upload.wikimedia.org/wikipedia/commons/d/dc/Chlorophyll_a.svg
We did HPLC separation of Bacteriochlorophylls before (see the paper Biochemistry 2011, 50, 3502). I agreed with Gaetan. Chlorophyll is strongly retained on the reverse phase. If you want to double confirm, you can measure the abs. spectrum of your fraction. The chlorophyll has unique abs.
I advise you to check the absorption spectrum. Probably is Chlorophyll or Pheophytin.
yes, it's a good idea to obtain the absorption spectrum of the compound.
i will perform this the next time i will purifiy my sample, since i have a DAD detector connected to my prep HPLC
Yes the above suggestions are really justified. Please do check your HPLC chromatograms too after sample preparation by extracting your plant extract by hexane. For identification please check the following articles also: http://pubs.acs.org/doi/abs/10.1021/jf00008a018 and also http://www.tf.ni.ac.rs/casopis/sveska1/c2.pdf
Are you using a gradient elution? What is your elution condition? My advice would be to check your chromatography condition, like what is your % organic on your starting condition? because it may be that during each injection (low concentration of organic) you precipitates molecules which are very poorly soluble in aqueous medium. If it's the casey, you'll get all this compounds in column header until the minimum required of % organic solution
You could easily determine the nature of your misterious compound by MS analysis if you have the possibility to use such an instrument. The molecular peak in the obtained MS spectrum should support your assumption (chlorophyll), or in the other case, discard it without any doubt!
Cheers...
yes I agree with Laura, your compound could be either chlorophyll or pheophityn. They are strongly retained on RP columns, however they can be easily distinguished by their MS or DAD spectra (see http://www.ncbi.nlm.nih.gov/pubmed/20814906 or http://www.ncbi.nlm.nih.gov/pubmed/23592206). Otherwise, if you isolated it, you can define its diagnostic abs spectrum by UV-Vis spectrophotometer.
You may want to consider extraction with ethyl acetate or n-hexane before you run your samples, to protect your C8 column from degradation. Then analyse the hydrophobic fraction by some other method, such as GC-MS.
Dear Richard,
I recommend to use the C30 Carotenoid coloumn for Isolation of chlorophyll content. I observed very good separation for chlorophyll and carotenoids in green seed samples of pulses. Extraction solvent is MeOH:ACN (3:1) ratio. Good luck.
- Badges
- Science method