Plant pigment analysis

[Peggsy]

G'day all

We are undertaking the analysis of xanthophyll and chlorophyll pigments in Australian native plants as was wondering if anyone here has anby experience with this methodology.

We are using the method of Bungard et. al. (1997) Aust. J. Plant Physiol. 24:205-214.

In short

Column: C18 Novapak 4um, 150*3.9mm Endcapped
ResA: 85:15 (MeCN:MeOH)
ResB: 68:32(MeOH:EtOAc)
ResC: 50:50(EtOAc:Hexane)
Flow: 1ml/min
Detection: PDA at 445nm

Samples are extracted in 100% acetone, kept on ice and in the dark until 10uL maximum injected.
Pigmets we are interested in are: neoaxanthin; violaxanthin; antheraxanthin; zeaxanthin; lutein; chlorophyll a; chlorophyll b; alpha- & beta-carotene.


I would like to converse with anyone else who has been exposed to this application on the technical side of things if possible.

Regards
Greg

[Peggsy]

.....anybody....Bueller....Bueller...... Ferris Bueller!!

[ling]

Hi,

I've worked with seperating Chls extracted from plants on a C18 column before. What would you like to talk about more?

[Peggsy]

Dear ling

thanks so much for your reply.

i have queries as to both the quantification of these pigments, as well as their stability. My query related more to the xanthophyll pigments than Chls, but are you aware of the stability of both Chl A. and Chl B.? Are they both stable to the same degree, ie one not degrading quicker than the other? Stability will be affected by both temperature and light . What about extraction systems, why is there not a uniform system?

Some reports in the literature use calibration factors (ie area/ng pigment), or even prepare their own standards using prep LC. I donlt like the area/ng approach, different systems with the same column can give different bandspreading due to column age, flow rates, etc... While the use of actual standards would be the preferred way, cost often reduces thier use. Alternative means are to use the calibration factors from Wellburn(1994) however no factors are reported for the solvent regime i have used in this analysis.

Have you ever heard of a suitable, stable internal standard(s) that has been used for this analysis??

would love to hear your thoughts.

Seasons Greetings
Greg

[Mark Tracy]

Well, when I did carotenoids, I assumed that the standards would not be stable. First, I looked up the extinction coefficients in the literature. I diluted my stock standards in the solvent used in the literature and measured the absorbance with a spectrophotometer. Then I made my daily working standards for HPLC analysis. That little project was published in American Laboratory News, November 2005.

[Peggsy]

Thanks for the input Mark, greatly appreciated!

Your preparation of daily working standards, where were the primary stds stored (-20 or -80C)? How long could you store the primaries before degradation? Did you constantly buy in (or prep more) new primaries, so as to keep preparing fresh working standards. Is the stability of all the carotenoids the same? In addition, what about "preferential" degradation of one other the other, or even transformation from neo-viol-anther-ze (axanthin)? Does the report you mention cover any of these stability/transformation issues? What about time delays between extraction and actual analysis, any numbers on that sort of stability?

After you measure the Abs in the solvent specified in the literature, how did you then use this info for the HPLC separation, where the solvent regime would be different? From my reading, the most common solvents for extinction coefficients are MeOH, 80-100% acetone, and DMSO. These are certainly not my exact preference for HPLC solvents (I initially use 15:85, MeOH:MeCN), thus the HPLC detector response would be different. How did you account for this with the unknown quantification? That’s to say, you have coefficients from literature solvents, then run unknowns in different solvent via HPLC and quantitate these unknowns.

As you can tell, I have many queries, so may not be justified, perhaps I am a little confused, but I do know that this method is quite fickle, the separation of lutein from zeaxanthin being one area of concern, although with a Nova-Pak, this is accomplished.

Thanks again Mark, looking forward to hearing your valued comments on the above.

Cheers mate!

Greg

[Mark Tracy]

The primary standards were in solid form stored at -20C; since it was a short project they never went bad before I was finished. The stock solutions were usually in ethyl acetate or ethanol; I stored them at 4C because otherwise they risk precipitation. I prepared dilutions in heptane for spectrophotometry and in ethyl acetate for HPLC calibration from the same stock solution on the same day. The spectrophotometer was calibrated with certified filters, not my stock solutions; that way I calculated the concentration of my stock solutions for that day.

I did not do any stability studies. The stock solutions would lose about half their absorbance in a weeks time, so I made them weekly. I did not have a refrigerated autosampler; I suspect that my %rsd suffered for that reason. I always analyzed the extract the same day. The guys who really do this for a living have special room lights and dark glassware to prevent photolysis. I just worked fast.

I must say I am impressed with the vitamin tablets that I assayed. All within 15% of label for year-old tablets.

P.S. You should check the AOAC methods for carotenoids; lots of useful advice.

P.P.S You probably don't get American Laboratory in the land down under. Send me an email at mark.tracy@dionex.com and I can send a reprint.

[Peggsy]

Thanks again for your comments Mark, I will be in touch via email shortly to grab that report from you (cheers for the offer).

Luckily we do have a refrigerated autosampler, and i have done quite a few little experiments as to stability within a day, evaporation from pierced and unpierced vials, storage at -4C vs -20C, extraction efficiency and even calibration from commercial standards. I eventually went down the road of running calibration curves for the carotenoids I had stds for (i.e., anther, viol, and zeaxanthin), however these were at low concentration in EtOH and not compatible with the HPLC solvent system, really bad peak shape mainly as would be expected, especially for larger volumes injected.

As you have stated, we also have been doing these extractions in the dark, on ice and all sample are analysed within 2 hours of the extraction. My main concern is that with ongoing pigment analyses in our research program, my ability to use standards and thus calibration curves will be lost due to their lack of stability once in solution and their cost. Of course, prep LC could be used to do this, and i think i should probably look at this further for the future.


Seasons Greeting!!

Greg