I have several problems that I hope someone can help me with!
1. In the lab we use a HP5890 GC with FID. The column is a DB-225. Carriergas: Helium, flowrate: 30 ml/min. Split ration: 1:100. I inject 0.2-0.5 ul of FAME standards in hexane. The solvent peak is very broad, and the peaks are not as crisp as they could be. What are the possible causes behind this and what can be done about it?

2. My goal is to analyze fatty acids in seed exudate (seeds are leached in water for up to 24 hours). So far I have tried a liquid extraction with Dichloromethane, SPE extraction (C18 column), and SPE extraction of lyophilized exudate redissolved in an acidic buffer. I seem to have problems extracting the fatty acids. Does anyone have any tips, or ideas on how I can solve this?
Also, we're mainly interested in unsaturated fatty acids - would a methyl esterification that is base catalyzed be recommended over an acid catalyzed approach?
Thanks!

Sofia,
Concerning the GC problem, my first thought would be that the column may have slipped in the inlet while it was being installed and is not at the proper depth in the injector. You might try injecting a microliter of two of butane to check the peak shape. If you get bad peak shape remove the column, cut a new square end on the column, and reset the distance before installing it again in the injector.

I'm doing some seed/fatty acid work. A U of Michigan professor recommended extraction using a hexane:isopropanol solution.
Heat seed tissue (up to 400 mg) in 3 mL of IsoOH at 80-85 degrees C for 5-10 minutes. Then grind the tissue using a homogenizer. The wash the grinder with another 1mL of IsoOH and 6 mL of hexane. After shaking and allowing to stand for a few minutes, add 5 mL of aqueous sodium sulphate solution (15 g anhydrous sodium sulphate in 100 mL of water), shake vigorously and allow for phase separation. Pipet top layer to clean test tube. Add 4 mL of a 2:7 IsoOh/hexane solution, and again pipet top layer and add to other top layer. Evaporate to dryness and the result is the free fatty acids.

Julie,
I'll try that with the lyophilized exudate. Do you know how well that approach works with seed exudate in an aqueous solution ( I normally reduce the volume from 400 ml to ~5 ml. This is the leachate from 48 g of seeds)?

OK, I'm confused: 30 ml/min is appropriate for packed columns, but you detail a 100:1 split. What are the dimensions of your column. DB-225 is fine for FAME. We do fatty acid methyl esters here all the time.

Consumer products guy,
the column is 20 m, 0.2 mm, and 0.18 um.

Sofia,
The procedure I have regarding the lipid extraction refers to residual water..not as much as 5 mL, but it might still work. You need to create a monophaisc solvent mixture with a ratio of 1:4:6 (water:IsoOH:hexane). Once the sodium sulphate salt is added, it will separate the solution into phases, and the lipds will go into the organic upper phase.
This procedure has the seeds soak in IsoOH for a while at the beginning rather than water so that some intact plant lipases can be inactivated.
Hope this helps.

30 mL/min for a db225 20 m, 0.2 mm, 0.18 um,column is simply too high !

Sofia,

Since fatty acids are not very water soluble, you must hae very low concentrations in your leachate. You have been given some good extraction suggestions, but you probably need to achieve some significant concentration of your samples once they are in organic phase.

Anonymous,
what flowrates would you recommend (as a good starting point) for that type of column with He as the carrier? Is this what is causing the broad solvent peak?

Julie,
for how long do you soak the seeds in IsoOH? OK, so I have some exudate lyophilizing right now, and will try it with lyophilized exudate (~ 400 mg)tomorrow. Thanks!
Also, does anyone have any input on the base vs acid catalyzed esterification in terms of derivatizing unsaturated fatty acids.

It is often forgotten that pH control during extraction can be very important regarding recovery of FA (J Chrom, 228, 75 (1982).

Sofia,
it seems that this matter of base catalyzed FAME production has been discussed before, I donīt see how this is possible in H2O/MeOH media. What do you mean by that? Base hydrolysis of lipids, then acid re-esterification?
Hans

Hi

1ml/min is a good starting point for this column parameters with He
I hope that you have works not too long time with 30mL/min,otherwise you have probably damage the column ...
Poor column installation in the inlet,purge inlet time after a too long time,causing solvent peak broadening.

Sofia-
Also, for your information on future projects, Agilent has a downloadable program on their website that will help you properly determine flow rates, pressures, split ratios, etc. based on your column dimensions, carrier gasses, and solvents used. I've found it quite helpful in the past.
-Jeff
P.S. I don't work for Agilent :)

HW,
after extracting, the extracts are dried down under a stream of nitrogen. Thereafter I redissolve in methanolic H2SO4 for the esterification. My question was whether the esterification after the extraction step would be better if performed in methanolic KOH since I'm mainly interested in unsaturated fatty acids. My main focus is on the free fatty acids in the exudate. I hope this clarfies what I'm trying to figure out!

Jeff,
I've been really confused by some of the answers, but then I realized that I gave the average linear velocity instead of the volumetric flowrate. u = 30 cm/s, and the flowrate is 0.5 ml/min. Does this make sense? Is this to low?

Sofia,
The soaking time in the IsoOH is 5-10 minutes at 80-85 degrees C.
Also, speaking of the esterification, I"m not sure how it'll work yet because I haven't reached that point, but Alltech offers a reagent that you mix with your fatty acid sample and it esterifies them when you inject the sample onto the GC. Meth-Prep I if you want to check it out...seems like it would save time and hassle...I'll soon find out.
Julie

Julie,
I've worked with BSTFA before, where you coinject the BSTFA with your sample for an on column derivatization. It works OK (Nimz and Morgan. 1993.J. Chromatogr. Sci., 3: 145-149).

Thanks a lot for all the feedback that I have received so far. Cutting the column and reinstalling it in the inlet, plus changing the purge time did wonders for the solvent peak.

Sofia-

30 cm/sec at with a flow of 0.5 mL/min is in the right ballpark for your system, although you could increase it a little. Another thing to think about; what are you GC temperatures (inj/det/oven)? Are they high enough to vaporize your solvent/analytes?

-Jeff

Jeff,
the temp program that I have been using has a start T of 150C, ramps at 10C/min, and the final T is 220C. The inj T is 240C, and the det T is 230C.

Sofia-
Your system seems as if it should be ok now, especially with your re-installed column corrected purge time. I do agree, though, with Anonymous May 6th, 12:47am above, that you could probably increase your flow up to about 1 mL/min and get even sharper peaks. Incidentally, you could also safely inject 2-5 uL of your FAME standards under the conditions you originally described if reproducibility and/or sensitivity is an issue for you at 0.2-0.5 uL. Good luck...
-Jeff

I do agree with Jeff,that your system should be ok.
I wait your feedback

Anonymous May 6th, 12:47am

Sofia,

you can not get FAME in KOH/MeOH (formally: RCO2- + MeO- donīt go to FAME), you will have to acidify after the KOH hydrolysis of lipids. Sometimes it appears to be advantageous to do the transesterification in two steps (base for solvolysis, acid for re-esterification) for a higher cleaner and faster yield.

I am always surprised about how people get good results with those quicky, in injector, transesterifications. In our hands they have always shown to be highly non-robust (put nicely).

HW,
Thanks! I think I understand what you are trying to point out. I've talked to others that recommended base catalyzed esterification for unsaturated fatty acids, but there must have been a misunderstanding. I didn't realize that it does not work for free fatty acids.
Well, the BSTFA works OK. I have not had good success in terms of a reliable quantification, but for purely qualitative purposes it's alright. But that is also why I'm opting to use regular esterification for my project. What would you recommend for esterifying free fatty acids ( i e what is your favorite recipe)?

To everyone, the GC works fine thanks to the input from all of you!

See ref given above (J Chrom). For "Free" (not esterified) FA in plasma/serum (thus for ~ everything else also) the diazomethane method is absolute top in my opinion.
For esterified lipids it is opportune to try BF3/MeOH first, if recovery is bad or it takes too long, one may need Na/MeOH, etc., followed by acidification.

Hans

I have to perform GC analysis of Mycolic acids from Mycobacterium. For this first Mycolic acids are to be derivatized to trimethylsilyl ether derivative. My question is about the conc of BTSFA reagent to be used for derivatization (wt/vol). Its not given in any reference i refered.
Kindly let me know if anybody has any clue
thanks

Sofia Hi,
What is the conc of BSTFA you have been using for derivatization (wt/vol) and what is the solvent in which BSTFA dissolve?