a nonroutine crude Fame Procedure

[MSCHemist]

Hi all I am working on a nonroutine crude/qualitative fame procedure. Because, I don't do it very often I can't keep ordering reagents with short shelf lifes like BF3/methanol or HCl/MeOH.

I have two possibilities I am considering. The mild methanolysis protocol.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817593/
where MeOH/HCl is produced from concentrated hydrochloric acid, diluted down with methanol, and incubated overnight with the oil.

Methyl chlroformate. Fatty acids are methylated with methyl chloroformate in acetonitrile with a small amount of pyridine and methanol. Water up to 10% does not affect the yeild. What would the best way to hydrolyze the fats be for this method? Could I just use aqueous HCl and then extract it into acetonitrile from water with high salt? Should I include BHT to protect the unsaturated fatty acids?

Thanks for any advice.

[Consumer Products Guy]

We have used BF3-methanol and sulfuric acid in methanol to esterify fatty acids and soaps for decades, no issues, and heat on steam bath 5 minutes.

For triglycerides, we typically just saponify 20 minutes in NaOH-methanol or KOH-methanol, then add the BF3-methanol or sulfuric acid in methanol to neutralize excess caustic left over and provide excess, and heat on steam bath 5 minutes.

[MSCHemist]

Thanks I would use BF3/methanol but it has a very short shelf life. Methyl chloroformate lasts much longer and I get very sporadic samples.

I just used some 0.5N NaOH in methanol (made from pellets and methanol) and heated it in a hot water bath in a 20ml closed tube and then cooled, acidified with ~0.3g 11.4N HCl extracted with hexanes.

then I had to blow it down to get into into my reaction mix with acetonitrile, pyridine, and methanol. I then neutrilzed the MCF with bicarbonate in methanol and extracted with hexane again.

It still needs some fine tuning as the blowdown is a pain. Perhaps I can just take some of the neutrailized methanol for the reaction mix.

[Peter Apps]

How about transesterifying with sodium methoxide ?

Peter

[MSCHemist]

I ran it today it worked out fine but it would be more convenient to have a quicker way to get from the saponification to the reaction mix for methyl chlroformate than extract with hexane, blow down, and dissolve in acetonitrile.

Basic protocol was
0.5g oil
5ml 0.5N NaOH in methanol
steam bath 80-90 deg 30 minutes
cool
add 0.3g 11.4 N HCl
add 1ml hexane
add sat NaCl
transfer appropriate volume hexanes to 1.2ml autosampler tube
blow down
add 220 ul CH3CN
20ul pyridine
10ul MeOH
add 20 ul Methyl Chloroformate cap vortex and losen cap
add 400ul hexane and 200 ul of MeOH saturated with NaHCO3 to neutralize the MCF (it forms dimethyl carbonate)
transfer hexane layer to autosampler tube and inject in GC/MS with wax column.

I was just looking for a basic profile to identify the oil type.

[MSCHemist]

I suppose I could also keep some solid sodium methoxide arround and prepare it in methanol when needed. The nice thing about methyl chloroformate is it is more insensitive to water than most reagents. Up to 10% won't have noticable impact on the esterfication.

[MSCHemist]

https://www.researchgate.net/publicatio ... s_in_serum

I just found another paper that states that not only is not having 2 layers (alkane recommended isooctane) not a problem it is recommended to do a phase transfer derviatization. therefore there is no need for me to blow down the hexanes. Instead just saponify, extract with isooctane and set up the reaction.

[MSCHemist]

It seems to work fairly well but I see some underivatized palmitic acid eluting after the fames representing from 3-11% of the methyl palmitate peak. Is that normal? The reaction is quoted as being 90-98% efficient. I was also using pretty high concentrations of samples to see the minor acids.

[ketej andej]

Hello MSCHemist,

I'm working on identifying FAME's in Olive oil, and i would really appreciate if you can give me some info about your GC method parameters. I am obliged to use Nitrogen as carrier gas, and TR-225 60m x 0,25 mm x 0,25 um column.

Many thanks in advance.

[MSCHemist]

I used a 30m .32mm 0.5um wax column on my GC/MS with a 1.3ml/min he flow and an oven ramp

50 deg 1min
+20 200 deg
+3 230 20 minute hold (that column bleeds like a cheap marker higher than 200)

[ketej andej]

I possess a TR-WAX MS 30 m x 25 mm x 0.25 um column. If i'm not mistaken i can use it for my GC-FID analysis. Is yours a WAX-FAME column?

[MSCHemist]

just a general RTX-Wax column. Though my purpose in fames was mainly to get a basic profile of % C16, C18 C18:1 C18:2 C20 C20:1... I did not need to resolve the cis/trans nor each individual isomer (double bond position). The wax sufficed for that purpose. I have an FID (5890 SII) but I run several methods using a db-5 on it and didn't want to devote it to a wax column (many are derivatization methods and most derivatizing reagents kill wax columns plus they are high temp methods up to 325).

I work in a flavors lab and do a wide variety of analyses though not a whole lot of any one (except 3-mcpd and basic flavor scans on the MSD). I have a GC/MS with 2 inlets and a quickswap for changing columns and a 5890 Series II FID and a stupid useless FPD on my GC/MS (I wasn't arround when they bought it).

I do SPME-flavor scans, 3-mcpd, BHA/BHT/TBHQ, Raw Material purities, headspace ethanol, diacetyl and other quants, and amino acids and TCA cycle acids on the GC/MS

On my 5890-FID I do Piperine, scoville heat units, sugars, free fatty acids, occasionally MSG when not part of a full amino acid scan.

[ketej andej]

For the moment i need t o do the same, mainly to get a basic profile of % C16, C18 C18:1 C18:2 C20 C20:1... I did not need to resolve the cis/trans nor each individual isomer (double bond position). But i guess you have not used the WAX column on the GC-FID, right? I'm thinking to use it although it is a WAX-MS column.

[MSCHemist]

I had it on the FID once. It probably should stay there as it has a pretty bad bleed but I need to use it for static headspace of polars like ethanol and diacetyl on the GC/MS because of the quickswap my columns are under positive pressure rather than vacuum on the GC/MS so I can use wider columns though the bleed on this column is pretty high above 200. Otherwise a column is a column. You can put a basic wax column on the FID and get separation between C18:0, 18:1, 18:2 just fine.

[skunked_once]

The qualitative method I use for C10 through C24 FAME analysis: 0.2 g of ground seed or 1 drop of oil is transesterified by vortexing for a few seconds in 3 mL of a solution of hexane/chloroform/sodium methoxide (75:20:5). The sodium methoxide is 0.5 M sodium methoxide in methanol from Sigma (#403067) and is stable in the refrigerator for at least two years. The sample can be injected directly without further cleanup.

Here is the reference:

"Two-Year Study on the Inheritance of Reduced Saturated Fatty Acid Content in Sunflower Seed”, Vick, B. A., Jan, C. C., Miller, J. F.
HELIA 27 (4), pp. 25-40 (2004)