Chromatography Forum

This is probably a stupid question but here goes:

I'm doing some methylation of a fatty acid to its methyl ester using a standard methylation method (4% HCl-MeOH). The method I am following is not very detailed and I am unsure if I am about to do it right.

The method says to add 2mL of HCl-MeOH to my sample and heat at 90-110°C for one hour. So my question is this:

Since the bp of methanol is ~65°C, if I heat 2mL MeOH to 100°C in a sealed screwtop glass tube, won't it shatter from the pressure? And if I leave it uncapped, won't the sample just evaporate away?

I guess I could just try it and see, but I wanted to avoid the embarrasment of everybody else watching me clean up my mess....

If you use a class container which will shatter it might be others that have to clean up the mess. These reactions have to be done capped in small vials made for this purpose (Pierce and some class companies have them). In our hands some of these blew the top (cap), so it should be done in a hood with the front down. One opens them only when cooled back down. Also, it might be wise to go to 90° max. only, and to check into reagents like MeOH/BF3.

Thanks for the advice, I'll look into those specialised containers. I've already ordered some BF3 reagent to play with.

However, I found this published article (BMC Biochem 2005; 6:5) by Kang and Wang that provides a more detailed method. They use glass tubes. If I follow this exactly will the tubes explode?

Fatty acid methyl esters were prepared... using BF3/MeOH reagent. Lipid sample was mixed with 1mL hexane in 16mL glass tubes with Teflon-lined caps. BF3/MeOH reagent (1mL) was added and the mixture was heated at 90-110°C in a metal block or a sand bath for 1 hour, cooled to room temperature and methyl esters extracted in the hexane phase after addition of 1mL H2O.

I used culture tubes with a teflon lined screw cap at 100°C isothermal oven for years.

Never had one explode or break.

I placed them into a beaker and the pyrex beaker into the oven.

Avoid scratching the pyrex glass.

Allow to cool outside the oven in a hood behind a protective shield.

Use thermal gloves and use care.

They really break explosively if dropped on the floor.

best wishes,

Rod

Jonny, the ref and also Rod probably used the same or very similar vessels to what I used. Some people call them tubes, some vials, others reaction vials. Just make sure the manufacturer states that they can be used for reactions under pressure. Normal vials just used for storage are thin walled and should not hold up.

Hmm thanks....

I've been searching for "reaction tubes/vials" and can't seem to find anything that would be suitable. Do you (or anybody?) have an example supplier & part number?

I was planning to use these below, but I guess I need something more pressure resistant?

http://www.vwrcanlab.com/catalog/produc ... =53283-686

I'm still confused why all the papers I've read just seem to infer standard culture/centrifuge tubes. Maybe I'll just try it and see....

If you have adequate headspace, regular pyrex screw capped culture tubes (*not* the flat bottomed ones) are up to the task. A few mL in the bottom of one that holds ~20mL or more isn't a problem at 100-105°C.

I suspect you'll prefer the BF3/MeOH reagent.

We formerly used BF3-methanol and now use about 14% concentrated sulfuric acid in methanol (careful with concentrated sulfuric acid). We add fatty acid or soap sample into 100 ml VF, stir to dissolve if it's a soap sample, then heat on steam bath 5 MINUTES unstoppered, then stopper, cool to room temperature, add about 15 ml petroleum ether and swirl, then add saturated NaCl to bring the PE to the neck, then stopper, mix, allow layers to separate, then inject PE layer into the GC.

Thanks everyone for the tips....

I did see those Reaction vials but must have overlooked the 10mL ones. In any case $260 for 12 vials seems a little steep. I have more than a few hundred samples to run, and a GC that can hold 80 samples at a time. So I'd really need about $1000 worth of vials so I'm not continuously cleaning. I'll have to check my budget...

I'm going to just throw some MeOH/BF3 into a screwtop culture tube and stick it in the oven at 90°C. I guess we'll just have to see what happens..... If it explodes in the oven at least the staff will be out of harms way. What fun!

Well good news.... 16x100mm standard glass culture tubes do not explode when heated in a water bath at 95°C for 2 hours with 1mL MeOH/BF3 reagent.

I think I'll go this route until things start exploding, in the meantime we'll just be extra careful...!

Thanks for the tips!