Amino acid composition - hydrolysis techniques

[SSGG]

I've gone through a lot of articles about amino acid analysis by LC. But I need to ask some really basic (and silly) questions about hydrolysis techniques, because I've never seen it done before, and I've never worked with gases.

1. I have read of people using vacuum hydrolysis tubes (from Pierce), vials with teflon valves (also from Pierce), and ordinary borosilicate glass vials. What kind of hydrolysis vessel is best?

2. How do you evacuate a vial/tube flush it with an inert gas? i.e. I have a nitrogen gas supply and a vacuum pump. What next?

3. How do you make sure that the vessel is air-tight, and yet doesn't crack in the oven?

4. I read somewhere (in this forum, I think) that vapor-phase hydrolysis is falling out of favour. Why?

Can someone experienced with this technique help me out?

[Kostas Petritis]

Hi SGGG,

In one of my previous posts I have given a reference to Fountoulakis et al that has written a very nice review in different acid hydrolysis methods of proteins/peptides...

I have personally performed in the past acid hydrolysis in solution and never vapor-phase hydrolysis so I can not answer all of your questions but I can tell you that the vials that I used (although I do not remeber the brand anymore...) were very very thick and could withstand sudden changes in temperature without cracking.


http://www.sepsci.com/chromforum/viewto ... untoulakis

[Mary Carson]

Ah, this post brought back old memories.

The point of the vacuum/nitrogen hydrolysis is to get rid of the oxygen so that you don't destroy the tryptophans during hydrolysis. If you don't care about tryptophan, you can just do a vacuum hydrolysis.

You can use regular glass tubes if you like glass blowing. This is probably best learned by watching someone else do it first. Basically, you add your sample and HCl to the bottom of a 12 x 75 tube, making sure not to get any liquid on the sides of the tube. Use a glass-blowers bunsen burner to create a 1-3 mm constriction about half-way up the tube. Don't pull it too thin. Let it cool a bit. Have your vacuum (not a very strong one or your sample will bump out--I'm trying to remember whether we just used a water aspirator type) set up with a 3-way valve. Have a trickle of nitrogen on the second position, and place tubing from the third position over your tube. Evacuate the tube briefly (may 10 sec or less), add the trickle of nitrogen, then close off the constriction completely with the burner.

Make sure when you are heating the sample that it is safely enclosed.

This is all in the technique. Practice A LOT with just HCl in the tubes until your "explode-in-the-oven" and "evaporate-to-charred-nothingness-in-the-oven" rates reach acceptably low levels.

Alternatively, just buy the Pierce reacti-vessels. A lot more expensive, and not nearly as exciting....

[SSGG]

Thanks guys, for the great tips.

How do you attach the vacuum tubing to the glass hydrolysis tube? Snugly over the mouth? Will that give rise to contamination or carryover?

Secondly, what can I do to protect my vacuum pump from the acid vapours?

[Mary Carson]

If memory serves, yes the vacuum tubing (relatively light duty vacuum tubing here) went right over the end of the tube--no cross contamination because your sample is down at the bottom of the tube and never came in contact with the top part. (If you try to heat up wet glass, it WILL crack!)
I forgot one little step above. Right after you evaluate tube and trickle in N2, turn the 3-way valve so the tube is completely sealed off, and then immediately flame the constriction closed. The top part of the tube stays attached to the vacuum tubing while you lightly pull the bottom part away with your hand.
Did I mention that this little procedure is probably a safety officer's nightmare--6N HCl, glass, vacuum, open flames, all in close proximity to the chemist

As to protecting the vacuum pump from acid vapors--frequent oil changes! (Again, you don't need a real strong pump for this-you're not trying to evaporate the sample, just get rid of the atmosphere.)

[Mary Carson]

You do own one of these, right? You have to be able to get into the tube after hydrolysis without spilling your sample. The easiest way is to just score across the top with a diamond pen. You can use one of the files, but they're not nearly as nifty.