[hazira]

in my project I need to hydrolze protein for amino acid determination using HPLC.
in most method found was to heated the sample with 6M HCl in hydrolysis tube at 110oC 24hrs in vacuo.
does the hydolysis tube kind of special tube? or can I just use any regular glass test tube?
and how to create vacuum in the tube?

did anybody have video regarding laboratory protein hydrolysis that I can refer?

TQ

[Arkcon]

I've only done it in a Millipore dedicated rig, but at school, it was done in a more manual sort of way.

You will probably want a specialized tube for this:  clean, free of contaminants, probably borosilicate.  Getting a smooth result will not be easy without a specialized vacuum rig.  See, you must remove oxygen, but if you mix 6N HCl, your protein sample, some phenol (more on this later) and just vacuum it and seal, you won't get good results.

When I used the Waters rig, I took the protein sample, and dried it as a stain in a tiny, scrupulously clean, borosilicate tube.  That tube, or a collection of 5 or six other ones goes inside another borosilicate vessel.  The 6N HCl goes into that one, it never touches the protein dried onto the tiny tubes.  The bigger tube has a Teflon seal and a sliding valve closure.

You apply vacuum gradually, until the 6 HCl boils, then you immediately stop, and purge with dry, clean nitrogen.  You perform 3 cycles, and leave the rig under positive nitrogen pressure before you close the valve on the rig's cap.  Too much vacuum, for too long, and you lose too much HCl.  The whole rig goes into the oven.  Only hot HCl vapor contacts the protein.

Phenol is an important addition.  We added a few crystals as an oxidation scavenger.  Once, I put a large (~2 cm) crystal of phenol, and got much better recovery of tryptophan.  If that's important to you, then you'll also want recrystallized phenol.  Again, this goes in the acid, not in the protein sample.

Without this sort of specialized rig, I don't see how you'll keep liquid HCl away from your protein, carefully evacuate the air out, infuse nitrogen gas, without losing HCl vapor, before you begin hours of heating.  If you're just going to mix it all in one tube, at least try not to lose too much HCl before you seal the tube for heating.

[Arkcon]

Here's a link to a PDF detailing a microwave hydrolysis rig:  http://www.cem.de/documents/pdf/publikation/protheinhydrolyse/RH004.PDF  This trend really seems to save time.  Again, HCl vapor is preferable for protein hydrolysis.

[Babcock_Hall]

It's been many years since I have done this.  I purchased or was given a specially designed tube with a side arm for pulling the vacuum and screw threads with a gasket to allow the vacuum to be applied.  IIRC I did a few freeze-pump-thaw cycles before heating.  The quality of the HCl is probably important.  Constant-boiling HCl, which is just over 6 M, is preferred.

http://www.piercenet.com/product/vacuum-hydrolysis-tubes

[Arkcon]

The rig in Babcock_Hall:'s link seems just what you need for a few samples.  You pull moderate vacuum like I said, just enough to evac air, without boiling off too much HCl.  Constant boiling HCl is also very important -- regular HCl is either too impure (contains traces of other chlorine compounds that oxidize the amino acids) or is too weak in strength.  Constant boiling means this is an azeotropic mixture of HCl and water -- with reasonable vacuum strength and duration, you lose almost as much water as HCl, so the strength is the same from experiment to experiment.  Its just coincidentally slightly above 6M, not that 6M is important for any other reason.

*[EDIT]*

And read this application note linked to the product in the link: http://www.piercenet.com/objects/view.cfm?type=page&ID=266C1558-BC35-4E01-993C-2354B8B5AF26  If you freeze the mixture, you can pull vac while minimizing loss of HCl.   That's a useful tip.

[Babcock_Hall]

My recollection is that I froze the mixture (and did cycles of freeze-pump-thaw), but my memory is not as reliable as it used to be.