Volatile buffers info (TEAB; TEAA)

Gregory Stuart gstuart at unixg.ubc.ca
Mon Oct 17 16:39:26 EST 1994


To: 092ellbj at chiron.wits.ac.za
Subject: Re: VOLATILE BUFFERS FOR COLUMN CHROMATOGRAPHY
Newsgroups: bionet.molbio.methds-reagnts
In-Reply-To: <092ellbj.5 at chiron.wits.ac.za>
Organization: University of British Columbia, Vancouver, B.C., Canada
Cc: 
Bcc: 

Two choices might be :
(1). triethylammonium acetate (TEAA) : prepare from glacial acetic acid and 
triethylamine : 

	Et3N, HPLC-grade	55.64 ml
	CH3COOH, glacial	22.89 ml

Add Et3N to 2.0L volumetric; add 1.5 L dH2O; add CH3COOH in portions with 
swirling. Make up to 2.0 L. Transfer to 4 L bottle; add 1.5 L dH2O. 
Adjust pH to 7.0. Make up volume to 4.0 L. 

This buffer is commonly used in HPLC (e.g. separation of oligonucleotides 
on reversed phase columns), and is completely volatile. Use high-purity 
reagents, and after lyophilization (or evaporation under reduced 
pressure), add a small aliquot of dH2O to your sample and re-lyophilize.

(2). triethylammonium bicarbonate
Prepared from triethylamine and CO2 gas. As above, the Et3N should be 
quite pure (colorless; redistill if necessary, or purchase HPLC-grade new 
bottle). 

My method for making this was to add a calculated amount of Et3N (I forget
the exact amounts - I believe I used to aim for a 1M solution, final) in a
tall graduated cylinder. Record the volume. Add a small sytirbar, then
insert a glass tube with a sparger (fritted glass bubbler on the end) in
to the Et3N solution, near the bottom. The tube is held in place with a
split rubber stopper; ensure the contents of the grad cylinder can vent).
Pack the outside of the grad. cylinder with plenty of ice. Attach a CO2
line to the sparger from a CO2 tank equipped with a regulator. Bubble CO2 
through the stirred Et3N solution for approximately one hour or so, 
checking th pH. When the Et3N has been converted to triethylammonium 
bicarbonate, the pH will be approximately 8.0-8.5. Record the volume (to 
correct for Et3N loss due to evaporation during the bubbling process, to 
correct the Et3NHHCO3 concentration); pour the buffer into a screw-cap 
bottle and store at 4 deg. C. Keeps for at least a month (can check pH - 
will slowly increase). 

	This is another completely volatile buffer - I used to use it for 
purifying end-labeled oligos. A small DEAE column would be packed 
(Pasteur pipette, glass wool plug). The resin would be converted to the 
bicarbonate form with the undiluted TEAB buffer, then washed with dH2O 
(check pH). The end-label reaction (oligo + kinase + 32P) would be 
applied to the column. Unincorporated 32P, PPi, etc. would be eluted with 
0.1M TEAB, then the oligo would be eluted with the undiluted TEAB buffer 
(typically, approx. 0.8M, or so if the original starting ET3N volume was 
calc'd for a 1M solution). The oligo/TEAB solution is lyophilized; the 
pellet is resuspended in a few ul dH20 and evaporated to dryness, to get 
rid of residual carbamates, etc. This can be repeated several times. 

-- 
--
Greg Stuart		   Occupational Hygiene Programme
gstuart at unixg.ubc.ca	   University of British Columbia
604-822-9573 (FAX 9588)	   Vancouver, B.C. V6T 1Z3
-- 
--
Greg Stuart		   Occupational Hygiene Programme
gstuart at unixg.ubc.ca	   University of British Columbia
604-822-9573 (FAX 9588)	   Vancouver, B.C. V6T 1Z3



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