Dear Debbie:
Your trityl problem could be related mainly to the use of Argon, but it
could also represent a problem with some valves (or lines) in its early
development. I am surprised to read in your message that humidity
higher than 50-60% precludes the use of Helium. Quite the opposite ñ
Helium proves that it is better than Argon at those conditions. True,
you will not find in the manual for the Expedite that Helium will work
at 60% humidity, but you will find such information in the manual of
Beckman for their machine Oligo 1000M, which also uses helium. Beckman
recommend more frequent priming when the humidity is higher than
80-90%. Priming more often, not replacing Helium with Argon.
I already posted a couple of days ago a comparison of the specific
gravity of Helium and Argon. What I meant is that Argon will easily mix
with humid air (Argonís specific gravity is 1.376) while Helium will
provide a better protection because it has specific gravity of 0.138,
hence does not mix that easy with air. The specific gravity difference
between air and helium is 7 to 8 times, while air vs. argon is only
1.3-1.4 times.
In regards to the higher room temperatures, the Expedite protocols,
including the use of Helium, are optimized for work at 20 to 30 degrees
Celsius, see page 1-24 of their manual (30 degrees Celsius should be
million degrees Fahrenheit, just kidding, it is actually 86). Your
place is mentioned to be in the upper 70s. Therefore, once again,
Helium will work at your conditions.
You are lucky to have only one relatively minor problem with the Argon
use (lower trityl values for column 2). The manual for the Expedite
(page 1-20) warns of a trityl failure. Our own experience is with pure
trityl failure. It happened to us once last year, when our helium
supplier had a problem that lasted several days, we ran out of helium,
and decided to try Argon on one Expedite. The trityl was OK, despite
the multiple failure warnings, judging from the trityl values that we
obtained using external fraction collector. However, instead of
disabling the failure alarm, we opted for shutting down the machine that
ran out of Helium until the new bottle arrived.
If you have a fraction collector at hand, it may be the simplest thing
to do ñ compare the values from the OD470 of the fractions from the
collector (see page F-3 from your manual) with the bar graphic from your
built-in trityl monitor (I presume you use the Expedite Workstation
software).
Cleaning the trityl monitor of an Expedite is not difficult, it is
easier than getting to the valve trays. However, such cleaning is
probably not necessary and not very much likely to help you, I am
afraid. Better get a bottle of Helium.
Best regards,
victor
www.alphadna.com
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Walther Debbie
Sent: Monday, July 03, 2000 1:18 PM
To: Recipients of ABRF List
Subject: Expedite Trityl monitor
Anyone out there ever clean their Expedite trityl monitor? I'm
experiencing different readouts between col 1 and 2. The trityl monitor
is approx. 2.5 years old (the rest of the machine was replaced in
March). The syntheses look excellent, averaging 60 X 40-88mers per
month at between 60-79% purity. I'm using Argon due to our high
humidity levels (40-50%, so helium is out) and the room temp is in the
mid to upper 70's. I am getting outgassing and it seems a bit more
prevalent on col 2 than 1, but even when I get solid trityls, the level
on col 2 is lower than 1 (even when I go to diagnostics, the level is
lower on col 2 than 1). I've already cleaned w/ MeOH. PE suggests
just doing a normal shut down wash (done monthly and will be done on
Wed), but this problem continued after last month's shutdown wash. I'm
thinking of attaching a syringe to the col 2 post column line and
pushing a few mls of 10% nitric acid followed by MeOH. I plan to let
the MeOH sit ON, then so another shutdown wash w/ ACN. Any other
suggestions? To reduce the outgassing, I've been thinking of degassing
the Deblock w/ Helium prior to putting it on the machine. I wish I
could lower the room temp a bit, but the AC unit is maxed out.
Second question. I'm on a quest to reduce n+ products. We're testing
the 20% diethylamine deprotection Pharmacia talked about at the Tides
2000 meeting. I'll be attacking the multiple "G" coupling issue soon (I
use Ethylthiotetrazole). My biggest problem is with dimer products.
I've seen this for years, it's a batch to batch problem. I usually buy
commercially pre-packaged CPG's, 1000A (I'm making a lot of 68-88mers).
I once had a significant dimer product, but was due to the protecting
group falling off. However, I've also seen this w/ "T" (i.e. no
protecting group). At the Tides meeting, none of the manufacturers
talked about this, but then again, they all seem to be making their own
CPG with proprietary linkers and analyzing on HPLC or CE. We see this
most often w/ PAGE analysis (EtBr stained), rarely on HPLC. Anyone see
a reduction in dimer formation by switching linkers? I'm leery of going
to polymer supports, they tend to give lower yields and I don't know how
well they respond to the synthesis of such long oligos.
Thanks in advance for the advice.
Debbie Walther
Scientist I
ValiGen
300 Pheasant Run
Newtown, PA 18940
(215) 504-4444 ext232
fax (215) 504-1546
www.valigen.net
This archive was generated by hypermail 2b29 : Thu Jul 20 2000 - 09:37:01 EDT