It will be awhile before I have the problem fully identified and solved, but I
thought everybody might want a sampling of the responses I received. The things
I am looking at are: bacteria in the degasser lines and a plugged needle
assembly. The responses are listed in the order they came to me and I've deleted
all names. Thank you for all of the help.
> If you have been leaving the system in 150mM salt all this time, your
> problem could be that the salt is slowly precipitating out on the interior
> surfaces of the machine. We have HP 1090s. Whenever we run salt in our
> RP-HPLC systems it is always washed out extensively every night with water.
> The salt-containing buffer reservoirs are removed and plain water is put in
> new reservoir containers. We wash the column also (if it can be washed with
> water) or we remove it if it can't be washed with water and the whole system
> is left in water overnight or slowly washing with water overnight. If we
> don't do that we see back pressure buildup and eventually total plugging of
> the system. Around here the motto is "salt is poison to HPLCs; use it and
> then wash it out immediately!". When you washed it with nitric acid you
> probably formed some nitrate salts; those usually precipitate out and plug
> the machine. Nitric is fine if your normal application is RP-HPLC since RP
> buffers don't usually contain ions that nitric acid will form salts with.
> But with size exclusion chromatography I would stay away from nitric.
If you are using PEEK tubing, you may have a line that is getting
stopped up due to the ferrule crushing the tubing, thus making the ID much
smaller, especially with stainless steel ferrules. Over half the time I have
a backpressure problem on my 1100, it turns out to be a plugged line at a
ferrule. When you have the backpressure problem, try loosening/retightening
all the fittings with the HPLC running, starting at the purge valve and
working your way to the detector. If the problem is at a ferrule before the
column, the pressure will not drop much when you loosen the problem fitting.
If the problem is at a ferrule after the column, you should see a major
reduction in pressure when you loosen the problem fitting.
It also just struck me that you might have something growing in your
degasser lines. The standard HP trick is to pump dilute bleach from the
pickup lines through the purge valve and then water wash the system. Just to
be safe, I would block the purge-valve-to-heat-exchanger line, just to make
sure nothing reached the column.
You didn't say whether your 1100 has a binary or quat pump. If it's a
binary, then it probably has the static mixing column. If you and/or
Agilent (boy, it's going to take a while to get used to that name) haven't
already done so, try taking that out of line and see if your backpressure
problems go away. You probably need the mixing column for your RP-HPLC
work, so you might need to get a new one (be prepared to get banged-up
knuckles when you make the swap). However, for SEC, it's completely
unnecessary; also, it might actually be detrimental if your mobile phase
salt is corroding the stainless steel in the mixer. Consider this if you
continue to alternate the 1100 between RP and SEC.
I had problems with my HP 1100 similar to what you described. We mainly
used it for reverse phase, but began having problems when we used it for
size exclusion with a buffer of similar salt concentration to what you are
running. Our's did not clog up, but we did have extra pump noise and
higher back pressures. The only obvious thing we could find was what
appeared to be rust on the gold seals on the check valves and in the valve
blocks. Our solution was to try to avoid salt buffers in the 1100 and
when we did run buffers with salt, to rinse the system thoroughly
immediately after.
Is the back pressure still in the system and not in the column? Have you
tried disconnecting sections to isolate where the back pressure is occurring?
(I don't have an HP1100 but this is what I do on my other systems. One system I
had had gotten enough seal material into a line that the clog would get
unclogged when I cleaned up the seal but then clog again as more was released
into the system up against some frit. Is there a mixer that could be clogging?
You could bypass it and see if that if where it is. Steve Tindall might be a
good one to ask this question of--he seems to know HP1100s reallly well,
That is the culprit I think. HPLC don't like salt (I had a method using
1.5M KCl) it just eat up slowly the pump seals and block the frits. The
buffer leak slowly at the back of the pump seal through the piston, the
solvent evaporate leaving rasor blade salt crystal. It normally leave a
black residues on the frits of the output pump valve. A way to avoid that is
to wash the back of the seal with a lot of water, daily. I don't know about
the HP pumps but with the 125/126 from Beckman they have a port to circulate
water at the back seal location. I used to use a peristaltic pump to have a
continuous circulation of water at the back of the piston seal.
Jeni - have you tried changing the active inlet valve cartridge on your
pump? I've had problems with inconsistent pressure that would go away once
I replaced the valve cartridge. It was not the high backpressure problem
you're describing, but it was a pressure problem which was confined to one
pump of our binary pump. We could get a stable back pressure on the A
channel, but not on the B channel. Replacing the active inlet valve on the
B channel has taken care of the problem more than once.
Are you using an 1100 autosampler, and if so have you checked the needle on the
injector or the plumbing on the injector for plugs? There may be a plug in one
of
these lines that is causing the high pressure.
I cannot say that this will cure your problems,but I had something like
this happen to me
a while back.I started disconnecting tubing,till the pressure went down to
zero.I pinpointed the problem to a piece of tubing which was coming out of
the injector.It looks
like some crud was around sightseeing.In one case the loop got completely
plugged up,and
another time the tubing that goes to the column.If you do only size
exclusion isocratically,there should be no trouble,unless something
precipitated while switching from
organic to salt.
The worst mess that I caused occurred when I had a huge volume to load,some
50 ml of it,
so very clewerly,I loaded the sample from the buffer A position,then
washed with the real
A buffer,and started an aqueous to non-aqueous gradient.Everything back to
the mixer was a
mess.So,there are many possibilities.
Good luck.
Is your system fitted with the thermostated column compartment?
In my 1100 all the columns are now fitted with in-line frits,
because I find that that even in 'normal' RP-HPLC use they tend
to block fairly quickly, necessitating a change about once a
week. Examination of the blocked frits often shows that they
have collected small pieces of 'rust'? I suspect that they may
be coming from the thermostated compartment - they look like
sprayed cast iron blocks to me. Running salt solutions through
these blocks is therefore bound to accelerate your problems - if
what I suspect is right that is - anyone from Agilent care to
comment?
Apologies. A quick test with a small magnet suggests that the
block is probably aluminium. However there must be something
pre-column that's causing these rust particles. Whatever it is
may be the root of your problem also. You could try tracing the
problem back by inserting pre-filters at various places in the
system, such as at the autosampler and pump outlets. The peek
frit-type filters have minimal dead volumes and so shouldn't
affect system performance too much;-)!
Janelle Lauer-Fields
Department of Chemistry & Biochemistry
Florida Atlantic University
777 Glades Road
Boca Raton, Florida 33431
561-297-2094/Fax 561-297-2759
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