> ABRFers using 1 mm columns:
>
> The recent dmessages concerning 1 mm columns has raised some questions
> in my mind. I tried using these several years ago without much success.
> I would like to start a discussion on this topic. If you are using
> these columns (or have tried to use them), and would like to participat=
e
> in this dialog, I offer the following questions as starting points.
>
> 1)What applications are being done on 1 mm columns?
> 2)What advantages do you obtain from the 1 mm column (vs. a larger
> diameter)?
> 3)What are the problems encountered? How are they solved?
>
Dear ABFRers,
I have followed the discussion about the 1 mm i.d. column problem with qu=
ite
some interest as I really believe that this kind of technique is standard=
, at
least in the field of protein chemistry. Most of the columns that you can
commercially buy are of good quality and will not differ in their
characteristics from the 4.6 mm format except that you operate them at us=
ually
around 50 =B5l/min instead of 1 ml/min.
Of course, this places some constraints on your HPLC system and the UV
detection. This, I believe, has been adequatly discussed in previous mail=
s. It
is clear that you are not going to run successfully a 1 mm column with a =
pump
that is designed for preparative flow rates ... or with a flow cell in th=
e 10 of
=B5l volume that will completely destroy your separation. I think most of=
us are
aware of these problems, as read and discussed in previous mails.
The choice to go for a 4.6 mm, an 1 mm or a smaller column is now mostly
directed by the amount of sample that you have at your disposal, by the k=
ind of
application that you have to do and by the kind of chromatographic system=
you
use. Every time you decrease the column i.d. by 2, you gain theoretically=
a
factor 4 in sensitivity. This law, in my experience, is holding pretty we=
ll down
to the sub-microbore format (500 =B5m i.d.). In parallel, the amount of m=
aterial
that you can load onto the column decreases concomitantly. So, if you hav=
e tons
of material available, you go for the large columns. For small amount of
material, you go for a small column. Once given this dimension, it is wis=
e to
adapt the chromatographic system: you need a preparative HPLC for a prepa=
rative
column and you need a microbore HPLC system for a microbore column. Given=
good
understanding of the system you use, it is always possible to adapt an HP=
LC to
perform in a wider range of application than it has been designed for: th=
us, it
is possible to run an ABI 120 with a microbore column (1 mm i.d.) althoug=
h this
will be never as good as having a real microbore HPLC system to do the jo=
b. I
think most of us will aggree with this statement.
So, in my view, the discussion limits itself to the right application wit=
h the
right equipment. As further analogy, you do not perfom ultracentrigation =
in the
100'000 g with a preparative centrifugation rotor; similarly, you do not =
try to
centriguge liters of cell culture with an ultracentrifuge rotor. In my la=
b, I
run in a standard manner a 0.5 mm i.d. column with a submicrobore HPLC sy=
stem.
The flow rate to operate the column, 15-20 =B5l/min, is delivered without=
flow
splitting because I bought the HPLC system to perform this task. The flow=
cell
has a volumne of 300 nl that is adequatly small to not destroy the
chromatographic separation. I would probably damage the whole system if I=
wanted
to run a 4.6 mm column. Of course, there are some increasing difficulties=
to
trouble-shoot such a system, but this are things that you learn along. I =
do not
see microbore (or sub-microbore) chromatography as more difficult than
analytical chromatography. It is just best adapted to the kind of samples=
I am
working with.
Best regards,
Axel
_________________________________________________________________
Axel Ducret, Ph.D.
Senior Research Biologist
Merck-Frosst Canada Inc.
Dept. Biochemistry and Molecular Biology
P.O. Box 1005
Pointe-Claire-Dorval PQ H9R 4P8
Canada
tel. + (514) 428-3428
fax + (514) 428-4900