Post by rddAnyone use dabsylation/HPLC for determination of aa's? If so can
you comment on success of method, column used, column life,
pitfalls, etc?
We use it. It has advantages and disadvantages, as always. Any decent
C-18 column will do. Most vendors provide conditions you can start
from.
Advantages:
The derivative seems quite stable on an analytical scale. The reaction
is easy to do. All you need is a UV/VIS detector. Pierce has a little
instruction pamphlet for how to do the derivatization. You can
download that from www.piercenet.com.
Disadvantages:
You mix an aqueous solution of your sample (buffered) with an acetone
solution of the reagent, evaporate to dryness, take up in ethanol-
water, heat and inject. You need a good understanding of how much free
amino group you have in the matrix or else you could use up your
reagent. Given that you have a need for evaporation of the sample this
may not be sufficiently quick, once you find out you have a problem.
The same time problem arises if you do not have sufficient
concentration of analyte. Our solution is to make a 10-20X dilution of
the sample in water, dilute 10X, 100X and 1000X in buffer and then
analyze all three diluted solutions, hoping that we have provided a
sufficient range of dilution to cover the needs. If you have any
meaningful number of samples that is a loony solution in terms of
management.
It may be worth mentioning that in any pre-column derivatization it is
essential to have sufficient reagent to cover the needs of all
compounds which can react with the reagent. In post column work all
you need is sufficient to cover what is coming off the column at any
given moment.
The reagent is not all that stable. It needs some care in storage.
Caution: You will see a huge peak for unused reagent (presumably). You
will see a smaller peak for hydrolyzed reagent (presumably). Run
blanks. Otherwise you will find yourself wondering why one or the
other does not behave well on a calibration curve. Also, make sure you
get both compounds off in the run, or you could have them messing up
later runs.
Derivatized amino acids will run before and after these peaks, so you
need to pay attntion. Not that that is anything new but we all have a
tendency to see a nice large peak and say, "Hey that's a great
response." I have not done that in at least a couple of days but I
expect I will soon enough.
We do very little amino acid work (basically for glutamate in foods)
but my preference would be to do chromatography as the amino acids and
then do post-column derivatization with a reagent which does not
itself have signal under the detection conditions. Ortho-
phthallaldehyde comes to mind. Pre-column derivatization is more
common but the methods say to inject OPA derives within a minute or
two of derivatizations. I'm darned if I know why - never gave it much
thought, and I am not inclined to worry about whether that is a
reasonable concern.
There are other fluorescence probes, too.
I think most people nowadays do reverse-phase separations but this is
not really my turf, so take that with a pinch of salt. Dionex has very
good ion-exchange columns, so I am tempted to do ion chromatography
with some variety of post-column derivatization. Amperometric or
conductivity detection may be sufficient at food levels, which would
simplify my life. With that solution or some post-column solution, the
overhead of pre-column (in fact pre-hplc) derivatization presented by
DABSylation goes away. We seldom have the luxury of scheduling an
instrument for tomorrow and not finishing with the instrument that
day. I could live with in-line pre-column derivatization but that
requires me to mess with my hplc systems more than I want to mess with
them.
Unless what you are trying to do is limited in scope, you may be
better off finding a good amino acid lab to do it. I can recommend one
in the US, if you like, where I send my more complicated stuff. That's
all they do.
- Shankar