Schaap at rullf2.medfac.leidenuniv.nl
Wed Jan 22 07:12:09 EST 1997
In article <5c4p3q$21e at mserv1.dl.ac.uk>,
"Michael Duggan" <michael.duggan at camr.org.uk> wrote:
:There are methods in the literature using HPLC of derivatised
:(o-phthalaldehyde, for example) samples but before we invest in enormously
: expensive fluorescent detectors, can anybody answer the following
:1. Are the hplc methods more sensitive than the enzymatic methods?
: If so by how much?
First of all, I have no experience with glutamate detection, but did a lot of
courses on analytical chemistry. So my remarks are more general.
HPLC has more potential to sensitive methods. Really depends on your pre-column
method. The cleaner the sample, the more sensitive.
:2. What are the best derivatisation reagents for this technique?
If you go into the trouble of derivatisation, why not consider GC? If you have a
nice matrix-free sample, you can derivatize glutamate readily for GC. Than you
have indeed a very sensitive method. Only, derivatisation is very troublesome.
You need a lot of time to optimize the method.
:3. Are there any hplc methods which would allow sensitive detection by
: absorbance rather than fluorescence (we have a UV detector
: already, so this would be a lot cheaper)?
A very nice approach is a inverse one. Glutamate (I believe)-does not absorb
UV-light. You can load an UV-absorber in your eluens, and you get inverse peaks!
Where glutamate repels your eluens.
:4. Does anybody have any other good ideas about how best to do
A funny approach is the one of Orwar et alii. Science, vol 272 (1996) 1779-82.
Patch-clamp detection and CE. But not sensitive enough. And laborious!
CE is a very nice method to consider anyway. You only need some cappilars, a
high-voltage power supply, you can use -i believe- your standard UV-detector.
Detection methods like HPLC. Sharper peaks, very small loading samples
(microliters)- indeed fine for ntrans detection. Only you need a lot of
background info. Consider walking by your analytical chemistry dpmt. Profit:
glutamate is a very mobile ion!
:Thanks for any help you can give us
I hope it's useful.
:Mike and John
Regards and succes,
A man conducting a gee-whizz science show with fifty thousand dollars' worth of
Frankenstein equipment is not doing anything scientific if he knows beforehand
what the results of his efforts are going to be. A motorcycle mechanic, on the
other hand, who honks the horn to see if the battery works is informally
conducting a true scientific experiment.
Robert M. Pirsig
== J Schaap =======================================================
Faculty of Medicine, Leiden University
e-mail: SCHAAP at rullf2.MedFac.LeidenUniv.nl
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