Glutamate release

Jeroen Schaap 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:
:Dear All,
[snip]

: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 
:questions:
:
: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 
:       this?

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,

Jeroen

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
phone:   (0)71-5276763
e-mail:  SCHAAP at rullf2.MedFac.LeidenUniv.nl



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