As previous mentioned here on the blog I have for many months being trying to measure the glutamate (or as it is also known as , glutamic acid) content in some food related materials. Until now, not with a lot of success. But yesterday I obtained the following from the HPLC:
What you see here is on the top pure glutamate and in the lower part the first 10 min of an amino acids standard mixture (Sigma Aldrich AAS18, if anybody is interested) . To get this I used the procedure published by Yang and Sepulveda in 1985 (see here ) wich is a standard way of analysing amino acids. In short you react the AA’s with
phenyl isothiocyanate and then you separate the derivatives on a standard C-18 column, by an acetonitrille gradient. It probably don’t get more basic than that.
So why, oh why didn’t i get this long time ago? Well one problem was that I needed to get my head wrapped around the fact the I need very small amounts on a HPLC. In the AA standard chromatogram there is only 650 pmol 6,25nmol, which for me is like noting ( remember I am used to use 0.5ml of 5mM lipid for the DSC). So for several of the experiment I have done I have probably overloaded the column so it all came out in one big peak at around 3min.
One other problem was that I early on tried to run a real sample on the column, but I didn’t get rid of the proteins, wich the denatured on the column and that generated problems for the next many months. Therefor I have now talked with a biochemist and I will do a protein precipitation, with trichloroacetic acid and just to be sure I will try to extract the lipids there might be present with hexane (I guess).
When I finally get it working I will update you all again.
