And the there is the small thing called a phd thesis I have promised to write. It need some serious attending ASAP.  With these I kind was kinda run out of words/energy/time/what ever and the first “victim” was  my blog.

But now I have had two weeks vacation and tanked some energy  by looking at things like this:

And training my engineering skills by building by building this:

For use the glutamate analysis I have bought a new evaporator as the one we had in the lad only could handle 9 samples, which for the glu project is like nothing. I chose to buy a Stuart solvent concentrator as it can handle 60  samples in one go, which, I hope is more than I need, but the alternatives I could find  could only handle 27 samples.

I was also t hiking about a speedvac (a centrifuge with vacuum, basicly) , but they where around 40-60 KKr, without vacuum and the Stuart unit was just below 20 KKr wich helped making the decision. And then this unit can also be used for preparing lipid samples.

The second toy is an Eppendorf Multipette Stream for adding the reagents for the Glu analysis. I hope it will make my life simpler, but I don’t know as I still haven’t tried it

Pretreatment:

- If the sample is solid then I will dissolve it in MilliQ water, or a phosphate buffer (I need to remember nor to use an acetate buffer)

- If I can see some solids in it I centrifuge the sample (app. 10 min 14.000 rpm in an 1.5 mL eppendorf tube, or more if the particle don’t sediment)

Protein precipitation

Next step is to get rid of the proteins in the sample. They need to go as they could be denatured on the column and thereby block the HPLC column. As advised by a biochemist I do a TCA (trichloro acetic acid) precipitation

1.TCA solution:

Simply dissolve 50 g of TCA in 35  mL MilliQ water. I then keep this a 4 deg.

2. Precipitation

- To 500 myL of  the sample I add 125 myL of the TCA solution. If there is a lot of protein in the sample,      it immediately turn milky and one can see the denatured proteins as a white fluffy stuf.

- The sample then goes to the fridge (or cold room) for 5 to 10 min.

- The samples are the centrifuged for 10 min at 14.600 rpm (max speed of our centrifuge) and the at 4 deg.

-400 myL is them transferred to a new eppendorf for next step-

Removal of lipids

The lipis also have to be removed as they would stick pretty tightly to my HPLC column.

- To 400 myL of the protein free sample (in an 1.5 mL Eppendorf)  I add 400 myL hexane and wortex it.

-The again 5 min in the centrifuge to separate the Hexane from the water

-300 myL is then transferred to a new 500 myL eppendorf . This is now ready for the analysis.

The glutamat anlysis

The method I am using is a general method for amino acid analysis based on the old Edman degradation, ie. reaction of the amino acid with phenylisothiocyanate (PITC). I use the method published by Chao-Yuh Yang and Felix I. Sepulveda in Journal of Chromatography, 346 (1985) 413-416. This method has been used for food stuff in this paper: Daniels DH, Joe FL Jr, Diachenko GW: “Determination of free glutamic acid in a variety of foods by high-performance liquid chromatography. Food Addit Contam. 1995 Jan-Feb;12(1):21-9. and probally in many other papers.

Derivatisation

This is a two step process. First a evaporation with an ammonia source present and then the reaction with the PITC.

The evaporation

Here we use a drying solution of triethylamine (TEA) Methanol and water. One part of each, usually 100myL of each. I make a new solution for every experiment.

- The sample (usually 3 myL of around .5mM or 30 myL of around  0.05 mM is used) and 30 myL of the drying solution is mixed in a glass vial.

- Then evaporate the sample to dryness on a Thermo Reacti-vap set at 60 deg and with a stream of nitrogen flowing over the sample. (Usually this takes around 20 min).

The actual derivatisation

The derivation solution is also mader fresh for each experiment and contains 100 myl TEA, 50 myL water, 50 myL PITC and 350 myL MeOH.

- to the dry sample 30 myL of the derivation solution is added. It then reacts for around 30 min at room temperature (Probably i could cut down this time, but It works and in 30 min I do something).

- The samples  are again evaporated to dryness (or almost, it is difficult to get the TCA away) on the Reacti-vap.

- They are then dissolved in 300 myL of the buffer A for the HPLC analysis.

- Finally they are filtered through a 0.22myM filter syringe filter into the final vial for the HPLC auto sample.

When dissolving the sample in the last step I get some white crystals forming. Of what I don’t know, but that is primarily why I filter them.

HPLC

So now we are now ready for the HPLC. I have access to a Dionex Ultimate 3000 equipped with a auto sampler, 6 different columns and all what the is of extras. The column I use here is Acclaim 120 C-18 Reverse Phase.

The HPLC Buffers

-Buffer A:  Is a sodium acetate Buffer (NaAc) with TEA and Acetonitrille (MeCN). In details for 5L: 57,25 g NaAc is dissolved in 3-4 L of MilliQ water. To this 2,5 mL of TEA is added and the ph is adjusted to 6.4. 300 ml of MeCN is added and the volume i made op to 5L.

-Buffer B: 600 mL of MeCN mixed with 400 mL og  MilliQ  water.

The HPLC gradient:

0-1.5 min 100% A
1.5-11.5 min Gradient to 88% A
11.5-22.5 min Gradient to 52% A
22.5-27.5 5% A
27.5-39 min 100% A

Adnd that is all, I need to do to determine glutamate content
-

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.

This was seen on our HP printer this week. And yes that is what we always do …

First, long time a go I picked up our old Microcal MC-2 DSC from the  Technical university in Copenhagen  and trying have been trying to get it to run from time to time. But I never got it to run and as I now need the lab space, this is where it lives now:

It is not supposed to be it’s final tomb, but who am i kidding. I will probably be in there for a long time.

Then there is my HPLC project, were I am trying to measure glutamate contend of some food stuff and related materials.  The one thing I have excelled in is producing strange errors on the HPLC. The latest being that the column is leaking from were it is not possible according to the owner.  I have still not succeeded in determining the glu contend of  anything. But I have bought the following:

As you probably can it is an Invitrogen glutamate kit. I it is so much nicer to work with than the Sigma kit i used long time ago. For one thing there are only 2 pipetting step per sample (besides the diluting steps).  Then  it is an fluorescence based assay, emitting at 590 nm, wich is so much nice that trying to do abs at around 300 nm in yellow and brown food stuff. On last thing is how it is packed. It looks so need:

Okay real life is calling. I will be back soon I hope wit more from the lab.

Dear Lars,

Your replacement MI-4156 Electrode is on its way. Please see attached invoice.
Thank you for your business – we appreciate it very much.

Sincerely,
Microelectrodes, Inc.
(603)668-0692

That’s really good costumer service! And they usually make great electrodes.