Chromatography Forum

Dear chromatographers,

I'm getting a headache with one peptide analysis, because the response is pretty poor for the lack of real chromophores. When thinking about derivatization, the possibilities seem to be endless. Anyhow, some of you might be able to say right away, what would work and what not.

The sample is a peptide having four AA's. One aa is Arg. The C-terminal is protected.
I've searched in the literature, and possible candidates in my opinion for reagent might be dansyl-Cl, FMOC-Cl, PITC, and probably NBD-F?

I'd like to know which one would work best when the aim is to have sample preparation nice and clean and the reaction being relatively fast. The sample is water solution. Will that be a problem? And finally the amount of side-products should be as small as possible, 'cause I'd like to use this method as a stability indicating.

For the best candidate, what would be the reaction parameters? How the trick is done?

Best regards,
JAM

Below is a chromatogram of methylated arginines determination in rat plasma using NBD-F:

http://www.silvertonesciences.com/files/TI371E.pdf

They separated the AA on Unison UK-C18. The derivatization
protocol / journal reference is there as well.

Check out the AccQ-Tag method from Waters. Reaction in seconds...
It is designed for amino acids, but I do not see why it should not work for your problem...

Dear both,

Thank for the tips. I just heard that my colleague in our subsidiary company had bad experiences with NBD-F for this problem. I have to ask more deeply what went wrong.

I've read about AccQ-tag method, but my understanding is that it needs special column for that analysis. Is this correct? I was dreaming about using the same amino column that is used for the assay at the moment. The compound and its degradation products are very hydrophilic, but it has been retained in some 100%aqueous RP-columns as well.

Thanks anyway!

You need the special column only, if you want to do a standard amino acid analysis. You want to run a derivatized peptide, so you do not need an amino acid column.

Jam,

Which detection wavelength have you chosen? Also, what is the column diameter?

Best Regards

Dear Uwe, Thanks for clarifying things. The AccQ-Tag seems to be promising having a short reaction time and limited amounts of side-products if any. Maybe I have to give it a try! I believe the procedure is well described in the instructions?

My chosen column dimensions at the moment are (250*4.6mm, 5µm) and the wavelength now is 205nm. If the derivatization will be succesfull, the detection wavelength then will be around 254nm. The elution might be changed also, so probably I will gain more resolution also...

JAM

Hi Jam,

I don’t know how much sensitivity boost you need, but if it’s not an astronomical number, you might like to try a couple of things, that are going to increase the sensitivity a number of times.

First: Move the wavelength to a longer one – 215 nm. The peptide bonds in any peptide absorb a lot of energy at this wavelength, whilst the background absorption will be reduced – compared to the 205 nm option.

Second: Change the column to a 3 or even 2.1 mm diameter column (provided you don’t inject huge volumes e.g. > 100 μL). This is the action/change that will boost sensitivity the most.

The latter will necessitate a reduction of the flow rate, so this could be an additional incitement – savings.


Best Regards

Dear Danko,

Thanks for these advices! I haven't played with peptides before, so all information is new. I will definetely try 215nm as a wavelength. The ID reduction sounds interesting too.

JAM