amino acids isolation-important

[mtar]

Hi,
I have a question about isolation of amino acids and its derivatives from aqeous solution containing a lot of sodium chloride, my idea was a cation exchanger binding amino acids, but is it compatible with high salt concentration? maybe other simple spe procedure is known(adsorption chromatography), or how to desalt the solution easily and don't loose amino acids. Please help:)
MT

[HW Mueller]

Can you do a dialysis?

[mtar]

yes, i could, but it is time concuming, and i am workin with volumes about 100 ml, so it is not so good for me, and i could happen that aninoacids can be lost on MWCO100 membrane, the smallest i have

[HW Mueller]

If you give more details like relative NaCl concentration it could be that some of the IX people can give you fairly concrete suggestions. Theoretically, if your NaCL concentration is not astronomical, relatively, you could get rid of Cl- by using a cation exchanger under acidic conditions, etc.....

[Kostas Petritis]

Again if your salt concentrations are not astronomical, you could still use a cation excanger in the following way. Add a "Tee" after your injection valve, and have another pump pumbing pure water. Arange then the flow rates at the desired dilution you want to achieve. If you need to decrease the salt concentration 100 times you will need to pump 99 volumes of water for one volume of your sample. It might take some time to load your column but it should work.

You may want also to introduce some additional void volume after your "Tee" for better mixing...

[Mark Tracy]

If your AA or derivative is hydrophobic enough, a reversed-phase extraction is easier. You need to adjust the pH to the pI of the AA; this is where its aqueous solubility is lowest. Adsorb the AA onto a solid-phase extraction device. Wash with water. Elute with methanol plus your favorite acid.

The tricky part about cation exchange is not getting it to stick. Dilute the sample and lower the pH below pI. The tricky part is getting it off without eluting the sodium that is also bound. You need to raise the pH of the eluent to just above the pI with a low-ionic strength buffer. You will still end up with sodium in the solution, just not as much.

[SIELC_Tech]

Mtar,

We were conducting experiments on the influence of high salt concentration on the separation of amines (dopamine) and were able to separate/retain it even at 1M NaCl solution on Primesep 200 column. Chloride is not retained on the column but amine and sodium are separated. You need ELSD or LC/MS for most of the amino acids. I will post the link here when we create a file for our website.
Right now we are doing the same thing with tyrosine, tryptophan and phenylalanine (part of the catecholamine pathway) on Primesep 100 column.

What amino acids do you have in you mixture? What is your detection technique?

We have a method for separation of underivatized amino acids on Primesep 100 column:

http://www.sielc.com/compound_002.html

It should work with NaCl too.

Regards,

Vlad

[mtar]

I have to describe my problem more clearly:
my sample is a aques natural extract (very complex), it contains amino acids, carbohydrates and other hihg molecular weight components, and a lot of sodium chloride (concentration about 50%);
I want to find in the extract Amadori compounds which are sugar derivatives of amino acids, but if these compounds are present in the extract, the concentration are probably very low, so I wanted to isolate the fraction containing amino acids and Amadori compounds on strong cation exchanger (sugars and all the rest i washed with water), and here is the problem-sodium chloride is also binding to the resin, I have to add that I would like to isolate the proper fraction from about 100ml of the extract, and then analyse it with hplc on aminopropyl column with postcolumn derivatization and uv-vis detection, and also by means of mass spectrometry.
my idea was dialysis, gel filtration, precipitating sodium chloride with absolute etrhanol, some kind od adsorption chromatography to bind amino acids, and ion exchange chromatography to bind amino acids, but none of them is good enough for me;)
maybe you can give me some idea
thanks
mt

[HW Mueller]

How about a pre-column derivatization? This should impart sufficient hydrophobicity to do reverse phase.

[dlliu]

Dear all,

I'm kinda curious what happens to amino acid separation in HPLC column when some salt (e.g. NaCl) is around, in addition to ion supression effect on the MS detection. Will the presence of salt affect chromatography too?

I'd appreciate your comments.

DL

[Mark Tracy]

Pre-column derivatization on 100 mL of soup? His sample sounds like a beef bullion concentrate. Barbecue sauce? One can waste a fortune on FMOC-Cl and not have reliable results; FMOC is not soluble in brine. OPA/Thiol chemistry is at least soluble, but not stable enough.

Have you tried chilling the sample to precipitate the NaCl?

[HW Mueller]

One could probably make the "essentials" in the "brine" (small aliquot of that) to solve in the FMOC-Cl solution, though. But I agree: I would not work on this without first getting rid of most of the NaCl. There should be some temps and organics (alcohols, etc.) concentrations where this is possible.

If mtar needs all of the 100mL to do the analysis he has a "slight" problem, indeed.

[mtar]

Dear Friends,
all of the methods with dervatization through reaction with amino acid amine group are not suitable for me, beacause in the derivatives which I am looking for the amine gruop is "blocked" by sugar moiety, and thats why I am trying to find easy solution for analysis; my scenario for now is like that: concentrate, gel filtration to desalt, preconcentratioon on cation exchanger, and at the end hplc with postcolumn derivatization or ms. what do you think about that?

[Mark Tracy]

You may want to consider using cation exchange HPLC for the determination. If your sample is sufficiently desalted, you can simply inject a generous amount of it. Your don't say what post-column chemistry you have in mind. Is it compatible with cation exchange mobile phases? If the derivative is fluorescent, you should have very good detection limits, and perhaps not need the concentration step.

[mtar]

Hi,
my postcolumn derivatization is rection with triphenyltetrazolium chloride, it is reduced by the sugars and my compounds containing sugar moiety and the color is produced for uv-vis detection (hplc on aminopropyl column), other possibility is ms without derivatization; i have tried hplc with derivatization without preconcentration and it wasn't working, so i am lookin for something else which would work in my conditions