Chromatography Forum

I have a amino acid library to purificate, these compounds have bad rentention behavior, them elute with solvent at the dead time on C18, CN column even using pure water, I know the common methods for amino acid analysis is derivation or ion exchange, but for preparative HPLC these methods are not available. which column or method should I choose? thanks!

In addition to the traditional approaches to retain amino acids (ion-pairing, HILIC, etc.) you can use our approach. We have several columns with embedded ion-pairing reagent (IPR), so you can use a simple ACN-water-TFA (formic acid, ammonium formate/ammonium acetate) mobile phase and basically have any retention (from no retention to indefinite retention) for amino acids and other ionizable compounds.
Here are the links:

http://allsep.com/makeChr.php?chr=Chr_009 underivatized amino acids with ACN-water-TFA mobile phase

http://allsep.com/Technology_RetentionO ... pounds.php Retention of Polar Compounds without Ion-Pairing Reagents

http://allsep.com/Technology_Preparativ ... graphy.php example on preparative chromatography of small peptide, including amino acid impuritis.

Of cause you can try traditional ion-pairing chromatography followed by ion-exchange to remove IPR or try HILIC. Some participant on this board will provide you with examples. Contact us if you need additional information.

Regards,

We have numerous of customers using ZIC®-HILIC for zwitterionic and amphiphilic compounds. We can try to support you with our zwitterionic stationary phase.

The task that you want to accomplish is not trivial. Ion-exchange (and related techniques) tends to give a low preparative loadability. HILIC or even normal-phase chromatography are potentially better from the standpoint of column loadability, but I expect that amino acids do not have a good solubility in organic solvents.
I would look first, if the solubility of the amino acids in DMSO, methanol or acetonitrile is adequate, and then I would try HILIC on a silica column.

Thanks a lot for all your opinion!

The statement about the lower loadability might not be true in all cases. With mixed mode chromatography you have both ion-exchange interaction and reverse phase (like on a regular C18 column). Our study showed that it is at least comparable to ODS AQ column for the retention/loadability. The separation is related to small peptide, amino acid and byproducts – “Optimizing Selectivity for Preparative Separations: Mixed-Mode Chromatography versus Reversed Phase and HILIC’

http://allsep.com/brochures/PrepOptimization.pdf

Regards To All

Blackdrum,

Some years ago I developed a method for very polar amino acid separation. The method separates the 10 more polar proteinogenic amino acids by using ion-pairing chromatography isocratically (Asp, Asn, Glu, Gln, Cys, Ala, Pro, Gly, Thr, Ser). In later years, where I had a bigger selection of amino acids I saw that is possible to separate simultaneously 15 amino acids.

Anyway, the method employs 1 mM only of pentadecafluorooctanoic acid (which is still realatively volatile) so I do not know what is going to give in terms of loadibility. The column used tooks 3 hours to be equilibrated so there is a certain amount fixed in the stationary phase. If you'll find out that the loading capacity or your preparative application is not enough, you could go one chain less (tridecafluoroheptanoic acid) and use a higher concentration of ion-pairing reagent.

The reference is:

Title: Ion-pair reversed-phase liquid chromatography for determination of polar underivatized amino acids using perfluorinated carboxylic acids as ion pairing agent
Author(s): Petritis K, Chaimbault P, Elfakir C, Dreux M
Source: JOURNAL OF CHROMATOGRAPHY A 833 (2): 147-155 FEB 19 1999

By the way I just saw that it is cited 34 times so it should be usefull to some people...

SIELC_Tech and Kostas Petritis,

Thanks for your references.

Maybe I should give more details about these compounds, they aren’t typical amino acid, but are synthesized by my colleagues. The matrices are complicated, containing impurities which polarities are in a wide range, so isocratic condition isn’t suitable. The mixed-mode chromatography is attractive, but I need more details, such as stability under gradient conditions, and how long need to equilibrate. YMC ODS AQ is in common use in my lab, it’s stable at 0.1%TFA(w/v), but it give little retention to those compound under this condition. I have received a post from waters recently, promoting its atlantis column, the chromatograms show it suit for polar compound. Has anybody used this column? Could you give any comment? Thanks!

Another column worth considering is Macherey-Nagel Nucleodur 100-5 C18 Pyramid. This column is designed for use in eluent systems of up to 100% water and has highly polar surface derivatisation which make the column act differently that other C18 columns.

Steve,

What is the highly polar surface derivatization used for the Pyramid column?

That's going to cost you. Why don't you order a column an take it for a drive.

Well, from your earlier suggestions one would surmise that your column does not have a reverse gear..... who would want to drive such a column nowadays??
Also, this proprietariness is getting to be a real nuisance.

HW Mueller

Welcome back… Long time since you have been around. Hope your vacation was refreshing. If was certainly a welcome for us!

We all here are just trying to help people out the best we can.

Blackdrum,

With our mixed mode columns you can do three types of gradient. Mobile phase usually has ACN, water and additive which provides ion strength to the mobile phase and facilitates ion-exchange mechanism. You can adjust retention time of compounds by changing organic content, concentration and nature of buffer or both. Based on the fact that a lot of compounds have different hydrophobicity and different ion properties you can separate very different compounds on Primesep columns.
Here are gradients you can use:

1. Organic gradient with ACN from 0% to 70%, keeping buffer concentration the same (TFA, formic acid, ammonium formate, ammonium acetate, phosphate buffers, etc.)
2. Constant organic concentration with gradient of buffer (0.05 % to 0.3% for acids, 5 to 100 mmol for salt buffer).
3. Double gradient (according to the combination of paragraphs 1 and 2)

In addition you can play with the gradient of pH and change ionization state of your compounds and Primesep stationary phases. Some of our columns are "Switch-mode" and you can turn on or off either hydrophobic or ion-exchange properties (like in SPE applications).

You can find a few applications in "Literature" and "Application" sections on our website for compounds with different polarity and hydrophobic properties (http://allsep.com/Posters_Home.php)

"Universal Stationary Phase for Reverse, Normal, Ion-Exchange and Ion-Exclusion Chromatography"

"Fast Separation of Vastly Different Compounds by Isocratic HPLC"

"Simultaneous Separation of Inorganic and Organic Compounds in Single HPLC Run"

(if you are unable to open links here just go on our website and check publications and applications sections)

You can download Primesep brochures at http://allsep.com/Brochures_Home.php

Contact us if you need more help

Regards,

Some of our columns are "Switch-mode" and you can turn on or off either hydrophobic or ion-exchange properties (like in SPE applications).

Sielc_Tech,

Do you really mean that you can switch on and off the hydrophobic properties of your columns? It sounds kind of unlike to me... I would guess that the hydrophobic properties would be always on...