How to seperate this kind of compound...

[bos]

Hi, experts!

Please give me some suggestion to seperate these two compounds! They are Europium Complexes from two similar tetraamide ligands. You can see that the only difference is A has one more methyl group on the arm.

I have tried C18 column (5 micron, 250X5) with various ratio of acetonitrile (0.1% TFA or 0.1% HCL) and water ( 0.1% TFA or 0.1% HCL), and I also tried MeOH. The load is about 200 microgram, I only got one huge peak at 2-3 min, which I assume is the solvent peak (the compounds were dissolved in pure water).

Any information or advice will be highly appreciated!!

[Uwe Neue]

1. This compound is very polar. I would expect that it will be barely retained in 100% water. This is also indicated by your description that you got a large peak that is essentially unretained. These are your compounds.
In order to get them retained in reversed-phase, you need to use a packing that is compatible with 100% water. The best choice is the Atlantis dC18 from Waters. Alternatively, packings with an embedded polar goupr are also compatible with 100% water (such as SymmetryShield RP18 or XBridge RP18, also both from Waters).

2. What do you know about the stability of the complex? Is it stable under your acidic conditions, or are you separating the organic part from the Eu.

3. You need to start with low loads, before we worry about how to separate a large quantity of the material. 200 microgram is a large load for a compound with this molecular weight.

It is also possible to get retention of the analytes in HILIC. I am fairly sure that I can get retention on a HILIC silica column. However, I do not think that HILIC is a good way to separate two compounds that are only different by a methyl group. My opinion would be to try to do your best with reversed-phase.

[bos]

Uwe Neue,

First, thank you very very much!

For your point 1, accturally, I am also thinking about change to a polar column. But since I am not a HPLC person, I don't know which kind of column I can start trying with...Thank you again to provide me some choices!

For your point 2, I am pretty sure that my complex is stable. But considering about this, if I have the ligands only (no Eu3+ complexed), what kind of condition you would suggest to separate the mixture?

For your point 3, I will pay attention next time when I do the HPLC. Accturally I was starting with 10 micrograms, but since I did not see peak other than the first solvent peak, i was wondering maybe the load was too low, so I increased it...

[bos]

Uwe Neue,

I searched the website of waters and could not get any information about XBridge RP18 column. Does it have any other alternative name?

Thanks a lot!

Bos

[Uwe Neue]

I believe that the Atlantis dC18 would be best for you.

If you are interested in the XBridge packing, try www.behtechnology.com.

[pawan ratra]

Following parameters can be optimized for the better retention and separation-

pH of the mobile phase
The compund have -NH gp and will be easilyf orm -NH+ at lower pH. The ionized compound elutes early. Thus the pH of the mobile phase can be increased to maximize retention.

Selection of Column-
Start with C18 clumn with the high carbon load ( e.g. Inertsil ODS, Luna C18, Kromasil C18) for the maximum retention.

If no change in the retention time is observed, try using polar gp embedded columns. Phenyl columns e.g. Novapak phenyl, Phemomenex phenyl hexyl, can be used as the compound contains phenyl gp.

Use of ion pair-
If above changes make no changes in retention time, try using ion pair reagent like octane sulphonic acid sodium at lower pH like ph-2.5. The sulphonic part will interact with -NH+ and the compund will become more non polar. Use high carbon load column.

Alternatively you can also try mixed mode column like Primsep columns. Use different concentrations of buffer like KH2PO4 or H3PO4 rather than organic gradient for retention of the compound.

Once you are able to retain the compund then work on the separation.

I hope this will help.

[bos]

pawan ratra,

Thank you very much for your answer!

pH of the mobile phase
The compund have -NH gp and will be easilyf orm -NH+ at lower pH. The ionized compound elutes early. Thus the pH of the mobile phase can be increased to maximize retention.

I tried several mobile phase system: a) various ratio of water (0.1% TFA) with CH3CN (0.1% TFA), b) various ratio of water (0.1% HCl) with CH3CN (0.1% HCl), c) various ratio of water (no acid) with CH3CN (no acid), d) various ratio of water (no acid) with CH3OH (no acid).

Although I did not purposely change the pH, the pH should be different when I am using pure H2O/CH3CN than the acidic H2O/CH3CN system. However, none of them work.

Selection of Column-
Start with C18 clumn with the high carbon load ( e.g. Inertsil ODS, Luna C18, Kromasil C18) for the maximum retention.

I tried luna C18, it does not help.

If above changes make no changes in retention time, try using ion pair reagent like octane sulphonic acid sodium at lower pH like ph-2.5. The sulphonic part will interact with -NH+ and the compund will become more non polar. Use high carbon load column.

is it easy to remove the sulphonic part after HPLC separation?

[bos]

for a C18 HPLC, can I use high pH mobile phase (eg. pH=9)? What kind of buffer can I use?

[bos]

I believe that the Atlantis dC18 would be best for you.

If you are interested in the XBridge packing, try www.behtechnology.com.

I am going to try an Atlantis dC18...

[SIELC_Tech]

Hi Bos,

There are a few references in the literature, which are describing separation of various complexes of this type of aza-crown ethers.

Bioconjugate Chem. 2005, 16, 237-240
Bioconjugate Chem. 12, 320-324
Bioconjugate Chem. 5, 101-104

Each reference has a lot of literature examples for the separation of compounds like yours.

We recently developed a method for one of our customers for very similar compound (DOTA and nitro phenyl derivatives). The compound is different then yours (has acetic acid fragments attached to nitrogen). We are finalizing results and I can send you a method description if you provide us with your email.

I also have an example for your review; you can apply same principles. The method shows good applicability of Primesep mixed mode phase for retention of polar ionizable compounds. I know that this is a different compound but it shows you can you use Primesep column for polar compounds with multiple amines.

http://hplcmethods.com/compound_140.php

For ELSD/MS detection you probably can use Primesep C column with ammonium formate/ammonium acetate buffers and different pH (3-6).
For UV detection phosphate and sulphate buffers.



You compound will retain by two mechanisms: reverse phase and cation exchange so you can adjust mechanisms independently by varying amount of organic, buffer nature and concentration, and pH of the mobile phase.

For the effect of pH on the retention on Primesep C column please see the following link:

http://hplcmethods.com/compound_166.php

Application shows you how by adjusting pH and concentration of the mobile phase you can move compounds along the column.

I am not sure that you complex will survive analysis, but there is a chance that you can see both Eu+ and your ligand with our Primesep approach (ELSD detection).
If you are willing to send us a sample we can try to do method development for you. This service is free of charge and you can see the results and then decide if Primesep is suitable for you.

Contact us if you have question or would like us to do method development for you.

Regards,

Vlad

[bos]

Vlad,
Thank you very much!

My email address is jw301@hotmail.com. It will be highly appreciated if you could kindly send me the method discription!

About method development, could you please tell me how much sample you need?

Regards,

Bos

[Uwe Neue]

Whether or not a pH manipulation could get you different results is all a question of the stability of the complex. I suspect that the complex is definitely stable at neutral to alkaline pH. Thus you will always deal with the Eu 3+ form, and not with an only partially charged amine. It is possible, that the complex is not stable under acidic conditions, but then you always deal with a positively charged form, whether due to Eu3+ or due to protonation.
Ion-pairing is feasible, but you are contaminating your analyte with the ion-pair reagent, which is not a good choice if you want to prep the material. Also see my next comment on ion-exchange.
Ion-exchange is possible, but I do not give it a high rating, since you want to separate two entities that are just different by a methyl group. This is the same argument that I used to dismiss HILIC as an option.

[SIELC_Tech]

We need a few mg of material. If you have markers for impurities that might help us to identify peaks in the mixture. In your case may be it is better to analyze ligands first.
Send us a sample and we will analyze it in a few days.
50% of the methods you will find on our website were developed for our customers. Usual turn around time is 3-5 days (unless separation is tough and we need more time). In case of successful method you will decide if you want to try it.
We are going to do it only on Primesep columns, you can always try traditional approaches (C18, ion-pairing, ion chromatography). My guess is that mixed mode chromatography will give you more leverage to separate two ionizable compounds with slightly different structures. You are using synergism of two mechanisms.
Here are the link for latest methods for mixed mode chromatography (you can sort them by compound, day, column type, etc.):

http://hplcmethods.com/Applications_By_Publish_Date.php

[HW Mueller]

Pawan,
the NH groups are amides, they will not protonate easily. If the amino groups (chelated to the Eu) protonate you have destroyed the complex (I agree with Uwe, it would be surprising that this suvives a lot of acid).
Bos,
Strangely, the literature is full HPLC or TLC of complexes with radioactive metals, these guys take it completely for granted that they retain. I have done some myself, still I am continuously surprised, but I find it almost impossible to predict whether a complex ion is easily retained or not. If you dislodge the metal you can get a mess, especially in base they form polymer- like oxides which stick everywhwere. So of prime importance is to know, not guess, about the stability. You will just have to fiddle around with it, this would be easy if there were a radioisotope in there. My guess is that if you get enough retention (adsorption type) you can separate them.
Hopefully you will let us know how you successfully solved this.

[bos]

Thank you guys very very much!
I will post what kind of result I get....