Unretained Compound

[vikmurush]

Compound: Bis(2-chloroethyl) amine HCl
Wavelength: 195nm
Detectors used: CAD and UV

I am trying to make this compound retained and I tried

1. C18, Phenyl Hexyl and Cyano columns using acetonitrile and water as MP. I also tried changing aqueous and organic in the MP and didn’t help.
2. Primesep with 0.1%, 0.05% TFA in water and Acetonitrile as MP
3. Normal phase with amino column (hexane and etoh as MP)
4. C18 with Ion pairing agent
5. Normail Phase with Supelco Supelcosil LC-SI

None of the above gave retention for the above compound. I would appreciate if you any ideas/suggestions.

[SIELC_Tech]

Use mixed mode approach. You can retain your comound based on cation-exchange and reverse phase mechanisms. Here are few examples. You will need ELSD, LC/MS or RI detection. Your compound has no UV activity even at 190 nm.

http://www.sielc.com/compound_107.html (ethanolamines are similar to your compound)
http://www.sielc.com/compound_005.html

[SIELC_Tech]

Just realized thta you tried Primesep 100.
You troubles come from detection. Run UV spectra for your compound and you will see thta it is not adsorbing even at 190.

[vikmurush]

Thank you for your suggestions.

I am seeing this compound using 195nmUV and on CAD with reverse phase mp. I also tried RI and not able to detect. I tried Primesep C.

[SIELC_Tech]

With Primesep C you don't have cation-exchange mechanism at pH-2 (0.1% TFA), because acid fragment on the surface of column is not ionized. You need pH above 3 (3-7) in order to retain your compound on Primesep C column. Try sodium phosphate pH-6.5. Expect K prime of 5-10 (8-15 minutes on 150 mm column).
Check applications for Primesep C column. You will find a lot of hydrophilic amines retained, but pay attention on pH of the mobile phase.

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

[Kostas Petritis]

What kind of ion pairing reagent did you use?

[vikmurush]

I have used 5 millimolar heptane sulfonic acid.

[maria.rey]

You could probably use a polymeric cation exchange column with suppressed conductivity detection.
Diethylamine is an analyte that can routinely be analyzed by columns such as the Dionex IonPac CS17, with a simple acidic eluent.
I would expect the chloro groups in your analyte of interest will make the compound more hydrophobic and "sticky", longer retained.
With a simple acidic eluent, the peak may be broader.
To sharpen it, I would first try elevated temperature for the column (40C), and as a last resort, I would add 5% acetonitrile to the acidic eluent.

[dave.cheng]

Compound: Bis(2-chloroethyl) amine HCl
Wavelength: 195nm
Detectors used: CAD and UV

I am trying to make this compound retained and I tried

1. C18, Phenyl Hexyl and Cyano columns using acetonitrile and water as MP. I also tried changing aqueous and organic in the MP and didn’t help.
2. Primesep with 0.1%, 0.05% TFA in water and Acetonitrile as MP
3. Normal phase with amino column (hexane and etoh as MP)
4. C18 with Ion pairing agent
5. Normail Phase with Supelco Supelcosil LC-SI

None of the above gave retention for the above compound. I would appreciate if you any ideas/suggestions.

You can use HILIC(Hydrophilic Interaction Chromatography) column, which could retain highly polar analytes that would not be retained by RP.
you can contact with tech_service@alltechemail.com for tech support and via alltech@alltechemail.com for ordering HILIC column.
Good Luck!

[vikmurush]

Thank you for your suggestions.

I have ordered HILIC column today and will screen once i got it. Also, thinking about ordering cation exchange column too.

any other ideas/suggestions will greatly appreciated.

[JA]

Wow, compared to a lot of posts on the forum that really was an easy sell, especially given Vlad's information where a solution may be found with your existing column hardware I had a feeling it should also be possible with options 4 (retention, but detector incompatibility with CAD) and 5 (maybe ion exchange or HILIC on this column too), given the right usage.

Quite confused how a non chromaphoric compound can be observed in UV but not by RI; one would of expected the opposite. open question: has chlorine, like bromine, got a noticeable UV absorbance?

[SIELC_Tech]

In my opinion whatever vikmurush sees at low UV is not target compound. You don't need too much of UV active impurity to mislead you. Neither amine nor chloride absorb in UV. Every approach you had tried should give you retention (IP, HILIC, Primesep). The problem is in detection and not separation. Try to take any other hydrophilic amine (Benzylamine, for example at lower pH will give you good indication) and you will see (UV) that it is retained with some of your 5 approaches.

Try to find suitable detector. ELSD is my choice.

[vikmurush]

Thank you for your suggestions dear Sielc_Tech

I have read about Primesep columns through the links you have provided and very interesting. Lot of applications used ELSD on Primesep columns. Do you have any information/applications on these columns using CAD?

Thank you for your help.

[SIELC_Tech]

It will work with CAD. I would inject your compound without column measure peak area (CAD) and then run it wit column and measure peak area with column, both areas should be within 5-10%.
You might need Primesep 100 and run it with ACN/water/TFA (similar to ethanolamines in my previous reference)

[vikmurush]

Thank you Sielc_Tech

May i know why do we need to compare peak areas with and without columns? We have Primesep A, B and C columns in house and what are your suggestions?

Thanks