Chromatography Forum

Can anybody recommend how to analyze cyanoacetamide by LC?
NP or RP?
UV or RID?

I would say HILIC with low-UV detection. 80% acetonitrile, 20% water. Atlantis HILIC silica.

Uwe Neue,

I do not have Atlantis HILIC culmn,but I do have a Cosmosil one.
In 80% AcN/20% 10 mM AmmAc buffer a peak of CAA is very close to front.

In 50% AcN/50% 10 mM AmmAc buffer there is no peak.

Can you explain this?

In HILIC, water is the *strong* solvent. If you had little retention with 20% H2O, try 15% or 10%.

My recommendation is based on HILIC done on a HILIC silica column. I have no clue how your analyte will behave on this Cosmosil column, which could very well be more of a RP column than a true HILIC column.

Cosmosil does offer a silica based HILIC column. Interesting, it has a triazole bonded phase.

If you do have the Cosmosil HILIC then when you inject your sample at 50% organic you should have an even taller, unretained peak in the void volume.

If you see a poorly retained peak with 80% ACN and then nothing at 50% ACN then you probably do have a C18 column.

yaakov:

Can you tell us more about why you're looking for cyanoacetamide? LC-HILIC-low UV and GC-NPD/MS have been suggested across both the LC and GC forums, both of which seem reasonable. However, if you gave us more information on matrix type, purpose, detection limit concerns, etc., there may be people here who could suggest more appropriate instrument conditions/parameters/detection techniques. More info almost always gets better answers.

bisnettrj2,

I have read your conversations with other participants and at long last understood that: "This aggression will not stand, man" is just your device...I'll try to be more forthcoming:

I need a method for Assay of Cyanocaetamide CyAcAm. The impurities are Acetic asid AcAs, Acetamide AcAm, Cyanoacetic Asid CyAcAs.

I guess GC/NPD is not useful for AcAs.

I have run Zorbax SB-Aq in 100% water buffer: a good resolution of AcAm and CyAcAm, but AcAs and CyAcAs were on tail of CyAcAm.

Anion Exlusion on Aminex-87H provides wonderful resolution of AcAs, CyAcAm and and CyAcAs, but no AcAm peak.

That is all,

yaakov

Mardexis,
The results described were obtained using Cosmosil triazole bonded column.
Regards,
yaakov

Dear Yaakov

Looking at your compounds I have to think to look at them according to their Pka.
acetic acid= 4.75
acetamide= 17.0
cyanoacetic acid= 2.45
cyanoacetamide= 2.95

In HILIC mode we can speak of 2 interactions that we can play around with
hydrophilic and ionic.
playing with them is done by changing the organic composition and the buffer concentration. I can send you information about the cosmosil HILIC and how it affects it's separation

Which detector did you use, was it UV?
by changing the organic and buffer ratio you have a different pH, this can cause a shift of the ABS for your compound. We had such a thing for an amino acid we were working on recently by HILIC

Dear unmgvar,

I completely agree with you regarding the pKa. That is why I have tried first an Anion Exclusion chromatography that is supposed to separate acids according to their pKa. I have got the following RT:
acetic acid= 4.75 RT~ 13 min
acetamide= 17.0 RT~ 42 min
cyanoacetic acid= 2.45 RT~11 min
cyanoacetamide= 2.95 RT~14 min
UV 190 nm was used.
Any information about Cosmosil HILIC will be greatly appreciated.
Thanks,
yaakov

Ammonium acetate at 190 nm??? Why not phosphate/phosporic acid?

Uwe Neue,
AmmAc was recommended by Cosmosil producer. 190 nm was not set.
In AnExcl Chrom 0.02 N H2SO4 was used.
yaakov

Looks like a combination of bad advice. I now suspect that you saw your analyte(s) in 20% of your ammonium acetate solution and not in 50% because your detector went completely blind with the higher concentration of ammonium acetate.

Since we now know that some of your analytes are ionic in nature, what is the pH of your ammonium acetate solution?

Yaakov

it does looks like you got a good separation with the anion exchange.
what was the problem there? why did you not optimise it?

with the cosmosil HILIC you can reduce the ionic interaction by increasing the buffer conc.
try to go to 50 even 100 mmol/l, try being at 85-95% ACN.
it is also one of the moves advised in case of mixed compound types, acids, neutral, based.

do a simple check with standards dissolve in MB in a spectrophotometer to see the more suited WL to look at them.
If you want to educate us tell us what happens when you look in 50:50 ACN-buffer and 80:20
BTW in what solvent do you inject them, and what volume?