Chromatography Forum

Hi Uwe Neue,

That was a Waters Atlantis Hilic column. It was named as such. I dont remember if it is a C18, but before I used it - I am pretty sure it is a Waters Atlantis Hilic column.

The brochure did not mention any pH compatibility. Thanks for sending me the link. I will keep this info for future reference.

Thanks Uwe.

Good information in the care and use manual. I appreciate for sending this valuable link.

Hi Uwe,

Your book on HPLC columns - is there a new edition to this. I am very interested to purchase this item.

Thanks Uwe for the information on Atlantis columns.

I dont remember if it is a C18, but before I used it - I am pretty sure it is a Waters Atlantis Hilic column.

Just keep in mind that a C18 is not HILIC.

Hi !

I think this molecule is ionizable due to an amine function. The pKa may be quite high. If your molecule isn't retained at low pH, why don't you try at hign pH.
Because if it's a basic compound (amine function) you must work with a pH above the pKa for basics.
I advise you to try at pH 9,10 or 11 with an XBridge column from waters.
(eg BEH Shield RP18)

You must perform a generic gradient :
Initial : ACN/Buffer (5:95 v/v)
15min : ACN/Buffer (95:5 v/v)

And then adjust your chromatographic conditions (T°C, gradient, flow rate etc...)

Enjoy !

Thanks for your suggestion adrien16

The paper/brochure Uwe links to, should give an idea of what some of us tried to communicate to Vikmurush. It should be mentioned too, that method development, optimization etc. would ideally be preceded by acquiring some basic knowledge of chromatography and in these particular case different types of interactions.
Mohan_2008, if I may I would point out that both methanol and TFA (as suggested previously) would eliminate the possibility of UV detection at this short wavelength. With regards to the chemical contribution in this context I really can’t see the rationales for using those chemicals.

Best Regards

Thanks Danko,

I said TFA by mistake. No we are not using TFA here. As per Methanol, it is a 20% methanol in water combination. I don't think this would lower the sensitivity of detection due to lower operating wavelength (lower than Methanol cut-off).

I can understand if Methanol is significantly present in the combination.
But, please correct me if I am wrong.

Vikmurush's Method development problem is pretty simple. I am trying to tweak the method to obtain an optimal retention time, say 3 min.

I had this problem occurred to me several times, and most of the resolution came through regular RP methods.

Thanks for your suggestions mohan. I tried with methanol and acetonitrile and with methanol i have noticed a significant loss in signal. At 195nm i dont think i can use methanol for this compound.

If your compound is not ionizable (as Vlad and Uwe said) ion-pairing chromatography won't help. As my post indicated you need to be able to ionize your compound at a certain pH in order for ion-pairing chromatography to work.

Can you do a quick PDA/DAD spectral scan for the compound preferably at your mobile phase composition (20% Methanol in 80% Water) and see if you can obtain an alternate absorption maxima.

If it over the Methanols UV cut-off, probably you can use that wavelength.

I would like to ask vikmurush by what means it was confirmed that the compound given in the opening post elutes at 1 minute on a 250 mm column.

Then for my benefit, I invite the other contributors to explain how this compound, in a neutral state, can elute before t0 on a "deactivated" C18?

Thanks.

My take on this long discussion which goes nowhere (I am sorry):
1. There is no proof that the peak at 1 minute is the target compound. The compound has no UV activity, any impurity of unknown nature will throw you off the track. You need an alternative technique to confirm the elution. At this point, you are guessing and it is not a good approach. ELSD or LC/MS can give more indications.
2. The compound is not ionizable, it is not amine but rather phoshonamide. It might be ionizable at a very high pH. So ion-pairting will not work. Polar embedded or HILIC will work
3. Methanol will totally mask any UV activity that this compound has (below 195 nm). You need to use VERY pure ACN and any additives you use (buffer, acid, etc.)
4. Take any silica column you have in your drawer and run this with 85% ACN and volatile buffer, try to use CAD or ELSD. More ACN will give you longer retention, pH 7 will give you slightly longer retention than pH 2 (due to more polar surface of silica, not due to ion-exchange between silanols and "amide").
5. For reverse phase, use any C18 column and run it with 100% water and buffer/acid, make sure that column is compatible with 100% water (most of the modern polar embedded columns are)

Hi Vlad,

You said, Phosphanamide group is not ionizable as compared to a regular -NH2 group and I am assuming you are talking about a low pH.

I am just trying to understand why phosphanamide acts rather differently than an amine. Is this because, even at low pH the phosphanamide will be sufficiently acidic and hence will not be protonated or ionizable.

I am curious to understand any technical reason.