ammonium bicarbonate Vs ammonia buffer

[subha]

Hi

Currently i have a gradient HPLC run which uses 10 mM Ammonium bicarbonate buffer adjusted to pH 10.0 with ammonium hydroxide.

MP A : the above buffer
MP B: ACN

When i run gradient

time A B curve
0 95 5
5 95 5 6
17 50 50 6
36 50 50 6
37 95 5 6


I see a negative baseline after 8 minutes. the baseline goes from 0 absorbance units to -0.01. and then goes back to 0 when the gradient reaches 95 :5.


When i use 10 mM Ammonia (PH is 10.6) as my MP A. I don't see this behavior . But my late eluters specificity interchanged.

Column used is xterra, RP8 3.5 u, 4.6x150mm
wavelength 235 nm.

at 225 and below negative baseline is worse.


My question is why i see negative baseline with ammonium bicarbonate and why not with ammonia at 235 nm wave length.

Buffer capacity of 10 mM ammonia (pH10.6) is good or not. I am wondering to get the same specificity as ammonium bicarbonate can i prepare 5 mM ammonia ( does it have buffer capacity or not).



Please explain

[Noser222]

The carbonate absorbs at low UV wavelengths. Since the concentration of carbonate during the run is decreasing, the absorbance of the mobile phase decreases.

Ammonium carbonate is a very nice buffer from pH 9-10 because you are at the pKa of both the ammonium and the carbonate. To get the same buffering capacity out of ammonia by itself, I guess you'd need at least twice the molarity.

To make the baseline a little better, maybe you could try adding ammonium bicarbonate to the acetonitrile as well.

[Uwe Neue]

If you have a low-pressure mixing system (which typically comes with more than 2 solvent inlet lines), you can keep the buffer concetration constant by making a 100 mM buffer and diluting it 1:9 with the acetonitrile-water gradient. This gives you a constant 10 mM buffer concentration.

You will need to stop the buffer line before you do the final flush to the high acetonitrile concentration.

[subha]

Hi

Thanks for the reply. I did try making line A 100mM ammonium bicarbonate and line B ACN and Line C 100% water. But to prepare 100 mM ammonium bicarbonate i have to add more than 25mL of ammonium hydroxide and critical pair separation got effected.

I am wondering if i have line A 95% buffer +5% ACN
Line B : 95% ACN and 5% buffer

Then the gradient will be

time A B
0 100 0
5 100 0
17 50 50
etc.,

I hope this way specificity may not get effected and easy to make it.

Please guide me through.

thanks

subha

[Uwe Neue]

Essentially, your observation means that the resolution of this particular critical pair is a function of the buffer concentration. It is not possible to predict, how to fix this, but you could try to use a lower buffer concentration, say 50 mM instead of the current 100 mM. in line A.

The other approach that you are thinking of will still give you a baseline drift, and you are back to where you started.

Or am I missing something? Why did you stress that you needed to add 25 mL of ammonia?

[subha]

Hi

Thanks for email. My active comes at 14 mins. Impurity 1 comes at 12.2 mins. Imp 2 is at 16 mins. Impurity 3 is at 24 and Impurity 4 is at 26 mins. and Impurity 5 is at 27.5 and impurity 6 is at 29 mins.

and a placebo peak at 30 mins. and lots other placebo peaks after 30 and another impurity 7 comes at 34 mins.


When i used 5 mM ammonium bicarb, baseline seemed good but Imp. 1 did not resolve properly fro Active peak at 14 mins.

Temp did not help either. So i had to stick to 10 mM buffer.

If i have both buffer and ACN in both A and B does not it help with the baseline.



thanks


But tomorrow i am going to look into 50 mM ammonium bicarb tomorrow.

Another thing is to adjust the pH of ammonium bicarb to 10 i have to add atleast or more than 25 mL ammonium hydroxide for (1 L of 100mM ammonium bicarb).

thanks