Chromatography Forum

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

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.

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.

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

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?

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