Chromatography Forum

I have a problem..i developed UV method using Dimethyhexylamine (10mM) and methanol in my gradient. My peak comes out at 4.0 min when ATP is diluted in water only. The sample is diluted in 100mM ammonimu bicarbonate buffer ,when sample is injected peak comes out at 0.9 min.

my column in C8,2.1x50mm,2.5 micron.

injection volume is 10 ul

what is ammounim buffer doing to make my peak elute so early

Ion-paring is generally very difficult to use in gradient separations.
Retention is dependent on ionic interactions so my guess is that the 100mM bicarbonate is to strongly eluting.
What is the pH in your eluent and sample?

I am not an expert in Ion-paring so I am sure there is a way of making it work but the easy way of doing this separation is in HILIC mode

http://www.sequant.com/files/documents/ ... nd_ATP.pdf

The ph of my mobile phase is 7.0 and sample ph is 8.0.

Hi,

You are using Dimethyhexylamine as a ionparing agent.
the problem is with your buffer, the amonium present there is compiting with the Dimethyhexylamine, as a result of that competition the amonia is a realy bad as a ion paring agent, so your peak elutes earlier than with Dimethyhexylamine.
what do you need is to change the buffer salt: try diluting with mobile phase or use Dimethyhexylamine as part of your buffer...forget about the bicarbonate, because the species buffering there are Dimethyhexylamine/ Dimethyhexylamonium.
the pka of the Dimethyhexylamine is arround 10.7, pH 8 could be a low pH to be a buffer with good capacity...but that is another topic...
I hope this help you.
Slds,

G

I can not remove bicarbonate buffer from sample..is there any other way to neutrilize its effect?

I can dry my sample plate and redissolve in organic only but i am afraid the ATP will stay stable during drying or it will change to ADP?

Hi again,

options: 1-reduce injection volume (and concentrating the solution)
2-substitute the cation in your bicarbonate buffer
3-dilute your buffer concentration
4-increase the amount of your ionparing agent

You can play with this options to your convinience...
The Drying step could affect the ATP, that should be your last option.

slds,

G