Chromatography Forum

Can i run a gradiënt from 0.1% ammonia in acetonitrile to 0..1% ammonia in water? I tried to make ammoniumcorbonate buffer in 5% waterin acetonitrile but buffer does not desolve. Is a buffer relay neacesary for hilic or will 0.1% ammonia so do.

The potential problem here is not whether or not the electrolyte additive is buffered. Rather, it's that retention in HILIC decreases as pH increases above pH 7 (see my recent papers and David McCalley's that document that). An aqueous solution containing 0.1% NH4OH has a pH slightly above 9. I'd recommend that you drop the pH to the range 8-8.5 at least. If you want a volatile solvent, then you might consider making an aqueous solution containing 20% NH4OH and another aqueous solution containing ammonium carbonate with the same molar concentration of ammonium ion in both solutions. Mix them together in whatever ratio is necessary to give you the pH that you want (8? 8.5?). Use the resulting solution as a stock solution for preparation of your HPLC mobile phases (and no, do not bother measuring or adjusting the pH after addition of the acetonitrile. Just prepare the mobile phases reproducibly and you'll get reproducible results).

Deze Andy,
Thanks for your answer. I did chromatography with 1mM buffer ph 10 and get good retention but bad tailing peak shape. I also had bad peak shape if i inject witout column. At 50 mm buffer in water i get good peak shape but 50 mM buffer does not disolve in water/acn 5/95. That is why i am thinking about 0.1% ammonia

We are missing about 80% of the necessary details here:

1) What are you analyzing?
2) What solvent is your sample dissolved in when you inject it?
3) What "buffer" did you have 1 mM of, how did you prepare it, and how did you prepare the mobile phase that it's in?

I and analyzing pyridoxin 5'-phosphate and thiamine pyrophosphate. As buffer i use ammonium bicarbonate. I prepare stock buffer 400 mM and add 25 ml stock to 175 ml water for eluens A and 25 ml stock to 175 ml ACN dit eluens B. If i inject without column with eluens A i get Nice peakshape but the buffer precepitate in eluens B. Max concentration buffer in water /ACN 12.5/87.5 IS APROX. 2mM but then peakshape of TOP is horible.

I tried different Sample solvents. Water is a problem becouse it is hilic.acetonitril os a problem becouse analytes disapear probably becouse of bad solubility of analytes. So dat i use methanol and i inject 0.5 ul. Later i trie different solvent s to inject lager volumes.

The way we analyze these B vitamins in different food matrices by LCMS involves hydrolyzing the possible phosphate groups by enzymatic digestion. By doing this, you can analyze thiamine and pyridoxine using reversed phase.

It might not be applicable for your analysis since you will be measuring the sum of phosphates and native vitamins.

I believe I see the problem. You are using a starting mobile phase (mobile phase A) that's nearly 100% ACN and a final mobile phase (mobile phase B) that's aqueous, and then generating your gradient by blending these two solvents. That is a bad idea for the following reasons:

1) As you've learned, 100% ACN is a poor solvent for electrolytes. It's also a poor solvent for B vitamins.

2) The mixing of water and ACN is an endothermic reaction; the combined solutions cool. This change in temperature could affect the chromatography. You would be better off having some water in the mobile phase A reservoir along with the ACN and having some ACN in the mobile phase B reservoir along with the water. Ratios of, say, 9:1 or 1:9 would suffice. That way, the endothermic reaction takes place prior to the chromatography and you can use mobile phases that are at room temperature.

3) It isn't necessary to use high levels of ACN in order to get B vitamins to be retained adequately in HILIC, per the following example:
http://tinypic.com/view.php?pic=287hyl1&s=9

To spell out the running conditions:
Column: PolyHYDROXYETHYL A, 200x4.6-mm, 5-µm, 60-Å
Mobile phase (isocratic): 40 mM ammonium acetate, pH 5.0, with 75% ACN
Flow rate: 1 ml/min
Detection: 265 nm

Retention times: riboflavin (B2): 3.6'; pyridoxal phosphate (B6): 4.8'; thiamine (B1): 9.5'; niacin: 13'.

Thiamine pyrophosphate will be even better retained.

You should make sure that you have at least 20 mM salt present in order to shield negatively charged groups in the vitamins from electrostatic repulsion, since most HILIC materials have at least some negative charge.

If you start with, say, 80 or 85% ACN in mobile phase A, then you will have adequate capacity for dissolving enough salt in it to serve your purpose. It is important to use this as the sample solvent as well, or at least a solvent that has the same concentration of ACN to within 10% of that in the mobile phase. Otherwise, you could get skewed or even multiple peaks for your analytes due to "fingering effects".