chromatography problems for phosphate containing compound

[williamsn]

Hi,

I'm new to this forum and a novice at chromatography. I'm a biologist/pharmacologist who has to develop my own analytical techniques. I have a hydrophobic compound which has been made more soluble by the addition of sodium phosphate (NaHP04). I can detect the parent (minus the Na) and multiple daughter ions by infusion on my mass spec (AB 3200 QTRAP) but my chromatography is terrible. I get multiple peaks. I have tried a phenomenex synergi fusion RP or just a C18 column. I use water and acetonitrile both with 0.1% formic acid as my mobile phase ( have tried multiple gradients). I finally succeeded in getting 1 broad peak but my column now seems to be retaining the compound as I get a similar peak with blank samples. The chemist who made the compound gets a single nice peak on a different mass spec using a C18 and water/acetonitrile + 0.05% TFA. I don't want to use TFA but am wondering if this could account for the difference in our results? If anyone has any suggestions, I would be most appreciative. Someone suggested ion pairing, but I'm not sure how I would go about doing that and what would be acceptable additives that would not have a lasting impact on my mass spec.

Thanks,

Noelle

[Uwe Neue]

What is the composition of the sample solvent, and what is the starting composition of your gradient?

[williamsn]

My sample solvent is water + 0.1% formic acid as is the starting composition of my gradient.

Thanks,

Noelle

[Uwe Neue]

I am not sure what is going on. My suggestion is to do two blank runs before you inject your sample again. Do not keep the column for an extended time in the water. If this gets you back the nice and narrow peak, you have had a dewetting problem. Otherwise check if your pump is working (disconnect the MS and measure the flow rate).

[Kostas Petritis]

I do not think that you would see dramatic differences between mobile phases containing TFA or formic acid, unless there is some solubility problems that are taken care with the addition of the TFA (due to the pH differences or ion-pair formation but I would doubt that too). As you are novice in chromatography, can I ask if you are using HPLC grade solvents and addtives for your mobile phase? And when you are infusing your compound, the daughter ions are once you select your compound for fragmentation or in full scan?

If I was you I would try to use the exact chromatographic conditions of my collegue by using high purity solvent and reagents... if you still can not get your peak let us know...