Chromatography Forum

Hello,

I have a problem with one HPLC method I use.

Eluent A Water +0.1% TFA
Eluent B ACN +0.1% TFA

C8 column

Gradient from water to ACN.

I inject a sample containing 1,2-dioleoyl-3-trimethylammonium-propane a cationic lipid and 28 times more sugar. The sample is dissolved in ACN/H2O.

The standard is only the lipid in ACN (same concentration).

At the 100 % concentration everything works fine.

If I dilute the standard 1:100 in ACN and the sample 1:100 in ACN/H2O I get the problem that the standard area is only 2/3 of the sample area.

If I adapt the matrix of the standard to the sample (put sugar inside), the area of the standard is as big as the area of the sample.

I can reproduce this effect.

Can anybody explain this effect?

Thank you in advance

A few questions:
1. What's your detector? PDA? ELS?

2. Why is the solvent you're using for your sample different from that for your standard? (I can think of a few reasons but I want to know yours).

3. What do you mean by "At the 100% concentration everything works fine?"

I'm sorry, but I don't think there's quite enough information (for me at least) to begin to give a cogent analysis.

Hi,

1. DAD

2. Because it is not possible to dissolve the sample in ACN and it is also not possible to dissolve the pure lipid in ACN/H2O

3. At the 100% level of my sample the accuracy is ok, the area of the sample and the 100% standard are nearly the same. At the 1% level the areas differ like described.

New information:
I don’t need the sugar in the standard, the water, the sugar is dissolved in, is enough to get similar areas between sample and standard at the 1% level.
But I still don´t understand the reason.

Consider adsorption of the sample to the wall of your vial, and that this adsorption may be modified by the addition of the sugar.