Understanding trace in chiral method

[Mike H.]

This is a chiral method using an AD-H column (25cm, 5µ) from chiracel. MP is 90:10:0.4 Hexane:EtOH:TEA, 1.0 ml/min, 40°. Concentration of main peak is 1.0 mg/ml. Injection volume is 10µl. Diluent is EtOH.

The question is pose is why does the background trend like a hill instead of remaining flat? (the are of concern is between 16min-22min. Any ideas? thx

[HPLCaddict]

Could this be an artifact due to the strong solvent effect? You're using pure ethanol as solvent and a mobile phase of 90% hexane...
Try diluting your sample 1:1 with hexane and inject 20 µL. What happens?

[Mike H.]

I'm not certain my sample will dissolve in 50:50 etoh:hexane, but haven't actually tried it. I will try this. thx

[itspip]

If your sample wont go into hexanes, or even the 1:1 EtOH:Hexanes, try dissolving in ethanol first then diluting to a final concentration of 1:1 EtOH:Hexanes.

[Mike H.]

It was soluble in 50:50 Hexane:EtOH at 1mg/ml, but the hump did not go away completely. I'm tried several diluent blends using hexane,etoh and IPA to varying degrees of success. What seems to have the greatest effect so far is to inject the smallest amount. The hump is ~2mAu injecting 0.25µl of a 10 mg/ml solution.

Is there any tricks that can be used to minimize it further?