Chromatography Forum

I loaded about 3-4 mL of ~5mg/mL crude onto a semi-prep column and performed mass spec on fractions collected, including solvent peak. I got the mass expected of pure compound in the spectra of the solvent peak and a peak a little later but not in the other peaks (One of the other peaks was before the peak that showed compound presence).

Anyone know why this is happening/what I can do about it?

Note: The column had been cleaned and the blank run before and after the run looked normal

Anyone know why this is happening/what I can do about it?

Not without more details about what you were doing:
- what kind of stationary phase?
- what mobile phase?
- how big was your "semi-prep column"? and what was your flow rate?
- what was your sample dissolved in?

c-18 column
Ran a water/acetonitrile gradient from 5-95% over 60 minutes.
Sample dissolved in 50:50 water/acetonitrile solution
Flow rate: 10mL/min

Not sure how big the column is but it's definitely big enough where injecting with 3-4 mL of ~5mg/mL solution would not overload it.

Try diluting your sample in 95:5 Water:Acetonitrile - you might be seeing a dilution solvent mismatch upon injection.

I'm sorry but I'm new to this. What exactly is dilution solvent mismatch and how does it affect my run?

You are injecting a "slug" of 50:50 ACN:water (plus sample) into a flow of 5:95 ACN:water, this will caused a localised compiosition change in the mobile phase flow into the column which may mean that the sample is not retained in the column "uniformly". This may cause the elution of the peak in several fractions.

You see similar effects in HPLC when the injection solvent and mobile phase have major differences in composition.

Check the chromatogram I posted in this thread:

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