Chromatography Forum

This is a overlay of 100ppm, 200ppm and 300 ppm of one of our compounds. What would cause a retention time shift that is influenced by analyte concentration? Note that the retention is dropping as the concentration increasing. Thanks.

Actually, the peak shape is surprisingly good. The shift in retention is more consistent with overload. See
http://chromforum.org/viewtopic.php?f=31&t=18426

And it depends on the time the column was flushed with MP to get to an equilibrium. Peak shape and symmetry looks excellent.

We use an Inertsil ODS-2 250mm x 4.6mm x 5µm column. Is it possible that the decrease in RTs with increasing concentration be caused by the sample diluent being different than starting MP conditions?
At the start of the gradient the composition is 98:2 10mmol potassium phosphate monobasic pH 3.2: ACN and our diluent is just plain 98 water:2 ACN. One interesting observation is that the RTs do not shift at a 1µl injection. Normal injection volume is 10µl

The peak shape’s not too bad, but it’s tilting forward more and more with concentration increase.
That means overload.

Best Regards

We use an Inertsil ODS-2 250mm x 4.6mm x 5µm column. Is it possible that the decrease in RTs with increasing concentration be caused by the sample diluent being different than starting MP conditions?
At the start of the gradient the composition is 98:2 10mmol potassium phosphate monobasic pH 3.2: ACN and our diluent is just plain 98 water:2 ACN. One interesting observation is that the RTs do not shift at a 1µl injection. Normal injection volume is 10µl

Are you injecting the same volume for each standard?

If you are injecting different volumes to achieve different concentrations as some do then it could cause such a problem if the standard is not made up in the starting mobile phase solution. It is best to always inject the same volume for all standards and samples if possible.

We inject the same volume for every injection.

I'm going to try a run with reduced injection volume. I actually thought about overload initially but 10µl of 0.5 mg/ml sample didn't seem like it would be enough to overload a 25cm column.

We use an Inertsil ODS-2 250mm x 4.6mm x 5µm column. Is it possible that the decrease in RTs with increasing concentration be caused by the sample diluent being different than starting MP conditions?
At the start of the gradient the composition is 98:2 10mmol potassium phosphate monobasic pH 3.2: ACN and our diluent is just plain 98 water:2 ACN. One interesting observation is that the RTs do not shift at a 1µl injection. Normal injection volume is 10µl

Retained peaks are not severely affected by the sample diluent. The peaks seem to show a non-classical fronting behaviour. If you overload further, then you would see either split peaks or much more clear fronting (shark fin type) peaks. If lower injection volume is not leading to mass overload, then why not try a lower injection volume?

Regards,
Farooq

my guess is that your compound is basic in nature and part of the retention comes from residual silanols cation-exchange mechanism. You don't see change in peak shape yet because you still have some capacity left, if you make 5-10 times higher concentration you will start observing tailing.

We inject the same volume for every injection.

I'm going to try a run with reduced injection volume. I actually thought about overload initially but 10µl of 0.5 mg/ml sample didn't seem like it would be enough to overload a 25cm column.

I'm thinking that most of what I inject is in the low ug/ml concentration range and I inject 10ul. Most of what we run here will give peak heights in the 30mAU and lower range instead of the 50-150mAU range like your peaks. If those three peaks covers the range of sample concentrations you need for your analysis, then you would easily get by with injecting 1-2ul of samples or even diluting the samples before injection.