Chromatography Forum

I just want to have simple question. I'm running my venlafaxine sample via HPLC using 250X4.6mm, 5microns particle size phenyl column. What would be the effect of changes in concentration on its retention time. Let say between 0.001mg/mL against 1mg/mL. Thanks. by the way, I'm using gradient condition where my mobile phasse are buffer pH 3.0 and 100% Acetonitrile.

So long as you are not overloading, retention time should be independent of concentration. At high levels, overloading *usually* results in decreased retention time and increased tailing. Occasionally (particularly if your analyte has limited solubility), retention time will increase and you will see fronting.

So long as you are not overloading, retention time should be independent of concentration. At high levels, overloading *usually* results in decreased retention time and increased tailing. Occasionally (particularly if your analyte has limited solubility), retention time will increase and you will see fronting.

originally, i prepared 1 mg/ml concentration of sample, afterwards i injected it and I was getting approximately 6.985 minutes retention time. From the same 1mg/mL sample, I diluted it to have 0.001 mg/mL and when I injected it, i am getting 7.3 minutes as retention time. Why on this case, the results seems dependent with concentration? As from my method, it was noted that 1mg/ml sample retention time should be having +/- 0.1 difference only from the retention time of 0.001mg/mL but base from what I'm getting, results did not follow the premise given. Any possible reason behind?

I'd make a linearity experiment, and inject concentrations from your lowest to highest (same injection volume), and then inject lowest at the end. If your retention times are less for the higher concentrations, likely would be that there's more molecules present for a finite amount of column particles/phase.

results did not follow the premise given. Any possible reason behind?

What you are seeing is perfectly reasonable: at 1 mg/mL, retention time was shorter than at 0.001 mg/mL, which implies that at 1 mg/mL you are seeing some overload effect. CPG's advice is spot-on: run a series of calibrators to establish the linear range.

CPG's advice is spot-on: run a series of calibrators to establish the linear range.

Thanks, Tom. Logical is 98% of the game in my estimation.

In my opinion, the logical approach is way better than the lethargic one.