Chromatography Forum

How efficient is a HPLC method when internal std elutes with same mobile phases but different mobile phase 'composition(%)'? Can I go ahead and use that method for my experiment?

e.g The product elutes with 75:25: 1 of mobile phase composition but internal std elutes with 55:44:1 of the mobile phase.

Thanks in advance

If I understand the situation correctly you’re running both sample (analyte) and internal standard in the same run/injection and the elution is carried out using a gradient.
If the above is true, you’re doing the right thing and the procedure isn’t unusual at all.

Best Regards

the less chemically similar the internal standard is to the analyte, the less point there is in running it: it will compensate less appropriately for differential losses in extraction between different samples.

If your internal standard runs under drastically different conditions to the analyte, it may not be a good choice of standard. If it just runs a bit later or earlier in a gradient, you may be fine.

By definition, an internal standard must be added to the sample before it is injected. The internal standard and your analyte must be quantitated in the same run. The idea is that errors that affect both peaks in the same fashion (for example, injection volume errors) will cancel out. As lmh and danko pointed out, as long as both compounds elute in the same run (which can be a gradient run), then in principle, you are ok. As lmh pointed out, however, it is a good idea to have the internal standard and the analyte elute fairly close together (so that errors will affect both peaks the same way).

The situation you describe has the two compounds very widely separated (20% difference in strong solvent), so it's not ideal, but that's not a fatal problem in a gradient separation. If you are doing isocratic runs, however, that is *far* too much separation.