Chromatography Forum

Hello,

In our HPLC application (size exclusion), we know that mobile phase pH and IS are important parameters and we have a tight protocol for making up our eluent. However, we have the current (lazy?) practise of injecting our samples (typical injection volume ~50 ul) from solutions that may differ in pH by 2 or 3 units and have much lower IS than the mobile phase. We have been able to get reasonable results usually.

My question is this. If you inject about 50 ul (microlitres) of sample that has a low IS, let's say 0.005 M into an eluent of IS 0.1 M, is that little bit of injected water able to equilibrate with its the eluent water (that is to either side of it) by the time is reaches the column?

Thanks,

Dave

By virtue of the chromatography, your analyte will be immediately separated from the low-concentration injection plug. In your case, i.e. in size exclusion chromatography, it will migrate down the column faster than the plug of water. (Of course, I assume that you have a standard rather fat SEC column, where an injection of 50 microliters is small compared to the column volume.)