Chromatography Forum

I am currently using Shodex normal reversed phase column RsPak DE-413L column for HPLC analysis. The compounds that I intend to analyze is PHENOL and GUAICOL (2-methoxyphenol).
The following conditions were used for the analysis: flowrate 0.7 mL/min; eluent 0.005 M HClO4 aqueous solution/CH3CN = 50/50; oven temperature 40oC; UV/vis detector 220 nm.
However, i find that the RsPak De-413L is not able to separate these compounds (phenol, and guaiacol) and when analyzed in HPLC, it will come out together as 1 peak.
I notice when i analyzed the standard phenol and guaiacol only by its own it eluate at 10.3 and 10.4 minutes. But when it is analyzed together it will come out as 1 peak at 10.44 minute. Is there any advice on how i can separate out these 2 compounds. I have tried to change the wavelength from 220 to 280 and still no effect.

Several comments here.

First of all, changing the wavelenth will *not* improve your separation. If you think about it, the detection is carried out *after* the analytes have left the column.

Second, as a very rough rule of thumb, it takes about a 5-10% difference in retention to completely separate two compounds (depending on the plate number).

Third, that column is far from a "normal" reversed-phase column. It is a polymethacrylate, whereas the vast majority of reversed-phase columns are bonded phases on silica. That column would not be my first choice (or my second or third, for that matter). There is nothing wrong with starting method development with "whatever column is in the drawer", but we should not be surprised if it doesn't work very well.

At this point, you can try decreasing the acetonitrile concentration by about 5%. That should increase your retention times, and *may* give you enough separation to work with. If that fails (which is what I expect), your best bet would be to start over with something like a phenyl bonded phase column (or, check with your column vendor of choice and see what they recommend).