So you’re thinking about doing HPLC (or UHPLC) and don’t have a method? You’ve never even seen a method for anything remotely similar. You might have even searched our HPLC chromatogram database for the application and haven’t found anything. (After all, that’s the quickest and easiest way to get some ideas.) What to do… Let’s start with what you know. If you know the solubility and polarity characteristics of your sample, you’re well on your way. The first thing is to determine what type of HPLC separation you should consider. Here are the choices:
- Reverse phase- analytes that are slightly to moderately hydrophobic, yet are still soluble in aqueous solutions
- Normal phase- analytes that are slightly to moderately hydrophilic, but still soluble in organic solvent
- HILIC or “aqueous normal phase”- analytes that are very hydrophilic or polar, but soluble in organic solvent to some degree
The main idea with HPLC separations is that “like attracts like”. As your analytes travel through the column, they will be attracted to the stationary column phase in varying degrees based on the similarities with that phase. If it is attracted significantly more to the stationary phase in the column and hardly at all to the solvent in the mobile phase, the analytes will be retained very well. Most HPLC analyses these days are done via reverse phase, using a C18 or Aqueous C18 column. Others like our Biphenyl phase are popular also. Columns like our IBD and PFP propyl are designed mostly for HILIC mode, but can be used in other modes also. Silica is exclusively for normal phase chromatography.
If your application is reverse phase and you think you may need to use 100% aqueous phase, select an Aqueous C18 column. Otherwise, a conventional C18 may work OK. Keep in mind that just about anything that can be done on a C18 will also work on an Aqueous C18. If your analytes are mostly aromatic (i.e., contain benzene rings in their structure) or contain conjugated double bonds, a biphenyl phase will probably provide better separation. Sometimes separations that don’t quite cut it on a C18 may work like a dream on a biphenyl column.
If my discussion here is a little too general for you, it may be time to dig into the details further. You’ll need to know the following to get to the nitty-gritty:
- Analyte structure, size, polarity and key functional groups
- Number of analytes, their similarities and differences
- Analyte solubility issues or conditions that trigger decomposition
- Analyte acidity or basicity, in other words, pKa
- Sample matrix and potential interferences, including extraction solvent(s)
You’ll need to select a phase that provides enough interaction with key functional groups so there is opportunity for analytes to be separated from each other. If you know the nature of potential interferences, you should consider that also. Limitations with mobile phase selection, miscibility and pH also need be considered. Ideally the primary solvent for your sample extract should be the same as your mobile phase (or mobile phase A if using a gradient). Solvents in your sample and your mobile phase(s) must be miscible.