Reversed-phase liquid chromatography (LC) is a simple and easy-to-use technique that will give satisfactory separations for
the samples that most of us encounter. Based upon column sales, somewhere around 70–80% of samples can be separated by reversed-phase
techniques. The high-purity silica-based columns available today are quite robust in the 2 < pH < 8 range and there are several
choices for columns that will work well up to pH 10. The first choice for most workers is to work at low pH, in the pH 2–3
range. Under these conditions, most acidic samples will have suppressed ionization and be well retained, as will be neutral
molecules. Basic analytes will be ionized, but if the bulk of the sample molecule is nonpolar, adequate retention often is
obtained. At pH < 10, many basic compounds will not be ionized, so they can be chromatographed successfully, but other bases
will still be ionized, so retention can be poor.
Columns that contain a polar embedded group, sometimes called "AQ" columns, have come on the market in the last 15 years.
These columns allow the use of 100% aqueous mobile phases in the reversed-phase mode. These conditions will sometimes give
enough retention of polar basic molecules that would be retained poorly at 5% organic, the lower limit for most reversed-phase
columns. In other cases, basic compounds still don't have enough retention, even in 100% aqueous mobile phases or at any pH
within the column's pH-stable range. In cases such as this, and when the sample contains a mixture of acids and bases, ion
pairing might be a viable option. Ion pairing has plenty of potential problems that discourage its use. These, plus the availability
of some specialty "mixed-mode" alternative columns, have made ion pairing much less popular today than it was in the past.
However, ion pairing can work magic for some samples. This month's "LC Troubleshooting" will review ion-pair separations.
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