Selection of mobile phase solvents for reversed-phase chromatography

Typical mobile phases for reversed-phase LC include water with either acetonitrile or methanol as the
organic modifier. Less common modifiers are tetrahydrofuran (THF) and isopropanol. We recommend
that you always work with HPLC grade or better solvents and modifiers. For UHPLC, Agilent
recommends that you only use LC/MS grade solvents or better. Selectivity differences and sample
retention will vary significantly between mobile phases. Sample solubility is also likely to differ and
dictate the use of a specific solvent or solvents. In reversed-phase chromatography, both pH and ionic
strength of the aqueous portion of mobile phases are important in developing rugged methods not
sensitive to small variations in conditions. With ionic compounds, retention of typical species shows
significant changes with pH. It is very important to control pH in such reversed-phase systems to stabilize
retention and selectivity. A pH between 2 and 4 generally provides the most stable condition for retention
versus small changes in pH, and this pH range is recommended for starting method development with
most samples, including basic compounds and typical weak acids. For reproducibility, the pH used should
be ± one pH unit above or below the pKa or pKb of the solutes being separated. You may not know the
pKas of your analytes, so testing more than one mobile phase pH may provide the best results. Most
reversed-phase columns can be used between pH 2-8 or more, allowing a wide range to find the optimum
mobile phase pH for your separation. Note that when you are determining the mobile phase pH, measure
and adjust it on the aqueous component, before mixing with organic modifiers for the most accurate and
reproducible results.
Working with mobile phases
When you begin using a new column right out of the box, you should only use solvents that are
compatible with the shipping solvent. To prevent the buffer precipitating in the column, the buffer should
not be pumped through a column shipped or stored in 100% organic for reversed-phase operation.
Instead, we recommend equilibrating the column first, without the buffer, then equilibrating with buffered
mobile phase. Both the CN and NH2 columns can be used with normal and reversed-phase solvents, so
you need to check that your solvents are miscible with the shipping solvents before equilibration. If you
want to convert a normal phase column to a reversed-phase column you may have to flush it with a
mutually miscible solvent, such as isopropanol. Then you may equilibrate with your desired mobile
phase. Check the Useful References section in back for a solvent miscibility chart.
Troubleshooting mobile phases and mobile phase modifiers
In Figure 1, you can see an example of an analysis that was initially done on an older column (column 1)
that gave acceptable performance. However, when the chromatographer put on a new column (column 2)
the resolution of key components was quite different and the new column was 'blamed' for the
discrepancy. However, the chromatograher made up fresh mobile phase buffer and the resolution returned
to normal, as can be seen in the right chromatogram. In this case, the problem was narrowed down to a
‘bad’ bottle of TEA or phosphoric acid. These solvents had been used for a while and changes or
contamination had occurred.
It is important to try to prepare your sample in the same solvent as the mobile phase. See the example on
page 30 for the band broadening and splitting that can occur when the injection solvent is much stronger
than the mobile phase.

1
 Figure 1. Variations in mobile phase can have a marked effect on results

Mixing mobile phases

Sometimes, a mobile phase differs because of something as simple as the way it is mixed in your lab. If
you are making a 50/50 methanol/water mixture offline, for example, it’s important to measure each
volume separately, in clean glassware, before mixing them together, because the volume of the
MeOH:H2O mixture is more than the sum of the individual components. If you mix them in the same
container, the mixture will differ in total volume. Therefore, these two mobile phases, prepared in a
different manner, are not the same composition.

Degassing mobile phases

Degassing your mobile phase is important, too. Dissolved gas in the solvents can come out of solution,
forming an air bubble in the flow path, and possibly interfere with the pump or detector’s performance.
Fortunately, nowadays, most LC systems have degassers built in, but if the degasser is bypassed, absent,
or not working correctly, be sure to sparge with helium or use some other means to degas.