In the first two instalments of this series, we've looked at different factors that influence selectivity in reversed-phase
liquid chromatography (LC) separations. First, we saw some of the benefits of changing solvent type,1 such as a change from methanol to acetonitrile or tetrahydrofuran. Next, we looked at how a change in the solvent strength2 — the percentage of organic solvent in the mobile phase — was an easier adjustment to make and often had suitable "leverage"
to pull apart problem peak pairs. This month's "LC Troubleshooting" will focus on another common technique used to change
peak spacing in a chromatogram: changing from one column type to another.
Not All C18s Are Equal
Figure 1: Comparison of simulated chromatograms for C18 columns from three manufacturers. Column dimensions: 150 mm × 4.6
mm, 5 μm particles; mobile phase: 50:50 acetonitrile–30 mM phosphate buffer (pH 2.8); temperature: 35 °C; column efficiency
N: 10000. Retention normalized to peak 1. See Table 1 for column information and text for details. Peaks: 1 = anisole, 2 =
n-butylbenzoic acid, 3 = toluene, 4 = mefenamic acid, 5 = ethylbenzene, 6 = trans-chalcone.
When I first started using LC, it was thought widely that all C18 columns would give equivalent separations. In fact, the
United States Pharmacopoeia (USP) in its column classification scheme classifies C18 columns as "L1 — octadecyl silane chemically bonded to porous silica
or ceramic micro-particles, 1.5 to 10 µm in diameter, or a monolithic silica rod".3 Over the past 40 years, however, we've all come to recognize that all C18s are not created equal — many will give dramatically
different separations. In the current listing, the L-classification scheme contains 74 different column types,3 including C18, C8, phenyl, cyano and many others. Even columns that have the same label, such as C18 or phenyl, may have
significantly different selectivity characteristics. It is not clear how many different reversedphase columns are available,
but by some estimates there are at least 1000.4 In our work at LC Resources, we've tested more than 500 different reversed-phase columns.
So how different are C18 columns from each other? To illustrate this, I've chosen a set of six test compounds that should
be fairly well-behaved (see caption of Figure 1 for their identities). That is, at low pH (pH 2.8), they are neutral or un-ionized,
so ionic interactions with the column should be minimal. I arbitrarily chose three C18 columns from our database.5 These are from well-known sources and are all type-B, high-purity silica columns. These are simulated separations on 150
mm × 4.6 mm columns packed with 5 µm particles and generating 10000 plates. For simplified comparisons, I've normalized retention
to the first peak (anisole). The first column [Figure 1(a)] is our reference column. You can see that peaks 4 and 6 overlap
and the resolution is marginal for peaks 2 and 3. If we change to another manufacturer's C18 column [Figure 1(b)], the separation
of peaks 2 and 3 is improved, and the separation of the last three peaks has changed, but it is no better than the reference
column. A third manufacturer's C18 [Figure 1(c)] is no better — it shows a good separation for peaks 2 and 3, but peaks 4
and 6 still overlap. You can see in Figure 1 that, although the first three peaks are separated by all three columns, the
selectivity (relative peak spacing) is different for these peaks. And none of the three columns can separate the last three
peaks — even the peak order changes! So you can't arbitrarily swap one C18 for another and expect the same results. But on
the other hand, the differences aren't enough that we can make the same change and expect to solve a separation problem. Although
there is likely at least one manufacturer's C18 column that will separate all six compounds, changing from one to another
in an effort to get the desired separation is not a very good strategy.
Perspectives in Modern HPLC: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Seven Common Faux Pas in Modern HPLC