Section 5 Column-to-Column Selectivity Changes A Quick Diagnostic

Changes in selectivity (relative peak spacing) that occur when an HPLC column is replaced may lead to unacceptable chromatography, i.e., loss in resolution. It may seem obvious that the change in selectivity, observed in Figure 5-1, is due to the replacement column. How can you tell if this loss in resolution is due to the column, rather than something else? What actions can you take to correct the problem?

Figure 5-1 Selectivity Change Column A = k2 / k1 = 1.23 Column B = k2 / k1 = 1.10

Effects of Column Selectivity Changes
When there is a change in selectivity one or more peaks will elute with a different retention time, and selectivity ( = k₂/k₁) between one or more peak pairs will change. Usually, other parameters, such as theoretical plates and back pressure, will not be affected by a change in the stationary phase selectivity, although differences in peak symmetry may occur.

Causes of Column Selectivity Changes
Changes in column selectivity are caused by changes in the interactions of the sample solutes within the bonded-phase or underlying silica support. Bonded-phase interactions are hydrophobic in nature. Consistent retention depends upon consistent carbon load and surface coverage of the silica support with the bonded phase (i.e., C18, C8, etc.). Interactions can also occur between solutes and acidic silanol sites or trace metals on the silica surface. Subtle changes in the silica support may have a profound effect on selectivity, as well as peak shape, for basic compounds in particular.

Classifying Selectivity Changes
Significantly different -values (>10%) for two hydrophobic solutes, e.g., anthracene and naphthalene, suggest column selectivity changes due to bonded phase differences. Differing -values for a hydrophobic and polar, basic-solute pair, e.g., toluene and dimethylaniline, suggest changes in the silica support. (See Figure 5-2)

Correcting Selectivity Changes
If the selectivity problem only occurs with polar basic compounds, adjust the mobile phase to pH less than or equal to 3.0 and/or add 10 mM - 50 mM triethylamine (TEA) to "mask" silanol or non-bonded-phase effects. This modification will significantly improve column-to-column reproducibility for these solutes. Highly purified silica, such as that used in ZORBAX Rx, StableBond and Eclipse XDB columns, offers dramatic improvement in reproducibility for polar compounds. These higher quality, more reproducible columns should be your first choice for the analysis of these types of compounds. Selectivity changes due to changes in bonded phase may be corrected by an increase or decrease in solvent strength (% organic). Selectivity changes due to pH sensitivity may be corrected by using a 10 - 50 mM buffer concentration of a buffer selected for its buffer capacity in the desired pH region.

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