An unexpected observation concerning the effect of anionic additives on the retention behavior of basic drugs and peptides in reversed-phase liquid chromatography

Abstract

Anionic species with ion pair forming ability are commonly used to enhance the retention and efficiency of basic analytes in RPLC separations. However, little is known about the interactions between organic mobile phase modifiers and such ion pairing anions. In this work, we measured the magnitude of the retention increase of basic drugs and peptides upon addition of strong inorganic ion pairing anions (e.g. perchlorate) as a function of the volume fraction of modifier in acidic water-acetonitrile mobile phases on two different stationary phases. We found that the increase in retention upon addition of various salts depended strongly on the eluent strength. In general, larger retention increases upon addition of the anion were observed at higher organic fractions. Regression of retention against the volume fraction of organic modifier indicated that the ion pair forming anions substantially decreased S values while only slightly changing $\ln {k^{\prime }}_{\text{w}}$ values. The decrease in S is the major cause of the retention increase of basic drugs and peptides when such anions are added to the mobile phase.

Introduction

The separation of basic compounds by RPLC is of great importance in both pharmaceutical analysis and proteomic research. Most RPLC columns are packed with silica-based alkyl silane bonded stationary phases. However, detrimental interactions between cationic analytes and surface silanols can occur which frequently lead to peak tailing and poor efficiency. Typically, mobile phases containing acids (e.g. phosphoric acid, formic acid or trifluoroacetic acid (TFA)) are used to suppress these interactions [1], [2]. However, in acid the positive charge of the analyte can cause low retention and thus poor resolution. To mitigate this problem, anions are often added to the eluent to adjust the retention, efficiency and sample loading capacity of basic analytes [3], [4], [5], [6], [7], [8], [9], [10], [11].

The study of the retention mechanism of basic analytes in RPLC in the presence of very hydrophobic anionic additives such as alkyl sulfonates was pioneered by Horvath et al. [12], followed by further studies by Bidlingmeyer et al. [13], Knox and Hartwick [14] as well as an extensive review by Weber and coworkers [15]. Both the ion pairing and dynamic ion-exchange mechanisms were invoked to explain the increased retention. Unfortunately, the highly hydrophobic alkyl sulfonates have a tendency to “stick” to the stationary phase making recovery of column properties difficult. Recently, less hydrophobic anionic additives (e.g. CF₃COO⁻, ClO₄⁻, PF₆⁻) have gained in popularity for use with basic compounds and their influence on the retention mechanism has been the subject of extensive studies [4], [5], [7], [9], [16].

In general, adding ion pairing anionic additives increases the retention of basic compounds. More hydrophobic anions such as PF₆⁻ cause the largest retention increase, followed by ClO₄⁻, CF₃COO⁻, Cl⁻, HCOO⁻ and H₂PO₄⁻. Roberts et al. vaguely referred to the “Hofmeister effect” to explain this observation [9]. Kazakevich et al. attributed the retention increase due to addition of inorganic anions to a “chaotropic effect” [6]. Gritti and Guiochon studied the role of buffer (e.g. phosphate and acetate) on retention and overloading behavior of cationic drugs on different stationary phases [17], [18], [19]. They suggested that ion pair formation could explain their experimental results. It is clear that there was little agreement and measurements of the extent of ion pair formation were necessary to definitively settle this complex phenomenon.

Dai et al. determined ion pair formation constants between various basic drugs and different anions under conditions representative of the eluents used in RPLC through independent measurements of the effect of anions on the mobility of the cations of interest by capillary electrophoresis [20]. Their measurements confirmed that at pH 4–5 ion pair formation in the eluent is responsible for the large increases (2–5 folds) of retention of drugs upon addition of anions to the eluent. It is important to note that at typical anion concentrations (<100 mM) less than 50% of the test bases were actually present as ion pairs in the mobile phase even with the strongest pairing agent (PF₆⁻) used. At pH 2 slight but still significant adsorption of anionic additives to the stationary phase was also confirmed by their effect on the retention of anions such as Br⁻; these adsorbed anionic species generate dynamic ion exchange sites for retaining the cationic analytes [4]. Thus, both ion pair formation and dynamic ion exchange, especially at lower pH, contribute to the overall retention increase of the basic drugs upon addition of anions. The relative contribution depends on the analyte and the operating conditions (e.g. mobile phase, especially the pH and stationary phase).

Due to both of the above mechanisms anions are also found to be extremely useful for improving the separation of proteins and peptides. For instance, TFA is routinely used to control the pH and improve the peak shape of peptides [21]. Hodges and coworkers have extensively studied the effect of different anionic additives on the retention and selectivity of peptides in gradient elution RPLC [3], [10], [11], [22]. The ability of different anions to increase the retention of peptides in gradient elution agrees well with the results of retention increases for basic drugs in isocratic elution. They found that the retention increase depends strongly on the charge of the peptides and the concentration of the additive. For example, the addition of 50 mM sodium perchlorate to 10 mM phosphoric acid buffer increased the gradient retention times of peptides with +4 charges by up to 15 min [22].

A very important feature of anion ion pair forming additives, besides the increase in retention, is their ability to decrease the peak width of both basic drugs and biological molecules. In the absence of such strong ion pairers, wide peaks for cationic analytes are usually observed compared to neutral analytes on most stationary phases; this is mainly caused by the fact that typical RPLC stationary phases are overloaded with the positively charge analytes due to their poor loading capacity. McCalley studied the effect of buffer type, concentration and ionic strength at low pH on the efficiency of basic drugs [23], [24] and peptides [25]. He showed that high ionic strengths and stronger ion pairing agents significantly increase the sample loading capacity of basic analytes. As a result, much better plate counts can be obtained when the same amount of cationic analyte is injected.

It is clear that addition of anionic additives is very beneficial for the separation of cationic analytes. Of particular importance is the ability of anionic additives to increase retention under both isocratic and gradient conditions. However, all previous work measured the magnitude of isocratic retention increases due to anionic additives at constant eluent strength (i.e. fixed organic modifier fraction in the eluent with different types and concentrations of additives). Thus, little is known about how the eluent strength itself affects the magnitude of the retention increase when anions are added to the eluent. Due to the electrostatic nature of ion-ion interactions, we should not overlook the fact that eluent composition could play an important additional role in RPLC by affecting the dielectric constant of the mobile phase [7].

The retention behavior of basic analytes in RPLC when the eluent strength is varied can be described, at least approximately, by linear solvent strength theory (LSST) [26]:

$$\ln k^{\prime }=\ln {k^{\prime }}_{\text{w}}-S\varphi$$

where ln k′ is the logarithmic retention, $\ln {k^{\prime }}_{\text{w}}$ is the extrapolated retention in pure water, ϕ (i.e. %ACN) is the volume fraction of acetonitrile (ACN) in the mobile phase, and S is the slope of a plot of ln k′ versus ϕ. Regression of ln k′ against ϕ using Eq. (1) gives the $\ln {k^{\prime }}_{\text{w}}$ and S values for different analytes; these are important parameters for predicting the retention time and peak width in gradient elution separations. Since gradient elution is increasingly used we studied the effect of ion pairing anionic additives on the retention behavior of basic drugs and peptides as a function of eluent strength using LSST. As will be shown later, the presence of the anion has only a small effect on $\ln {k^{\prime }}_{\text{w}}$, but it significantly decreases the S values of basic compounds (both low molecular weight drugs and higher molecular weight peptides); the decrease in S is clearly the main factor which drives the increase in retention of basic compounds.