Assessing Column Comparison Studies: Modified-Euerby Comparison of Discovery Zr-PBD and Discovery C18

Reporter EU Volume 10

3 Your Problem Solving Partner in Chromatography Many reports discussing column screening procedures for purposes of stationary phase selection have recently become prominent in analytical journals. This report discusses the use of a modified-Euerby column classification study as a means to gain further understanding of a polybutadiene-coated zirconia stationary phase. Although silica-based stationary phases remain the workhorse for high-performance liquid chromatography (HPLC) analyses, separations based on modified zirconia phases are fast becoming a popular alternative. The interest in zirconia columns stems from their ability to withstand extreme pH and temperature conditions as well as their offering of unique selectivity and retention for various classes of compounds. The merits of the adopted procedure and the information it provided are discussed.

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To best utilize the unique offerings of any stationary phase it is paramount that the underlying mechanisms of interaction be understood. Many reports discussing column classification studies can be found in recent literature (1,2,3). These types of studies may:

  1. Demonstrate chromatographic orthogonality between stationary phases of differing bonding chemistries and substrate structure (a method development aid)
  2. Demonstrate similarity in columns (a method transfer aid)
  3. Help elucidate molecular interactions available on a given phase chemistry

There are several test procedures in analytical journals with significant data to assess their applicability. The NIST Standard Reference Material® 870 consists of uracil (to marker), toluene, ethylbenzene (hydrophobic retention markers), quinazarine (activity toward chelators) and amitriptyline (activity toward bases). Sander and Wise have evaluated the test and found it suitable to compare common reversed-phase systems such as C18 and polar embedded phases(1). Euerby, et. al. recently reported the use of the well-known Tanaka test procedure to evaluate 135 stationary phases of differing chemistries(2). The results of this testing are discussed in terms of hydrophobicity, shape selectivity, hydrogen bonding capacity, total ionexchange capacity and acidic ion-exchange capacity. A third report recently published by Neue, et. al., discusses still another set of test conditions(3). This latter report again describes parameters such as hydrophobicity and shape selectivity. In addition, silanophilic interactions as well as a “phenolic selectivity” parameter are discussed. Each of the test procedures noted have advantages and disadvantages. The fact that good retention data were obtained for the vastly differing phase chemistries using the Euerby procedure suggests that the conditions are universal enough to cover a wide range of chemistries and supports. Both the NIST procedure and the Neue procedure measure silanophilic interactions near neutral pH. Since silanophilic interactions are pH dependent, the additional set of conditions of the Euerby procedure at acidic pH is expected to add significant information regarding these important interactions. One disadvantage of the Euerby procedure is the lack of complete ionization of the benzylamine analyte used to determine ionic interaction at pH 7.6 (4). This incomplete ionization may cause some irreproducibility, therefore the addition of the quaternary amine, berberine, has been added to eliminate the dependence of hydrophobic interaction on pH. The addition of berberine was expected to better isolate the changes in phase characteristics as a function of pH. The other missing component to the mixture, which may provide significant information, is an acid. Since many analytes contain carboxylic acids, the addition of such a compound should provide essential data for method developers as well as for stationary phase research and development efforts. The carboxylic acid moiety generally exhibits a pKa value of about 4.5. This value is reasonably centered between the 2.7 and 7.6 pH conditions used in the Euerby tests and thus was expected to provide information in both the ionized and neutral states. The term modified-Euerby is used in this report to describe a procedure where the stated analyte additions and a few minor changes in mobile phase to increase analyte retention were made to the literature procedure.

In this study the modified-Euerby procedure was used to examine the retention characteristics of Discovery Zr-PBD as compared to Discovery C18. Hydrophobic selectivity, shape selectivity, hydrogen bonding capacity, and total ion exchange capacity at both pH 2.7 and 7.6 were determined. The values obtained were evaluated qualitatively against the observed chromatograms. The merits of the procedure and the information it provided are discussed.

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Results and Discussion

Figure A shows the structures of the hydrophobic selectivity probes, pentylbenzene (PB) and butylbenzene (BB) as well as the shape selectivity probes triphenylene (T) and o-terphenyl (O). The observed chromatograms are shown in Figure B and the numerical results are listed in Table I. Similar hydrophobic selectivity values were obtained for each of the phases, however, much greater resolution of PB and BB is observed in the chromatographic trace. As discussed in the Case Study of this Reporter, low retention, as is observed for these analytes on Zr-PBD, results in irreproducible values. The calculated values for shape selectivity indicate that the Zr-PBD column is more shape selective than the C18. Examination of the chromatogram, however, shows that the C18 exhibits greater resolution of the probe analytes. In this case, the capacity factors for the analytes, T and O, on the Zr-PBD are greater, however, they may still be too low for accurate parameter measurement.

Figure A. Hydrophobic Selectivity and Shape Selectivity Analyte Structures

Figure B. Hydrophobic Selectivity and Steric Selectivity on Discovery C18 and Discovery Zr-PBD

Table I. Hydrophobic Selectivity and Shape Selectivity Results

Figure C shows the structures of the hydrogen bonding capacity probes, caffeine (C) and phenol (P). The obtained chromatograms are given in Figure D and the numerical values obtained are listed in Table II. Low retention for both C and P on the Zr-PBD phase results in low confidence in the calculated values. The chromatograms show little significant difference in selectivity other than that attributable to lower hydrophobic interaction of the Zr-PBD phase.

Figure C. Hydrogen Bonding Analyte Structures

Figure D. Hydrogen Bonding Selectivity on Discovery C18 and Discovery Zr-PBD

Table II. Hydrogen Bonding Capacity Results Selectivity Results

Figure E presents the structures of the ion-exchange probes used for both the pH 2.7 and the pH 7.6 studies. The chromatograms obtained at pH 2.7 are shown in Figure F and the numerical results are given in Table III. The orginal Euerby method defines the ratio of benzylamine and phenol capacity factors as the total ion-exchange capacity. The data in Table III shows that the retention for benzylamine and phenol are quite low for both phases. As indicated before, this produces questionable results. The retention for berberine and benzene under the same conditions appears to be adequate for determining this parameter. In addition, the use of a quaternary amine negates effects due to incomplete ionization. The values obtained using k’BB/k’BZ indicate that the Zr-PBD exhibits greater ion-exchange capacity under these conditions when compared to C18. Examination of the chromatograms supports the obtained values. The Zr-PBD shows greater retention for the basic analytes benzylamine and berberine. The ion-exchange character, combined with the lower hydrophobicity of the phase, results in selectivity differences between the two columns. Neutral benzoic acid is preferentially retained on the C18 phase, corresponding to the greater hydrophobicity of the C18 stationary phase.

Figure E. Total Ion-Exchange Capacity at Both pH 2.7 and pH 7.6 Analyte Structures

Figure F. Total Ion-Exchange Capacity at pH 2.7 on Discovery C18 and Discovery Zr-PBD

Table III. Total Ion-Exchange, pH 2.7 Results Selectivity Results

The chromatograms obtained at pH 7.6 are presented in Figure G and the numerical results are listed in Table IV. Once again the retention of benzylamine is observed to be very low. Both berberine and benzene, however, are again well retained. The larger total ion-exchange value obtained for the Zr-PBD phase is supported by the preferential retention of benzylamine and berberine observed in the chromatographic traces. At pH 7.6, benzoic acid is expected to be fully ionized. The benzoic acid analyte appears to be excluded from the pores of the Zr- PBD phase (elutes before the void volume), which further supports that the surface of the Zr-PBD is negatively charged.

Figure G. Total Ion-Exchange Capacity at pH 7.6 on Discovery C18 and Discovery Zr-PBD

Table IV. Total Ion-Exchange, pH 7.6 Results Selectivity Results

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  1. L.C. Sander, S.A. Wise, Journal of Separation Science 26 (2003) 283.
  2. M.R. Euerby, P. Petersson, Journal of Chromatography A 994 (2003) 13.
  3. U.D. Neue, K. VanTran, P.C. Iraneta, B.A. Alden, Journal of Separation Science 26 (2003) 174.
  4. U.D. Neue, E. Serowik, P. Iraneta, B.A. Alden, T.H. Walter, Journal of Chromatography A 849 (1999) 87.

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