When developing a new method, one of the most important goals for the chromatographer is to achieve a consistent, reproducible separation. The selection of a highly reproducible and well-characterized HPLC column is essential if this goal is to be attained.
The physical testing of the chromatographic media that is packed inside an HPLC column, while undoubtedly important, will reveal only some of the characteristics of the column. The true nature of the column and how it will interact with a wide range of analytes can only be understood through rigorous chromatographic evaluation.
The retention properties of a reversed-phase packing material can be categorized into hydrophobic interactions, which include the measure of the hydrophobicity of the ligand and its density; steric or shape selectivity; and secondary interactions such as silanol and surface metal activity. The impact of interactions between analytes and silanols on the chromatographic performance depends on the pH of the mobile phase. Silanols on the silica surface can hydrogen-bond (both as a donor and acceptor) and dissociated silanols can ion-exchange with protonated bases.
To ensure consistent, predictable separations, the chromatographic media packed into Syncronis HPLC columns (Thermo Fisher Scientific, Runcorn, U.K.) is extensively characterized using a series of diagnostic chromatographic tests, based on those developed by Tanaka.1 These tests rigorously probe interactions between carefully selected analytes and stationary phase, characterizing the stationary phase with regard to hydrophobicity, shape selectivity, and secondary interactions with bases, acids, and chelators.
Test 1: Hydrophobic interactions
The first hydrophobic interaction that is measured is hydrophobic retention (HR). The capacity factor of a neutral, hydrophobic hydrocarbon (pentylbenzene) gives a measure of the hydrophobicity of the stationary phase and therefore a measure of the carbon load on the silica. Syncronis C18 columns are densely coated and double endcapped, resulting in a carbon load of 16%, therefore exhibiting very strong hydrophobic retention.
The second hydrophobic interaction measured is hydrophobic selectivity (HS). The selectivity factor between pentylbenzene and butylbenzene provides a measure of the surface coverage of the phase; these two alkylbenzenes differ by one methylene group, and their selectivity is dependent on the density of the bonded ligand.
Figure 1 - Example chromatogram for test 1: Hydrophobic interactions. Column: Syncronis C18, 5 μm, 100 × 4.6 mm. Mobile phase: water:methanol (35:65). Flow rate: 1.0 mL/min. Temp: 40 °C. Detection: 254 nm. Injection volume: 10 μL. Sample: 1) theophylline, 2) caffeine, 3) phenol, 4) butylbenzene, 5) o-terphenyl, 6) amyl(pentyl)benzene, 7) triphenylene.
Steric selectivity (SS) is the ability of the stationary phase to distinguish between molecules with similar structures and hydrophobicity but different shapes. The selectivity factor between o-terphenyl and triphenylene is indicative of steric selectivity, since the former has the ability to twist and bend, while the latter has a fairly rigid structure and will be retained quite differently.
Hydrogen bonding capacity
The hydrogen bonding capacity (HBC) of the stationary phase is probed by determining the relative retention of caffeine with respect to phenol. This provides a measure of the number of available silanol groups and the degree of endcapping. A low value indicates that the stationary phase has a low level of silanols available to hydrogen bond with caffeine and is therefore an indication of the thoroughness of the endcapping (see Figure 1).
Test 2: Interactions with basic and chelating compounds
Activity toward basic compounds
The presence of dissociated silanols at pH values greater than 7 can cause poor peak shapes of protonated basic compounds such as amitriptyline. Secondary ion exchange and silanolic interactions can cause shifts in retention and asymmetrical peaks. The capacity factor and tailing factor of amitriptyline are indicative of the overall performance of the column. Syncronis columns exhibit very good peak shape for amitriptyline, demonstrating a highly deactivated silica.
Figure 2 - Example chromatogram for test 2: Interactions with basic and chelating compounds. Column: Syncronis C18, 5 mm, 100 × 4.6 mm. Mobile phase: 10 mM phosphate (pH 7.6):methanol (20:80). Flow rate: 1.0 mL/min. Temp: 40 °C. Detection: 254 nm. Injection volume: 5 mL. Sample: 1) theophylline, 2) benzylamine, 3) phenol, 4) quinizarin, 5) amitriptyline.
Activity toward chelators
Silica surface metal interactions can cause changes in selectivity and peak shape for analytes, which are able to chelate. Changes in the capacity factor and tailing factor of quinizarin, a chelator, are indicative of secondary interactions with metallic impurities in the silica. The peak shape observed with Syncronis columns demonstrates the exceptionally low metals content and compares favorably with other chromatographic media.
Ion-exchange capacity at pH 7.6 (IEC7)
The selectivity factor between benzylamine and phenol is used to estimate the total silanol activity on the surface of the silica. At pH values above 7, the silanol groups are dissociated and, combined with the ion exchange sites, can influence the retention of benzylamine (see Figure 2).
Test 3: Interactions with acidic compounds
Activity toward acids
The inertness of the stationary phase toward acidic compounds is also important for consistent retention and selectivity, since acidic compounds may become adsorbed onto reversed-phase packing materials. The capacity factor and tailing factor of 4-chlorocinnamic acid are measured to test the applicability of the stationary phase to acidic analytes. Syncronis columns exhibit good peak shape, indicating a high degree of inertness toward acidic compounds.
Figure 3 - Example chromatogram for test 3: Interactions with acidic compounds. Column: Syncronis C18, 5 mm, 100 × 4.6 mm. Mobile phase: 10 mM phosphate (pH 2.7):methanol (45:55). Flow rate: 1.0 mL/min. Temp: 40 °C. Detection: 254 nm. Injection volume: 5 mL. Sample: 1) theophylline, 2) benzylamine, 3) phenol, 4) chlorocinnamic acid.
Ion-exchange capacity at pH 2.7 (IEC2)
Tanaka1 showed that the retention of protonated amines at pH <3 could be used to obtain a measure of the ion exchange sites on the silica surface. Silanol groups (Si–OH) are undissociated at pH <3 and therefore cannot contribute to the retention of protonated amines, but the acidic silanols in the dissociated form (SiO–) can. The latter contribute to the retention of the protonated amines. The contribution of the free silanols to retention can be estimated by the selectivity factor between benzylamine and phenol at pH 2.7. As can be seen from Figure 3, benzylamine is virtually unretained on the Syncronis C18 column, indicating that the surface of the silica is essentially free of acidic silanols.
Demonstration of column reproducibility in pharmaceutical analysis
By rigorously characterizing the stationary phase for primary and secondary interactions, the variability in column performance can be minimized. As can be seen from the data in Table 1, Syncronis C18 columns demonstrate very good column-to-column reproducibility for the USP method for zidovudine. The variation in retention time and peak area is less than 0.5% column-to-column, comfortably exceeding the method requirements (2% RSD).
A reduction in the lot-to-lot variation of the chromatographic media can be achieved by rigorously characterizing the column packing material using specific analyte probes and placing tight specifications on the test results. As a result, the chromatographer can be confident of achieving consistent chromatography, column after column.
- Kimata, K.; Iwaguchi, K. et al. J. Chromatogr. Sci. 1989, 27, 721–8.
Dr. Milton is Product Manager, Thermo Fisher Scientific, Tudor Rd., Runcorn, Cheshire WA7 1TA, U.K.; tel.: +44 1928 534 334; e-mail: firstname.lastname@example.org.