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Reversed phase HPLC analyses are predominantly performed on C18 columns, which, in most cases, work well. The stationary phase of a conventional C18 column is strictly comprised of linear, nonpolar alkyls, which are well-suited for stable and predictable separations of hydrophobic compounds. There are, however, situations in which a conventional C18 column produces less than optimal chromatography. For example, C18 columns have little retention for hydrophilic compounds, basic compounds often exhibit peak tailing, and highly aqueous conditions can cause inconsistent retention or even phase collapse.
One way in which column manufacturers attempt to address some of these issues, and yet maintain the favorable hydrophobic interaction of a C18 column, is to impart polar functionality into an alkyl phase. One such example is the Ultra IBD column. This column is a specialty phase that is comprised primarily of alkyls with a secondary polar functionality within, or intrinsic to, the hydrocarbon-bonded phase. This particular bonding chemistry is commonly referred to as “polar embedded” because a polar group is embedded within the linear alkyl chain. Compared to a C18 column, the polar functionality incorporated into the Ultra IBD column offers enhanced retention and selectivity toward a wider range of compounds, orthogonal separations, improved base-deactivation and compatibility with entirely aqueous mobile phases.
Polar embedded columns do not all produce the same chromatography, which can be confusing when developing methods. Depending on the type of polar group incorporated, as well as how and where the group is positioned in the stationary phase, chromatographic properties can vary, sometimes quite dramatically. The bonding chemistry used in the Ultra IBD column makes it a very adaptable column capable of unique separations. In the experiments listed below, we investigated the versatility of the Ultra IBD column and its ability to overcome some of the common drawbacks to conventional C18 columns.
In terms of overall stationary phase polarity, the bonding chemistry of the Ultra IBD column exhibits a high degree of polarity, relative to conventional and aqueous C18 phases. It is this heightened polarity that makes the Ultra IDB column so versatile and unique. Because the Ultra IBD column possesses both nonpolar and highly polar characteristics, it can be used in normal phase mode as well as in reversed phase mode; it is also rugged and reproducible in completely aqueous mobile phases. The analysis of cephaloridine, an antibiotic drug compound containing both polar and nonpolar moieties, demonstrates this concept and reveals the underlying property driving the usefulness and versatility of this phase—its high retentiveness for both polar and nonpolar compounds (Figure 1).
| Figure 1: The Ultra IBD column is comprised of nonpolar alkyls with an embedded polar group making it useful in both normal and reversed phase modes. | |||||||||||||||
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LC_0101-102
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The bonding chemistry of the Ultra IBD column alleviates one of the common problems associated with alkyl phases—peak tailing of basic analytes. Amitriptyline is a highly basic, tricyclic antidepressant that commonly tails on silica-based alkyl phases. Peak tailing is minimal on an Ultra IBD column without the use of mobile phase modifiers and regardless of mobile phase pH (Figure 2).
At pH 3 and below, almost all silanols are protonated and do not readily ion exchange with protonated bases (BH+). Modifiers like TEA (triethylamine) can be used to sharpen peak shape as they interact with ionized silanols, masking the silica surface. Even at a neutral pH and, importantly, with no modifiers, the Ultra IBD column exhibits excellent peak shape of basic compounds. This is potentially advantageous because it provides needed flexibility for method development, especially for analytes that are labile at acidic conditions. In applications where Gaussian peak shape is needed for accurate integrations, such as potency assays, or when tighter system suitability criteria are required, an intrinsically base-deactivated stationary phase offers a benefit that a conventional C18 column cannot—exceptional peak shape with a simplified analysis.
| Figure 2: Ultra IBD offers more flexibility in method development giving excellent peak shape for highly basic compounds — even without mobile phase modifiers and regardless of mobile phase pH. | |||||||||||||||||||||||||||||
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LC_PH00443-444 |
In contrast to conventional C18 columns, the Ultra IBD has a polar functional group embedded within the alkyl chain. Retention, therefore, is attributed not only to hydrophobic interactions (the major retention mechanism of the C18), but also to polar attraction between the analyte and stationary phase. This mixed-mode mechanism, amplified by the type and position of the polar functional group in the Ultra IBD, results in high retention for hydrophilic compounds or compounds with polar moieties. The analysis of purines demonstrates this point (Figure 3).
Another advantage, because the separation mechanism differs from that of a C18 column, is that alternate and improved selectivity can be achieved. Two sulfonamide antibiotics, sulfadiazine and sulfathiazole, were assayed isocratically on both a conventional C18 column and an Ultra IBD column of comparable surface area and carbon loading. Under identical conditions, the heightened polarity of the Ultra IBD column was capable of resolving these two polar drug compounds while a C18 column could not (Figure 4). Orthogonal separations also can be achieved through the Ultra IBD phase chemistry. A small group of hydroxybenzoic acids were also assayed on the same column pair under identical conditions. It was observed that the elution order of the analytes differed, showing an increased retention for the dihydroxybenzoic acid on the Ultra IBD column (Figure 5). Additionally, the unique phase chemistry of the Ultra IBD column makes it suitable for a simultaneous analysis of a wide range of compounds—acidic through basic, as well as zwitterions (Figure 6).
| Figure 3: The Ultra IBD column exhibits high retention for hydrophilic compounds, or compounds with polar moieties, and is compatible with up to 100% aqueous mobile phases. | |||||||||||||||||||||||||||||||||||||
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LC_0129
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| Figure 4: The embedded polarity of the Ultra IBD column resolves drug compounds that a conventional C18 cannot. | ||||||||||
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LC_PH00445-446 |
| Figure 5: The Ultra IBD column gives needed flexibility for polar compounds; it increases retention, enhances resolution, and creates alternate selectivity. | ||||||||||
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LC_PH00447-448 |
| Figure 6: The versatility of the Ultra IBD makes it well-suited for analyzing a wide range of compounds — acids through bases, and zwitterions. | |||||||||||||||||||||||||||||||||||||||||
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LC_0057
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The Ultra IBD, through unique bonding chemistry, is an extremely versatile HPLC column. It offers alternate selectivity and a high degree of both polar and nonpolar retention, making it a powerful tool for analyzing a wide range of compounds. The Ultra IBD also addresses the inherent problems attributed to linear alkyl phases, providing excellent peak shape for basic compounds and heightened retention of hydrophilic compounds. The versatility of the Ultra IBD makes it an excellent tool for the practicing method developer.
