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essential include plant, animal, human and drug metabolomics, clinical analysis, impurity testing, food safety and nutrition, forensics and environmental monitoring.
Reversed Phase
All TYPE C stationary phases display some reversed-phase behavior. Even the unmodified material can retain nonpolar compounds because the hydride surface is slightly hydrophobic. As the hydrophobicity of the stationary phase is increased by having greater surface coverage of bonded organic moieties, retention of nonpolar compounds increases as with all other reversed-phase materials. Selectivity for common phases such as C18 and C8 is somewhat different because of the underlying hydride surface and the resulting lack of water on this material.
Figure 2. Analysis of guanidine in a 100% aqueous mobile phase on a Cogent Bidentate C18 column. A. Repeatability of analysis using guanidine Standard. B. Comparison of retention times of standard and real sample. Mobile Phase: 100% DI water + 0.5% phosphoric acid + 1.5g/L pentane sulfonic acid (isocratic). Column: 4.6 x 150 mm. Flow rate: 1.0 mL/min. Detection at 200 nm. Standard Sample: 100 ppm. Injection Volume: 20 µL.
surrounding of the mobile phase. TYPE C stationary phases are an entirely different material with a slightly more hydrophobic surface that does not generate a dense water layer at the particle/mobile phase interface. While the mechanism of separation is not yet completely understood, the actual retention and separation capabilities for hydrophilic compounds have been extensively demonstrated [3-7]
.
The most rapidly growing and the most extensively investigated area of polar compound retention is in the aqueous normal phase (ANP) mode. Under these mobile phase conditions the stationary phases have properties that are similar to the characteristics of HILIC phases (increasing retention with increasing amount of organic component, usually acetonitrile or acetone). In contrast to many HILIC phases and applications, the TYPE-C silica material is robust (lasts for hundreds of injections), very reproducible from run-to-run (% RSD values generally 0.5% or less) and equilibrates rapidly (five minutes or less) after a gradient method. Applications where the analysis of polar compounds is
For retention of hydrophilic compounds under RP conditions with TYPE C materials, mobile phases are used that typically contain 90-100% (v/v) water. Stationary phases based on silica hydride are especially suited to these conditions since they do not undergo “dewetting”, sometimes referred to as phase collapse or phase fold back. In highly aqueous environments, many hydrophobic bonded phases such as C18 will try to minimize their contact with the polar environment by forming highly associated packets of organic moieties. This process reduces the total hydrophobic surface available for solutes to interact with and thus retention can drop drastically in high aqueous mobile phase environments. A few examples utilising high aqueous content mobile phases will be presented to illustrate this capability.
Figure 1 shows five overlaid chromatograms of a sample containing urea obtained on a C18 TYPE-C stationary phase using a 100% aqueous mobile phase. This highly polar compound is adequately retained under these conditions (k ≈ 1.5) demonstrating that the TYPE-C materials are particularly useful for hydrophilic compounds even in this mode. In addition, there is a remarkable degree of repeatability in these five runs which is another feature of the TYPE C stationary phases. Since this column material has an octadecyl bonded moiety, it can function like other reversed-phase stationary phases and can be used for the analysis of a wide range of hydrophobic compounds as well.
Another example of the retention capability for hydrophilic compounds in the reversed- phase mode is the analysis of guanidine shown in Figure 2. Guanidine is a strong base and is protonated at all pH values below 12. The low molecular weight of guanidine, its positive charge, and lack of a significant chromophore, make the analysis very difficult. Since guanidine is such a polar compound, it also requires a 100% aqueous mobile phase to be retained in reversed- phase on the TYPE-C C18 column. Normally thisanalysisanalysis would require a specialty column such as an ion-exchange phase to enhance retention of this polar molecule but there areother issues using this approach [8]
including long run and
re-equilibration times. Since the Cogent Bidentate C18™ (TYPE-C with C18 bonded moiety) does not suffer any loss of retention from run to run which is commonly known to be due to dewetting or “phase collapse” and it is not a “specialty column”, it can be used
Figure 3. Separation of melamine and cyanuric acid on the Cogent Diamond Hydride column using an acetic acid mobile phase and a gradient from high to low concentration of acetonitrile in the mobile phase. Mobile Phase: A: DI water + 0.1% acetic acid; B: Acetonitrile + 0.1% acetic acid. Gradient: 100% B to 50% B in 15 min. Column: 2.1 x 150 mm. Flow rate: 0.4 mL/min. Sample Concentration: Cyanuric acid 1.5 µg/ml and melamine 3 ug/mL. Detection: m/z 128 in the negative ion mode for cyanuric acid and m/z 127 in the positive ion mode on an Agilent 6210 MSD TOF spectrometer.
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