Aqueous Normal Phase: Not Just for Hydrophilic Compounds

                In HPLC analyses of hydrophobic molecules, reversed phase is the most commonly used separation method. For hydrophilic analytes on the other hand, Hydrophilic Interaction Liquid Chromatography (HILIC) is used in some cases. However, the drawback to these two retention modes is that they cannot simultaneously retain both hydrophilic and hydrophobic analytes in the same run.

                This is in contrast with Cogent TYPE-C Silica™ columns, in which can retain both types of compounds with the same column. In addition to conventional reversed phase, the columns can retain by Aqueous Normal Phase (ANP) as well. What may be surprising is that even hydrophobic analytes can be retained by an ANP mechanism. Cyclobenzaprine for example has a reported log P of approximately 4.6–4.9 and yet still shows excellent ANP retention, as shown in the chromatogram below. This behavior has been observed for hydrophobic peptides as well and has been published in a 2011 article in the Journal of Chromatography A.

                What this means for the practicing chromatographer is that in some cases it is possible to retain both types of compounds not just with the same column but in the same run. This feature is very useful for any analysis that involves compounds encompassing a wide range of polarity, such as metabolomics. The work of two columns could be done in this case with only one.

How Choice of Autosampler Vials Can Affect Quantitation

                The modern analytical laboratory makes use of more sophisticated analytical instrumentation than ever before, leading to lower detection limits and hence a need for more accurate quantitation.  One aspect of the analytical process that is often overlooked is the importance of the autosampler vial. Basic solutes are known to electrostatically adsorb with surface silanols on conventional glass and therefore affect quantitation. This effect becomes significant at lower concentrations.

                So while instrumental detection capabilities have greatly improved in recent years, autosampler glass technology has often not kept pace in many cases. However, unique glass chemistry is available from MicroSolv which is referred to as Reduced Surface Activity (RSA™) Glass. This material is virtually free of surface silanols and therefore better preserves the quantitative characteristics of the analytes.

                A study compared the peak areas of the same 5ppm thiamine standard solution in two different autosampler vials. One was made of conventional glass sold by a leading supplier and the other was MicroSolv RSA™ glass. The solutions were injected immediately after transferring to the vials and again after four hours. The conventional glass showed a 32.6% loss in peak area compared to only 3.1% in RSA glass (see below peak overlays). Thiamine was selected due to the cationic nature of the compound, which would have a high affinity for electrostatic interaction with any silanols on the glass surface. Compounds without basic functional groups did not show loss in peak area with either vial.

RSA Glass Vial Product Page