A great way to do phospholipid analysis

 In a recent article published in the Journal of Chromatography A, Cífková et al. have investigated the chromatographic behavior of various phospholipids such as (lyso)phosphatidic acids and (lyso)phosphatidylserines. If you have ever done these kinds of compounds then you know they present a challenge to say the least; they contain polar functional groups which make them less suitable for chromatographic methods used to retain more hydrophobic lipids.

Furthermore, identification of discreet lipid classes is greatly facilitated by the high specificity of LC-MS, but previous methods reported in the literature tend to use ion pair reagents, which are not amenable to MS.  This can be painful.

The authors present a comparison of columns for the LC-MS separation of these compounds in real world samples (porcine brain and kidney extracts). In terms of peak symmetry, the Cogent Diamond Hydride™ produced tailing factors in the range 1.0–1.6 for all the studied analytes. Two analytes in particular tailed so severely on HILIC columns that a tailing factor could not be calculated; in contrast, the same two compounds produced only moderate tailing (1.4 and 1.6) on the Diamond Hydride™.

If you struggle with phospholipid analysis, this paper is well worthwhile reading as it is full of new insights.

REFERENCE:

E. Cífková, R. Hájek, M. Lísa, M. Holčapek, Hydrophilic interaction liquid chromatography–mass spectrometry of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipid classes, J. Chromatogr. A (2016), in press.

http://dx.doi.org/10.1016/j.chroma.2016.01.064

How you can get better precision and faster runs using Cogent TYPE-C Silica™ columns

I have heard many chromatographers describe problems with HILIC methods they use. Common complaints include long equilibration times and inconsistent retention when using gradients. Given the nature of the HILIC retention mechanism, this is not surprising. In HILIC, hydrophilic silanol groups on the stationary phase surface cause a thick water layer to form, which allows for the partitioning responsible for analyte retention. The problem with this retention mode is that the water layer is not a static environment but rather is constantly changing as the mobile phase changes over the course of a gradient. Then, during re-equilibration, it takes a significant number of column volumes to fully regenerate the hydration shell to its original form. It is this variability in the water layer that gives rise to both long equilibration times and poor retention precision.

So what solution is there to this kind of problem? It would be better to rely on a retention mode that does not involve partitioning with a water layer. With Cogent TYPE-C Silica™ columns, hydrophilic silanols are replaced by silica hydride groups. Because the latter are moderately hydrophobic, they do not result in the formation of a thick water layer, as in conventional silica based phases. Polar compounds can still be retained using these columns in a mode called Aqueous Normal Phase (ANP). Mechanistic studies involving measurements of zeta potentials have demonstrated that the retention in ANP is primarily adsorptive in character rather than due to partitioning. Indeed, the greater precision and lower equilibration times observed using ANP methods compared to HILIC are consistent with these findings.

What this means to you is that you can obtain more reliable data with greater throughput. Solvent savings can also be realized due to the minimal equilibration. Furthermore, the same column can be used in some instances for both ANP and reversed phase chromatography, allowing for more streamlined analyses. With HILIC columns, only polar analytes may be suitable for retention while more hydrophobic species may elute at the solvent front. Hence, you can get more versatility out of one column with Cogent TYPE-C Silica™ phases.

Ever-increasing globalization in many industries that rely on HPLC has created more competition than ever before. In today’s business environment, laboratories need to maximize their resources in order to stay competitive. Using Cogent TYPE-C Silica™ columns is one way to help you achieve these goals.