New Uses for Cogent™ Columns: Ketorolac and Sucralose

Here at MicroSolv, we are always investigating new and exciting applications for our Cogent line of HPLC column products to address the ever-expanding needs of today’s analytical chemists. I am particularly pleased with these two latest application notes from our laboratories. Check them out, as you may indeed find them interesting as well!

In the first one, we examined the selection of the extraction solvent in influencing the extraction efficiency of ketorolac pharmaceutical formulations. We observed that use of DI water led to superior efficiency compared to ethanol. If you have encountered similar problems of low recovery in your sample prep of pharmaceutical extracts, you may pick up a pointer from the application. As for the chromatography, the compound was well-retained in reversed phase using the Cogent Phenyl Hydride™ column:

http://kb.mtc-usa.com/article/AA-02936/0/

The second application is for sucralose, commonly known by its brand name of Splenda®. In addition to being significantly sweeter than ordinary table sugar, sucralose has the further advantage of not contributing to elevated blood sugar levels among diabetics. If you’ve analyzed sucralose in your laboratory, you may have had problems with obtaining adequate sensitivity. UV absorption is low in this compound but it can be detected at sufficiently high concentrations and low wavelengths. Alternatively, you can try a universal detection method such as refractive index (RI). We tried both approaches in our application:

http://kb.mtc-usa.com/article/AA-02934/0/

I am looking forward to seeing what unique applications will be devised with these columns next!

Cogent TYPE-C Silica™ columns and ANP methods keep your MS source cleaner than HILIC methods

Does this situation look familiar?

Corroded Ion Source for Agilent TOF when used with HILIC Columns

It’s a common problem for many of us working with LC-MS and with polar compounds in particular. Our method calls for a mobile phase with a high concentration of buffer, and bam, your MS source quickly becomes contaminated (60 mM concentration and 500 injections in this case). The frequent required cleaning of the ion source reduces our throughput and takes up valuable time. What can we do to avoid problems like this?

The solution is to avoid using these high salt concentrations. The Cogent TYPE-C Silica™ materials allow you to do this by way of a aqueous normal phase (ANP) retention. Due to differences in the stationary phase properties compared to HILIC columns, a high salt mobile phase is simply not required. Typically we use 5–10 mM buffer where a HILIC column might require 50 or 60 mM for the same compounds. What this means for us the chromatographers is that the MS ion source will not be contaminated with salt precipitate as readily due to the lower concentration.  More uptime!, Less costs!

The difference is due to the stationary phase being more polar in HILIC phases than Cogent columns. Therefore a higher amount of salt in the mobile phase may be needed in HILIC to get compounds that are retained on the stationary phase to elute in a timely manner. By the way, this is also a great feature when doing prep HPLC as well.

Save yourself from a few headaches and try one for your next LC-MS method! You will be pleasantly surprised with the results!

Cogent TYPE-C™ Silica Columns and Mesquite Flour Separations

A new research article using Cogent TYPE-C Silica columns has been published in LC-GC Magazine. Our study investigates separation and identification of components in mesquite flour. Using these unique HPLC columns, we were able to identify a wide variety of compounds in these natural products, some of which have been unreported in previous works. Some analytes were quite polar and could only be retained in the ANP mode, while others were more amenable to reversed phase. Use of both of these complementary retention modes allowed for a more complete characterization than might be possible using either mode alone. Further study on the analytes could help elucidate their role in some of the health benefits of mesquite flour consumption, such as their antioxidant properties.


I feel very privileged to have the opportunity be a part of such innovative research. It is wonderful to see how many novel applications these columns have in disparate industries such as foods & beverages, pharmaceuticals, and clinical studies. I look forward to seeing what our research in the future may yield in addressing some of the challenges facing the modern analytical chemist.


You can read the full article here:
 
http://www.chromatographyonline.com/lc-ms-characterization-mesquite-flour-constituents

Exciting updates for the Cogent™ HPLC column product line

I have been working for MicroSolv for over five years now and have had the privilege of seeing our Cogent brand of HPLC column products expand significantly during that time. For example, we have introduced near-UHPLC small particle phases to complement our standard 4 µm columns, allowing users to combine the benefits of high efficiency particles with the unique retention and selectivity of TYPE-C Silica™. Several new types of bonded phases have also been added to our catalog in recent years, such as the Cogent Diol™ and Cogent UDA™.

                The future of Cogent TYPE-C Silica is bright as we anticipate several upcoming additions to the product line. Firstly, we will be releasing the Cogent Amide™ phase, which I have found to have tremendous potential for retention of polar sugar compounds. This material avoids many of the common pitfalls associated with amine phases, often considered the workhorse column for sugar analysis, such as poor robustness and low column lifetime. The Cogent Amide offers the best of both worlds because the bonded ligand can help retain sugars while not sharing the undesirable chemical reactivity of an amine column.

Another upcoming product we look forward to introducing is useful for compounds in which stainless steel column hardware can create detection/recovery problems. Now for the first time, you can choose PEEK hardware for your Cogent TYPE-C Silica column instead of stainless steel. Our laboratory has performed comparisons of anionic compounds such as nucleotides using both hardware types and has noted greater LC-MS detection levels using PEEK hardware.

              In light of these additions to the Cogent catalog, we are excited to announce a new logo for our Cogent line of products! Sleek and modern, this new look reflects our focus on updating the Cogent brand to better serve our Community and the growing needs of today’s practicing scientists. For more information, we invite you to check out our webpage and keep up with the latest news on these upcoming Cogent products:

www.Cogent-HPLC.com

Red Wine and the French Paradox

France is renowned for its wine cultivation and ubiquitous incorporation of the beverage into its everyday culture. Interestingly, the French have been noted for their low incidence of coronary heart diseases, a phenomenon popularly termed the “French Paradox.” Some have speculated that this may be linked to the relatively high consumption of red wine, which is known to have a number of antioxidants present. Among these is a stilbenoid called resveratrol. You may have read about resveratrol in the news in recent years. Ever since an influential 1997 article published in the prestigious journal Science demonstrated its chemopreventative activity, there has been ongoing research to explore its therapeutic effects on human health, addressing such disparate conditions such as heart disease, neurological disorders, and senescence.  

                Although there remains much to be done in resveratrol research, particularly in translating efficacy results to in vivo human clinical trials, this intriguing compound seems to show promise as a therapeutic agent. For this reason, I thought it would make a pertinent area of focus for research using the Cogent TYPE-C Silica™ columns. In the study, I addressed two major problems with resveratrol quantitative analysis in red wine samples: low sensitivity and poor specificity.

                Some laboratories may use UV detection while others require LC-MS. Using either detection method, these obstacles can be overcome. With UV detection, I employed a sample cleanup/preconcentration technique using Cogent TYPE-C Silica™ phases packed in a microextraction bed. This helped remove interfering peaks in the resulting chromatograms while also providing a 2X concentration increase, affording higher sensitivity. For LC-MS, you can obtain excellent sensitivity due to the nature of the MS detector, which is far greater than UV for this type of analyte. Furthermore, use of extracted ion chromatograms (EICs) allows you to obtain a clean chromatogram corresponding to the m/z of the resveratrol [M + H]⁺ ion.

                You can use these method strategies in your investigations of resveratrol in not just wine, but a variety of other matrices such as plasma samples. In particular, LC-MS is a powerful tool that will undoubtedly be a part of these quantitative studies more and more in the future. Cogent TYPE-C Silica™ columns offer advantages for these types of analyses, which I describe in detail in a research article published in LC-GC North America magazine.

Click here for the article!

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.