HPLC Troubleshooting Tutorial: Problems with the UV Baseline

I was using a Cogent Bidentate C8 2.o™ column in my HPLC method recently when I observed a strange phenomenon. Instead of a normal, flat baseline in the UV monitoring readout, there was a highly erratic pattern; the signal would descend and jump back to its starting point in a recurring “sawtooth” like waveform. Meanwhile, the pressure was stable and all other instrument parameters indicated normal operation. My isocratic mobile phase was fairly simple (50% DI water/ 50% acetonitrile/ 0.1% formic acid) and other columns I tried did not have the issue. No amount of equilibrating seemed to remedy the situation.

                I really wasn’t sure if the column had somehow been damaged but had no way of running a standard QC test if I could not observe analyte peaks amongst the unstable UV baseline. Next I thought maybe there was some contaminant on the column and it needed to be washed out. Initial trials using solvent systems such as 1:1 methanol: DI water proved unsuccessful, and then I had the idea that perhaps there was some trace immiscible solvent in the column and use of an aqueous-based mobile phase would not be appropriate.

                So I switched to pure isopropanol and gave it a try. Miscible with both reversed phase and normal phase solvents, isopropanol can often be an effective choice in general column cleaning. Soon after introducing the new solvent to the column, the UV baseline resolved into a typical, flat signal. Now the real test would be to see how my original solvent system behaved. Upon reintroducing the original mobile phase of 50% DI water/ 50% acetonitrile/ 0.1% formic acid, I was pleased to observe that the baseline had returned to normal.

                How can we account for this behavior? The outcome of the experiment seemed to indicate to me that the column did indeed have some minor amounts of a nonpolar solvent in it, which would create immiscibility issues with the reversed phase solvents I was using. These could have been residual packing solvents and will pose no problems once completely removed.

This just shows you if you think a column may be defective, try to investigate all your possibilities before prematurely concluding that it has been damaged. You can save your laboratory money by getting the most out of each valuable HPLC column instead of replacing it. I find issues like this can crop up in one form or another quite often; a column is suspected to be defective when in reality, there is a simple solution to resolve the issue. So the next time your HPLC column exhibits some unexpected behavior, try to ask yourself what might be causing it and whether it can be fixed.

 

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