Preparing HPLC Mobile Phase Solutions Consistently

Have you ever read what seemed like simple method conditions and found yourself pondering how exactly something was done? For example, what does “0.1% ammonium acetate” mean? Is the 0.1% in terms of w/v? What about w/w or v/v percent? I always try to avoid ambiguities such as these when describing some method conditions. What may be obvious to assume for one person may not be so with another. This becomes especially crucial when you want a method that’s reproducible across different laboratories and analysts. Ideally, anyone should be able to read the method conditions and arrive at the same end result as the person who wrote it.

What about premixed mobile phases with an aqueous buffer and an organic solvent? I often have some of the same questions about these. If a 90 : 10 acetonitrile : aqueous buffer is said to be “10 mM ammonium acetate,” is that just for the buffer component or for the total solution? It would actually make a big difference in the ammonium acetate concentration depending on how it was interpreted (a factor of ten in this case).

For this reason, we thought it would be helpful to show you a simple step-by-step walkthrough on how to make a premixed 90 : 10 acetonitrile : 10 mM ammonium acetate buffer. This mobile phase solution is used quite frequently in ANP methods and LC-MS, so it would be good to have a handy resource to refer to when you need it.

See the link below for our tutorial:

http://kb.mtc-usa.com/article/AA-03145

New Location for Manufacturing/Applications Laboratory

I am pleased to announce that MicroSolv’s laboratory division, previously located in upstate New York, has now relocated to Wilmington, NC. Situated in the CREST Research Park of UNCW, this new facility will allow for collaboration between MicroSolv and those at the university. In addition, the new lab is very well-equipped and up-to-date, which will allow our operations to be very streamlined and productive.

                I have spent seven years at the previous laboratory and watched it develop over the years, ever since its very inception. It has been very gratifying to have seen the laboratory acquire new instruments and capabilities while at that location, and I think our new facility here in Wilmington will allow for even more of this kind of growth in the future.

                I look forward to seeing how the new location will enhance our processes in both manufacturing and research applications. Stay tuned for more updates on our progress with the new laboratory!

Eastern Analytical Symposium 2017

Recently I had the privilege of giving a lecture at the annual Eastern Analytical Symposium in Plainsboro Township, NJ. This year, the symposium was held in an elegant new venue, the Crowne Plaza Princeton-Conference Center. I have been attending EAS since 2011 and I think this has been my favorite one so far.  

                My presentation was at 11:00am on Wednesday the 15^th. The subject of my lecture was about a method my colleagues and I had been working on for chromatographic assay and organic impurities detection of chlorpheniramine maleate formulations. The current USP method calls for a time-consuming multistep liquid–liquid extraction procedure for the assay, and no method is provided for impurities analysis. Consequently, there was a lot of room for improvement in bringing these methods up-to-date.

As a basic compound, chlorpheniramine may be prone to prone to chromatographic tailing via undesirable electrostatic interaction with residual silanols present on the surface of some traditional silica-based stationary phases. Hence, the moderately hydrophobic Si–H-based surface of Cogent™ columns was postulated to be advantageous in terms of minimizing tailing contributions from this phenomenon.  

                We encountered a few obstacles in method development. The first involved peak splitting for the first two peaks. I had previously selected a diluent with 50% acetonitrile in an effort to ensure solubility of the more hydrophobic analytes, but this choice had the undesired effect of peak splitting. Reducing the organic content in the diluent to 10% resolved this issue. Another encountered problem dealt with trying to use a high enough extract concentration such that we could detect impurity peaks at the 0.1% level while being careful not to overload the column so much that we lost resolution of the critical peak pair. In this case, adjusting the gradient and using a lower mobile phase concentration of trifluoracetic acid (0.05% instead of 0.1%) helped to improve selectivity enough that resolution was sufficient at the higher extract concentration.

                Despite these setbacks, we produced a nice method for subsequent validation studies and I think the audience was quite pleased with the end result. This was my second year as a speaker at EAS and look forward to the opportunity to possibly present again at future meetings! Hope to see you there in 2018!

Method Validation Tip: Do Robustness Studies First

After method development, I find that a lot of chemists will delve into method validation studies like accuracy, precision, etc. but leave robustness for the end. However, I think it is better to do robustness first. The reason for this is that the results can help you further refine the method in ways that you may not have considered during the method development stage.

                Let’s say for example that you do your method development have a perfectly satisfactory separation where your critical peak pair is baseline-resolved. You move on to validation and go through all the necessary studies (e.g. accuracy, linearity, repeatability, intermediate precision, LOD, LOQ, etc.). Then in the robustness studies you find that alteration of one of the method variables actually leads to a superior separation. This was what I found when I varied the TFA concentration. I chose a 0.1% concentration originally during the method development stage but hadn’t given much thought to it at the time. Then in robustness studies, I found that the critical peak pair, which had been just baseline-resolved with 0.1% TFA, became vastly better separated upon a decrease to 0.05%. Before proceeding with the rest of the validation, I could use the 0.05% concentration in my method.

                I hope this information helps you as much as it helped me. I am always learning new tips and tricks to make chromatographic analytical techniques more streamlined and efficient.

UV Trace Drift in Gradients

Gradient elution mode is an invaluable tool for us as analytical chemists, but the slope of the UV trace that results can present issues. You can understand why this happens by considering how each of the solvent systems has its own UV absorption profile. Some of the drift is due to differences in the refractive indices between the two solvents as well. Because of these differences, the UV readout will change continuously over the course of the gradient, producing the slope that we observe. If it is too steep, it can obscure eluting peaks, reducing sensitivity. It would be ideal to have the slope as shallow as possible if we can do so.

                The other day, I was working on a method development project for the USP that called for low pH. We observed tailing for some of our peaks and speculated that the lower pH was needed for this reason. To this end, we tried an additive of 0.5% formic acid in the mobile phase solvents but found a rise in the UV trace of 200 mAU over the course of our 30 min gradient. In an impurity method, where low detection levels are of critical importance, this degree of noise would clearly not be practical. By using TFA instead, however, we were able to reduce the additive concentration by a factor of five and still obtain the good peak shapes that we sought. With the lower additive concentration, the trace slope had risen by about 20 mAU, a mere tenth of the slope obtained previously! This clearly represented a much more viable option for a prospective USP method.

                You just have to remember a few things with TFA, though. It can be prone to oxidation from the atmosphere, so extraneous peaks or a steep UV trace can result if it degrades. In that case, you would have the same problem as before! Your best bet is to try single-use ampules of TFA, since these will have minimal contact time with air. If you use a resealable bulk bottle, you can try adding a blanket of argon after every time it’s opened to ensure it is kept free of air. This oxidation is a slow process, so it only becomes an issue after storage of the opened bottle. Then of course, another thing to keep in mind about TFA is its MS-incompatibility, as it contributes significantly to ion suppression. So if you develop a nice method for UV-based analyses with TFA, just be aware that its applicability will be more limited than with something like formic acid. Even so, TFA can do wonders for some tailing peaks of basic compounds, so it is a good tool for the analytical chemist to keep at the ready.

                I hope these are some helpful tips for you to try in your own method development process. I was always a fan of TFA for those situations where symmetrical peak shapes may be difficult to obtain otherwise. The effect of the trace slope can be one of the more tricky aspects of a UV-based gradient method. And if you can, maybe also consider using a more gradual solvent gradient. This will have a direct effect on how steep the resulting UV trace is.