An HPLC user meeting in the heart of Berlin

HPLC Praxistag is a user meeting for chromatographers in Germany. At the charming Hollywood Media Hotel in the middle of our home town Berlin, lab technicians, experienced HPLC specialists as well as manufacturers of different instruments and safety equipment were getting together. Many participants also used the chance to visit our production and assembly site and the application lab.

Julia, Silvia and I enjoyed meeting the people who are operating our instruments in the field and to learn what’s on their hearts.…

Which topics were discussed in the user meeting?

Many of the very informative talks were about HPLC troubleshooting, or even better: how to get ahead of trouble. A stable HPLC system can most often be guaranteed by using clean solvents and regularly exchanging (not refilling!) solvent bottles. Proper storage of the system during downtime is also important, avoiding buffers or acetonitrile inside the system when it is not running. Common issues are algae growth in water bottles, but pure organic solvents can also harm you HPLC by corrosion or polymerization inside the system.

All these effects can be slowed down by premixing your solvents, e.g. by using 10% organic solvent in water and 90% organic as mobile phase A and B, respectively, instead of pure water and pure methanol.

Eluent safety cap with air filter
Photo by KNAUER

For consistent results, the solvents should be pre-mixed correctly. Always measure the volumes of two solvents separately and mix inside the bottle. Do not combine by adding both solvents into the same measurement cylinder. A eluent safety cap with an air filter can help to maintain the correct solvent composition and contributes to the safety of everyone in your laboratory.

If you love your HPLC, never let acetonitrile, buffers or pure water sit inside your system without flow.

Hot Topic: Sustainability in HPLC

Many speakers discussed how HPLC can not only be safe and trouble-free, but also more sustainable. Besides energy consumption and waste produced during sample preparation, the solvents used in HPLC are the main environmental concern. Using biobased solvents, e.g. ethanol or propanol, can reduce the carbon footprint of your analysis. But increased pressure can be a problem, and developing a new method can be difficult, especially in a regulated environment.

Safe time and resources with the KNAUER HPLC method converter
Collage by KNAUER 

A much easier approach is to use smaller stationary phase particles and convert your methods to a smaller column diameter and flow rate. Our KNAUER HPLC method converter. will help you with all the calculations needed. The reduced solvent consumption is a win for the environment, and the faster analysis time is a win for you!

What did KNAUER present at the user meeting?

We proudly presented the KNAUER application notes about lipid nanoparticles (LNPs). The users were excited to learn how Covid-19 vaccines are formulated. Rapid mixing of lipids and oligonucleotides inside the impingement jets mixer of the KNAUER Nanoscaler guarantees the self-assembly of LNPs.

Fig. 1: The KNAUER IJM Naoscaler produces vaccines and other LNP formulations in research and preclinical scale Fig. 2: Isocratic separation and sensitive detection of LNP lipids with KNAUER HPLC-ELSD (Graphics by KNAUER)

With KNAUER Azura HPLC and an evaporative light scattering detector (ELSD), we also developed a method for measuring the lipid composition of LNPs. Here, the poor peak shape using water:acetonitrile gradients and incomplete elution in methanol didn’t stop us: by combining both eluents in the same gradient, both issues are resolved. But do we even need a gradient? Remarkably, the four lipids of a vaccine can even be separated in an isocratic run in just two minutes. With a method like that, the ELSD offers competitive sensitivity, adequate for LNP stability studies or quality control. The short analysis time with a low flow rate of only 0.4 mL/min is a good example of a win-win situation where the throughput is dramatically increased in comparison to traditional methods, while the solvent consumption is reduced by the same factor. If you are interested in this exciting method development, don’t hesitate to download our AppNote VPH0078.

Who even needs a gradient? Lipid composition in LNPs can be analyzed sensitively in less then three minutes with KNAUER HPLC-ELSD.

We also gave a sneak-peak into our upcoming AppNote. Coupled with a powerful quadrupole MS/MS, we can detect the identity and purity of the used lipids. Stay tuned to learn more! And last but not least, we also showed how to perform quality control for oligonucleotides by ion pairing reversed phase HPLC-MS. We presented the method published in the AppNote VBS0083. [DG1] as well as practical tips for analyzing oligos by HPLC in general. Especially for ion pairing methods, adjusting to a higher pH and always using freshly prepared mobile phases is crucial. Because volatile additives are used, we also emphasized the importance of closing your bottles carefully with a safety cap.

Azura UHPLC and Sciex triple quadrupole MS are used for the quality control of lipids and oligonucleotides (Graphic by KNAUER)

The HPLC practice days showed how we can keep our methods safe and robust – and what can be achieved in the development of new methods. Get inspired by our AppNote VPH0078 and speed up your LNP analysis!

For further information on this topic, please contact our author: pruefer@knauer.net