Why Biphenyl is a very interesting primary choice as stationary phase in LC.

Being a GC chemist for almost 38 years I always wondered why most LC separations are performed on a C18. A C18 is an extremely good non-polar stationary phase and is the foundation of reversed-phase chromatography, where polar mobiles phases are used with non-polar stationary phases. But there are so many C18 phases, how do you choose the correct one? There are also a lot of separations where analytes are polar, one of the big advantages of LC is that you can analyze much more polar analytes than GC without derivatization. For these types of analytes why do you choose a C18 at all?

A basic rule in chromatography is that you choose a stationary phase that shows interaction with your analytes. In a GC hydrocarbon separation, for example, a polydimethyl siloxane like Rtx-1 is used. For alcohols and glycols, a polyethylene glycol like Rtx-Wax is used, or for optical isomers a chiral stationary phase is used that shows specific affinity to one type of isomer.

In LC, multi-ringed structures, substituted ring structures, and small polar analytes are frequently analyzed. These analytes will interact with C18 through dispersive forces, but they may also interact with the silica substrate through hydrogen bonding or cation-exchange. These interactions are often referred to as “silanol” interactions and they are often thought of as undesirable. In fact, with many separations these silanol interactions contribute significantly to the retention of the analytes. This means silanols can be both beneficial, by creating retention; and detrimental, if they are not controlled or if the silica surface is not consistent. Differing silica substrates is one of the main reasons all C18’s are not the same.

Fig,1 Surface of C18 silica.

So the question that came to my mind is how do you take advantage of additional retention mechanisms for polar analytes in reversed-phase? One way is with the Biphenyl stationary phase which was originally developed at Restek.

The Biphenyl stationary phase retains compounds through the same dispersive forces as a C18 but it also allows for more polarizable substances to be retained. Basically, the pi electrons available on the Biphenyl phase create retention with analytes which are electron deficient. Because there are so many pi electrons in conjugation you get much better retention for small and polar analytes than on a phenyl-hexyl or diphenyl type phase.

Fig. 2 Biphenyl

I would argue this actually make the Biphenyl an even BETTER choice than a C18 when starting your method development. An example I saw at a recent food meeting showed >600 pesticides in one run using LC/MS/MS on Restek’s Raptor Biphenyl phase.

As with any phase chemistry there is always some type of downfall and in the case of the Biphenyl it is UV bleed. A dirty little industry secret is that ALL phases bleed whether you know it or not, it just so happens that with C18 you don’t see the bleed with UV or MS detectors. In the case of the Biphenyl you can see phase bleed in certain instances by UV. If this is the case contact Restek Technical Support and they can coach you through how to minimize or eliminate it.

So where did the Biphenyl phase come from? A number of really intelligent people were involved in making it happen at Restek and it included GC polymer chemists, LC R&D chemists, and Applications personnel. It’s a great story about experimentation, fundamental chemistry, and teamwork making a really great and unique product.

Special thanks to Ty Kahler and Paul Connolly for their input in this blog