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Normal? Reversed? June 26, 2008

Posted by fetzthechemist in Uncategorized.
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After talking to various people recently, I have been thinking on the basics of chromatography and even some history.  First, chromatography was originally used by Michael Tswett a little over a hundred years ago. He used things like powdered clays and other fine earths (afterall silica and alumina were first minerals before they became packings) to separate plant pigments. He used petroleum ether, the liquid distillate cut that includes the light hydrocarbons from about C5 to C10, as his extract solvent and mobile phase. The pigments separated, gave colored bands, and thus chromato-graphy – colored pictures.

This use of a polar adsorbent and a less polar solvent was what people did in chromatography for fifty years or so, thus it was the normal phase version. Somewhere along the line someone decided to coat the adsorbent with a wax or oil. This allowed the use of polar phases, like water. Thus, the phases were reversed from the normal mode.

In my research, I got into some exotic examples of both normal and reversed phases. These used mobile phases way beyond what other people liked to do. Once, I used a perfluorenated alkane based mobile phase and an extremely polar column to separate cyclic saturated hydrocarbons from each other. Yes, cyclohexane has a tenny-weeny polarity. It can be separated from decalin, two fused cyclohexanes, which is more retained.

I was doing really large PAHs, so to me the regular mobile phase in reversed-phase HPLC, like acetonitrile or methanol, were woefully weak. I used pure dichloromethane as a mobile phase, which is a couple of orders of magnitude stronger.

Even that was sometimes too weak, so I used toluene or chlorobenzene. You see those commonly used now to separate C60 fullerene. That was a twist I used based on my PAH work. Nobody else had ever dared to used these exotic phases, but toluene is more polar than a C18 phase. It is reversed-phase. HPLC experts used to want to debate whether it was or not when I first presented results about twenty years ago.

The bottomline is effective separation, which depends on understanding phase behavior, interactions, and partitioning. It doesn’t depend on what is in your textbooks.

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Comments»

1. Ψ*Ψ - June 27, 2008

Coooool. I’ve never done anything with reversed-phase chromatography.

2. fetzthechemist - June 27, 2008

It is a lot easier than fifty years ago. The coated particles are now bonded phases. That was the drawback for years, the coatings slowly dissolved or were unstable. There are now even shape-selective C18 phases that separate long, skinny molecules from more compact ones – these are great for separating PAH isomers or any other molecules that differ by shape. It is a piece of cake to separate the three C78 fullerenes from each other, even though they are variations on egg-shaped.

3. Milo - August 18, 2008

In my thesis research, I had the need to run a few columns using pyridine as the eluent. For those things that were particularly polar, NH3 in Methanol (9M) worked really well (normal phase SiO2).

4. fetzthechemist - August 18, 2008

Few people extend the range of mobile-phase solvents or solid phases to try to get the interactions that are needed. Chromatography is all about getting the interactions to compete in certain fashions of varying degrees to get varying retention. I loved doing these odd combinations. It got the separations I wanted; it showed I understood the interactions well; and it was things that nobody else had ever done.


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