When Your C18 Column Doesn't Work
In the last newsletter we talked about what I expect from a "good" LC method. Two important criteria were:
- Produce adequate retention (k> 2)
- Not operate near the limits for the column or instrument
How can you predict if a molecule is likely to be difficult to separate on C18? Problem analytes usually have one or more of these characteristics:
- Highly water soluble
- Ionic or a strong acid or base
- Carbon/heteroatom ratio less than 3.
- log P (octanol-water partition constant) is negative
The water soluble vitamins include several compounds that are difficult to separate on a traditional reversed phase column. Ascorbic acid (Vitamin C), for example, is highly water soluble and difficult to retain on a C18 column. I have seen many tricks (high salt or acid, no organic, ion pairing), but all are problematic in one way or another.
No matter what you try, you are likely to experience difficulty because, quite simply, you are trying to make a very polar molecule stick to a very non-polar surface. They don't like each other.
The alternative? Use chemistry! Attract your polar molecule with a polar phase. Examples include diol/OH, cyano, and pentafluorophenyl (PFP). There are other possibilities, but these are often now referred to as "HILIC" or aqueous normal phase. Here is an example:
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OK, it's a little messy, but let me point out some important results. The lines connect the same molecules on each phase, and crossing lines indicate a change in relative retention order (selectivity change). There are many, which is to be expected when you switch to a very different stationary phase.
Now, focus on ascorbic acid and nicotinic acid, both barely retained on C18. They are the last to elute on the polar column! Notice that the other acids, pantothenic and folic, are also highly retained.. So, if you need to analyze these, or other organic acids, this column looks like a much better option. Of course, there are still some problem analytes,thiamine, nicotinamide, and pyridoxine, but we are working on those now.
The take home message here, is simple: match the polarity of your analytes and stationary phase, and you will probably have much better luck with retention!
In the next newsletter we will look at general retention trends on these HILIC columns.
As always, feel free to contact us if you have any questions.
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