K' and Ion exclusion Chromatography

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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We are running an analysis for fructose, glucose, maltose, sucrose, and sorbitol on a "sugar" column that consists of a DVB matrix and makes use of Ca++ for ion exchange. I noticed that the K' for all of our analytes was not very impressive based strictly on a calculation of hypothetical t0.

I put in a call to the manufacturer and inquired as to how to possibly improve this as there is no gradient possibility in this analysis and the only MP that is documented as being suitable for this column is water.

They sent me one of their chromatograms and it had a K' situation that was nearly as bad with the lowest being 0.1. And then a somewhat humerous thing happened - I asked if perhaps a K' that low is acceptable in IEC and the response was to ask me for more details on my run conditions as they would like to help me improve that. And then I told them, no, I was talking about the K' for their run in their tech paper where t0 was based on an unretained marker compound.

And then I never heard back.

So, assuming a K' this low is equally unacceptable for ion exclusion chromatography as it is for RP chromatography, how would one go about improving it without a gradient option and being limited to pure water as a MP?
If I recall the DVBS-Ca Carbohydrate column (Sugarpak and equivalent) was developed before K' equation was even developed. It was at the time the 'only' HPLC column able to baseline separate the mono and disaccharides. It was the only column to baseline separate the sugar composition of 'high fructose corn syrup (HFCS)'. In fact it was approved by ICUMSA (International Commission of Uniform Methods of Sugar Analysis).
You're comparing a 'Tin Lizzie' to a 'Mazeratti'. I used to pack my own HPLC columns but I don't recommend it!
You have opened up a large, economy-size can of worms.

For the record, the concept of k' far precedes carbohdyrate separations on cation exchange resins. The catch is that the computation depends on knowing the value of t0.

The typical "rule of thumb" that t0 is approximately 0.5 x L x dc^2 (L is column length; dc is column diameter) depends on the assumption that mobile phase comprises about 60 - 65% of the internal volume of the column (roughly 25-30% is between the particles and 30-35% is inside the pores of the particles). While this is a good estimate for the porous materials used in most types of HPLC, 0.25 x L x dc^2 would give a better value for a non-porous packing like an 8% crosslinked cation exchanger.

Even taking that into account, retention for some of the monosaccharides *is* marginal; HPLC chemist is correct that that column was originally designed for separation of oligomers in high-fructose corn syrup.

Retention (k') can be decreased to some extent by adding acetonitrile to the mobile phase, but it can't easily be increased. The only real way to change the retention and selectivity is to use a column with the resin in a different ionic form. Ca++, Pb++, Ag+, and H+ forms (at least) are commercially available.

Finally, (to be a bit pedantic!) "ion exclusion" is not really a good description of the mechanism. Back in the late 70s when BioRad introduced the first commercial versions of these columns, we used the term IMP ("Ion Moderated Partition"). It never caught on, but I still think it's fairly accurate.
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
I did hear back from the manufacturer's tech team and they told me that:
1 - IEC or IMP is different from RP - indisputably true

and

2 - a K' of 0.1 is fine for IEC (or IMP) - OK, fine

Thanks for the input - I have recently made a big deal out of K' for our reversed phase methods which were very problematic in some cases and when the K' for our sugar method looked like a turkey I needed answers for myself and our team.
I am glad to hear that you are questioning this. So many ignore the importance of K prime values. However, it is true that its importance and relative value depend on the mode of chromatography being used. IE and SEC are two examples where it is less relevant.

Of great importance is to first correctly measure the Column Void Volume so you can correctly determine the Column Void TIME, using your specific method and thus T ZERO (what we want). I stress the word 'Measure'. Estimating is great to get started, but then it needs to be measured. Here is a link which may help to do this: https://hplctips.blogspot.com/2011/05/determination-of-hplc-column-dead-time.html

Next, once we have Tzero, then we can calculate the K prime values for each sample. Here is a link for that too: https://hplctips.blogspot.com/2015/06/k-prime-also-known-as-capacity-factor.html Note that it mentions how the mode of chromatography used can effect K prime. While K prime is always important (to show you have retention), in a few cases, K prime is less relevant than it is for traditional NP and RP methods (where it is far more important).
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