Cation ion exchange chromatography

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prabhakarthota
prabhakarthota's picture
Cation ion exchange chromatography

Hai,
I am doing the analysis of sorbitol using cation exchange chromatography
the column conditions are as follows
a stainless steel column 0.3 m long and 7.8 mm in internal diameter packed with strong cation excahange resin (calcium form) ( 9µm ) and maintained at a temperature of 85 ± 1 ° C
Mobile phase is water
Detector RID
Now my question is
On what principle RID works? and How to decide the detector at the stage of method development (for RID)? what is the importance of temperature of the column? why so heigh end temperature?
and how the column equilbrated after elution of the sample?

Sami Tuomivaara
Sami Tuomivaara's picture
prabhakarthota,

prabhakarthota,

First of all, it sounds like you are using the resin not as a cation exchanger, but affinity column. This is based on weak complexation of divalent cations with polyalcohols.

RID (refractive index detection) relies on the change in the optical refractive index of the flowing fluid through the detector cuvette. With an optical arrangement, you can detect the RI of the fluid in the detector and hence a substance if it has different RI than the buffer. At best RID is way less sensitive detection method compared to fluorescence or UV absorbance (at their best), but in some cases the only way to go, especially with sorbitol which is not strong chromo or fluorophore. RI is incompatible with gradient elution because the change of the RI of the buffer in the background would most likely swamp the RI signal from the analytes. RI detection can be also quite fussy about ambient temperature and flow rate changes... among other things.

The high temperature might be a relic from sugar separations, where is increases the mutarotation rates and hence gives sharper peaks. Sorbitol being an sugar alditol, it doesn't have alpha and beta anomers, hence there is no mutarotation. If I recall correctly, the effect of temperature is more complicated in these separations, in some cases you can get better separations by using low (lower than rt) temperatures. Unless you find a good rationale from the literature why this temperature is good, you could try other temperatures.

Pure water is used in cleaning the column between runs. In some cases calcium chloride or calcium acetate can be used to wash/regenerate the column but in routine use, ultrapure water is used. Because the interaction between calcium and polyol is weak, no other eluent than water is needed to elute the analytes. In any case, the column should be equilibrated before each run with the elution buffer, water in this case.

Check the work of Stephen Angyal, he pioneered much of the cation-sugar interaction research, including separations of sugars and sugar alditols using calcium-loaded columns...

Cheers,

Bryan Evans
Bryan Evans's picture
Below is a method for

Below is a method for sorbitol on Unison UK-Amino:
http://www.imtaktusa.com/site_media/files/technical_information/TI313E.pdf

It is LC-ELSD, but it is isocratic so you use this on LC-RID as well.
Just to add, for RID, it's helpful if you can minimize temperature gradient from column to detector.

prabhakarthota
prabhakarthota's picture
Hai ,

Hai ,
thanq very much for you reply.
regards,
prabhakar

prabhakarthota
prabhakarthota's picture
thanq very much for your

thanq very much for your valuble reply