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which column have very different selectivity from RPLC
Posted: Tue Jan 12, 2010 3:33 am
by jiang295
besides ion exchange, size exclusion, i need it for 2-D lc separation.
Posted: Tue Jan 12, 2010 5:49 am
by Vlad Orlovsky
The answer is mixed-mode:
http://www.sielc.com/Technology_2D_Properties.html
Few newsletters:
http://www.sielc.com/pdf/SIELC_August_2008.pdf
http://www.sielc.com/pdf/SIELC_May_2007.pdf
Contact me if you have questions. We have bimodal (RP+cation or anion exchange) and trimodal columns (RP+cation+anion exchange)....with endless possibilities to control selectivity. The ligand is assembled on silica and has all functionalities. It is not a physical mixture of two or more silica gels packed in the same column.
Posted: Tue Jan 12, 2010 1:28 pm
by Uwe Neue
My suggestion is to NOT dismiss different options in reversed-phase chromatography. The most drastic changes in selectivity that I have seen are associated with a pH change from acidic to alkaline. In addition, you can also get significant changes in the retention pattern by changing from a standard C18 to one with an embedded polar group, but admittedly these are smaller changes. The latter is roughly equivalent to the changes observed going from acetonitrile to methanol.
Posted: Tue Jan 12, 2010 2:15 pm
by Bryan Evans
Can you tell us more about which type of solutes you'll be separating?
Posted: Wed Jan 13, 2010 2:06 pm
by Bintang
If you do not want to run IC or SEC, that sort of leaves only another RP (at a different pH) or HILIC.
A good HILIC/RP ref is (JOURNAL OF PROTEOME RESEARCH Volume: 6 Issue: 3 Pages: 937-946 Published: MAR 2007)
Or visit or web page
http://www.sequant.com/default.asp?ML=11733
Posted: Wed Jan 13, 2010 4:33 pm
by pharmaguy
Consider using the following tool for RP columns, you can get drastic changes in retention by utilizing the different factors driving your separation. This work is well referenced and independent. I know a full day will be devoted at this years Pittcon to the topic.
http://www.usp.org/USPNF/columnsPQRIapproach.html
Posted: Wed Jan 13, 2010 4:51 pm
by HW Mueller
An affinity column can be quite different to anything else.
The classical "orthogonal" case would be a silica column with low polarity solvent (NP) + a RP column.
I used the stark differences caused by pH to get rid of acidic and basic matrix components by using acidic, then basic washes of biological samples on a C-18 column. Finally the neutral analyte was eluted with a neutral mobile phase.
Did I see correctly that zirconium based columns can be quite different to silica analogs (never did much with a zirconium column)?
Re: which column have very different selectivity from RPLC
Posted: Tue Jan 19, 2010 7:15 pm
by Jiri Urban
besides ion exchange, size exclusion, i need it for 2-D lc separation.
you should probably look more deeply on your samples and their orthogonality/dimensionality
see P. Sandra, Analytica Chimica Acta, 641 (2009) 14.
Retention in orthogonal dimensions may still be weakly correlated when a series of analytes have similar physicochemical properties and behave similarly in both dimensions. As an example, one could consider the SEC × RP-LC combination as orthogonal, but for certain mixtures, the separation by size may be correlated to RP retention. Likewise, ion-exchange (IEX) LC separates analytes mainly on the basis of charge, but might show hydrophobic interactions.
In contrast to the more obvious orthogonal combinations, intuitively, one would expect hydrophilic interaction liquid chromatography (HILIC) to be inversely correlated to RP-LC i.e. compounds displaying a large k value on HILIC are not well retained by RP-LC and vice versa. Investigations have, however, shown that this is not the case, leading to the conclusion that the interactions in HILIC cannot solely be explained by hydrophilic interactions. Therefore, the definition of orthogonality must be focused not only on the differential selectivities of the two dimensions, but perhaps more accurately on the independent retention of the analytes.
Re: which column have very different selectivity from RPLC
Posted: Wed Jan 20, 2010 6:19 am
by jiang295
problem is that i dont know my sample's dimensionality.
besides ion exchange, size exclusion, i need it for 2-D lc separation.
you should probably look more deeply on your samples and their orthogonality/dimensionality
see P. Sandra, Analytica Chimica Acta, 641 (2009) 14.
Retention in orthogonal dimensions may still be weakly correlated when a series of analytes have similar physicochemical properties and behave similarly in both dimensions. As an example, one could consider the SEC × RP-LC combination as orthogonal, but for certain mixtures, the separation by size may be correlated to RP retention. Likewise, ion-exchange (IEX) LC separates analytes mainly on the basis of charge, but might show hydrophobic interactions.
In contrast to the more obvious orthogonal combinations, intuitively, one would expect hydrophilic interaction liquid chromatography (HILIC) to be inversely correlated to RP-LC i.e. compounds displaying a large k value on HILIC are not well retained by RP-LC and vice versa. Investigations have, however, shown that this is not the case, leading to the conclusion that the interactions in HILIC cannot solely be explained by hydrophilic interactions. Therefore, the definition of orthogonality must be focused not only on the differential selectivities of the two dimensions, but perhaps more accurately on the independent retention of the analytes.
Re: which column have very different selectivity from RPLC
Posted: Wed Jan 20, 2010 7:21 am
by Jiri Urban
problem is that i dont know my sample's dimensionality.
And can you try runing several isocratic analysis to find out the differences in selectivity and orthogonality, respectively?
I think there are some papers dealing with orthogonality even for two reversed-phase systems.
Posted: Wed Jan 20, 2010 4:13 pm
by Bryan Evans
You can try Scherzo SM-C18 (ODS + anion + cation ligand). Below is the tech report:
http://www.imtaktusa.com/site_media/fil ... eport_.pdf
Scherzo SM-C18 is very efficient (3um silica) and highly reproducible. A lot scientists
working with metabolites / bioligicals are finding it useful for their research.
Posted: Thu Jan 21, 2010 3:03 am
by jiang295
i can only use volatile buffer, is it compatilbe.