Chromatography Forum

will it cause irreproducibilility as unmodified silica column?
what is the major difference between HILIC and other bonded phase NPC (Cyano, amino and diol)? Can separtion achieved by HILIC also can get it from cyano or amino or diol column with water as mobile phase?

Why do you want to do NPC? If you select a bonded phase, why not doing RP, or HILIC?
In HILIC, you use water in the mobile phase, so all difficulties with water will go away when using silica as the mobile phase.

primary reason is to achieve different selectivity from C18 and sample is only water soluble, so I can only use water as mobile phase.

And I think I am confused with definition of HILIC. Can diol column with aqueous mobile phase called HILIC. I do see someone called NPC with aqueous mobile phase as HILIC. but I also see some HILIC with special stationary phase. thanks a lot if you can clear my confusion.

Why do you want to do NPC?

If you select a bonded phase, why not doing RP, or HILIC?



In HILIC, you use water in the mobile phase, so all difficulties with water will go away when using silica as the mobile phase.

HILIC is chromatography with a polar bonded phase and commonly acetonitrile with a low water content as the mobile phase. Retention decreases as you increase the water content of the mobile phase. The lowest lowest content of water into he mobile phase is about 5%. The reason is that pure HILIC needs a water layer on the surface of the packing. Packings used are silica, amino packings (the classical approach for sugar analysis), amide packings and to a lesser extent diol packings. Waters developed a new amide packing for HILIC last year. A special branch of this technique are chiral separations (and practitioners of this version of HILIC often call this "aqueous normal phase" or ANP).
Due to the fact that water is used as a component of the mobile phase, all the concerns in NP about the water content of the mobile phase go away.
While the classical retention mechanism is partitioning of the analyes into the water layer on the surface of the packing, reality is much more complex, since silica and amino phases also work as ion exchangers, and the polar packings add on their own some elements of hydrogen bonding with suitable analytes.
Otherwise, read my book on pages 217ff.

as I am doing offline two dimension prep scale chromatography,
do you think HILIC and C18 will be somewhat orthogonal column couple. As you know, most RPC are more similar than different, do you think HILIC and C18 will be more different than two PR columns?

another question, since diol, amide, cyano column with aqueous mobile phase called as HILIC, so what is the pakaging material for real "HILIC" columns, which I mean columns use "HILIC" as brand name.

HILIC is chromatography with a polar bonded phase and commonly acetonitrile with a low water content as the mobile phase. Retention decreases as you increase the water content of the mobile phase. The lowest lowest content of water into he mobile phase is about 5%. The reason is that pure HILIC needs a water layer on the surface of the packing. Packings used are silica, amino packings (the classical approach for sugar analysis), amide packings and to a lesser extent diol packings. Waters developed a new amide packing for HILIC last year. A special branch of this technique are chiral separations (and practitioners of this version of HILIC often call this "aqueous normal phase" or ANP).
Due to the fact that water is used as a component of the mobile phase, all the concerns in NP about the water content of the mobile phase go away.
While the classical retention mechanism is partitioning of the analyes into the water layer on the surface of the packing, reality is much more complex, since silica and amino phases also work as ion exchangers, and the polar packings add on their own some elements of hydrogen bonding with suitable analytes.
Otherwise, read my book on pages 217ff.

Yes, RP and HILIC are rather orthogonal. Toluene is well retained in RP, and unretained in HILIC.

I do not place cyano materials into the HILIC category. (Well, maybe with one exception: Nova-Pak CN RP.)

Real HILIC columns contain - as mentioned before - silica (or the hybrid equivalent), amino phases, amide phases, and diol phases. Manufacturers may have given versions of their packing materials that are used in HILIC the name HILIC. This means that the material has been qualified for HILIC, is used in HILIC and has HILIC application literature. (Other packings with the same surface may not work to the same quality or have problems under HILIC conditions.)

There are other ways to get to orthogonal separations as well. You can - for example - use low pH and high pH (pH 10) - to get orthogonal separations in RP. I can send you my presentation from the last PittCon which shows how much difference one can get with this approach. Not a single peak remained in the same spot...

RP and HILIC and RP at low and high pH have their own pecularities. Since HILIC does not work for purely hydrophobic compounds, it is not an alternative to RP for this set of analytes. A change in pH will only work for ionizable analytes, and will do nothing if the charge of the analytes does not change between pH 2 and pH 11.

thanks a lot if you can send me slides. that will be very instructive. My email is jiang295@umn.edu.

I think most challenging analytes for achieving orthogonal selectivity would be neutral hydrophobic analytes.
do you think LH-20 and RP can be good combination fro this set of analytes?

Yes, RP and HILIC are rather orthogonal. Toluene is well retained in RP, and unretained in HILIC.

I do not place cyano materials into the HILIC category. (Well, maybe with one exception: Nova-Pak CN RP.)

Real HILIC columns contain - as mentioned before - silica (or the hybrid equivalent), amino phases, amide phases, and diol phases. Manufacturers may have given versions of their packing materials that are used in HILIC the name HILIC. This means that the material has been qualified for HILIC, is used in HILIC and has HILIC application literature. (Other packings with the same surface may not work to the same quality or have problems under HILIC conditions.)

There are other ways to get to orthogonal separations as well. You can - for example - use low pH and high pH (pH 10) - to get orthogonal separations in RP. I can send you my presentation from the last PittCon which shows how much difference one can get with this approach. Not a single peak remained in the same spot...

RP and HILIC and RP at low and high pH have their own pecularities. Since HILIC does not work for purely hydrophobic compounds, it is not an alternative to RP for this set of analytes. A change in pH will only work for ionizable analytes, and will do nothing if the charge of the analytes does not change between pH 2 and pH 11.