I have found good success with Symmetry columns C18, but I was wondering if someone can tell me other columns types (Brands) which they have practically found good, specially for isomer separation in reverse phase.

thanks for your responses

Reversed-phase is inherently non-selective for isomers (compared to, say, normal-phase). Graphite columns seem to give much better isomer selectivity while still using "reversed-phase" retention mechanism (this is from reading the literature, not from first-hand experience). Downside is cost and maybe fragility.

-- Tom Jupille / LC Resources

Tom is correct, the graphite columns work well for resolving isomers. In my experience with them (about 3 yrs), they also appear to be very rugged, as long as you are careful. They are quite expensive however. By the way, if you go this route, talk to the tech reps at Thermo Hypersil (I'm not affiliated) concerning column size before you purchase. These columns tend to be extremely retentive and I've found that you generally need to go with shorter (and less expensive!) columns than what you are normally used to in reverse phase. Good luck.

Joe Runkle

Hypercarb sounds really good. How about its life time? Thanks.

Hypercarb has been mentioned before in this forum.
I just loaned a couple of Hypercarb columns to a friend to separate some isomers of nonylphenol. The columns had been stored for some years after analyses of oxalic acid in urine (mobile phase: aqu. TFA). They are still doing a splendid job. Apparently, they really do have the inertness of graphite. Mechanically, I wouldn´t pressure shock or pulse them excessively, though the manufacturer says that the columns are packed at 400 bar (6000psi).

This is for you Tom: Can graphite columns be used in reverse phase (20 to 50% acetonitrile in water). As you know the main problem with normal phase is reproducibility and baseline as well as detector response (In our company we have to detect a drug at 0.05% of impurity assay concentration of drug, so if response is not sufficient, the concentration has to be increased, which again can lead to solubility problems). Are there any other column types for isomer separation which can be used in reversed phase??

What columns would you suggest for normal phase separation of isomers (Brands and mobile phase composition)

Anybody wants to take this one:

Suppose one is not able to separate an isomer from main drug peak, and they analyze this isomer with say an NMR method, and also prove on chemistry basis that the isomer (e.g. structural isomer) cannot be generated on standing (stabilty studies), should it be considered a stabiltiy-indicating method.

And a little variation of the above case is where another where one cannot separate the isomer but also cannot on chemistry rationale prove that the isomer (enantiomer or diastereomer) will not generate on standing (stability studies), is it acceptable as a method for stability studies??

Thanks and appreciations!!!

Sean;

It seems like you have very interesting problems. Graphite columns can be used in reversed-phase mode, however, because of the highly hydrophobic surface they show strong retention and sometimes this makes necessary to use mobile phases with a very high % of organic solvents. Also, they are frequently used in the "non-aqueous mode".

I disagree somewhat with one of the opinions above about RP being inherently non-selective, it can be very selective when the right groups are present. If the isomers you are trying to separate have some aromatic group or are highly conjugated you may want to try the "fluorinated" columns from Keystone. I have used the Fluophase PFP ones with great success. When no aromatic centers are present perhaps you will do better with Graphite columns or some sort of silica column such as Zorbax-Sil.

You must also remeber that many Chiral columns have been used for the separation of non-optical isomers. However, in this case is difficult to predict wich would be best to try without more information.

NMR or Infrared should be able to distinguish the isomers you are interested in, but the sensitivity is likely to be low. I see no problem in using these techniques to assay isomers if you can distinguish them. However, the detectability of the off isomer should be tested with authentic samples.

I hope this helps you;

Benjamin

Thanks Benjamin, Do you think chiral columns can be used in normal phase and reversed phase both. Particularly for separating isomers, is it better to use chiral columns in normal phase or reversed phase. I think some chiral columns prohibit the use of chloroform.

Thanks

Sean;

Many types of Chiral columns can be used in either mode. I remember such are the cases of the Chirobiotic ones. Some others they exist in two versions, one for each mode, there are some Chiralpak and Chiralcel ones like these.

It is also true that some of the columns mentioned above do not tolerate solventes such as THF, CHCl3 or CH2Cl2. I feel that you can decide the best mode to separate your compounds just on the basis of their solubility in water. If they are insoluble you will do better trying normal phase.

Benjamin

can someone help me with some of these questions?
for teh quantification of D-(+) and L-(-) phenyalanine in urine,
what chromatographic technique and detector is used, and what is the mobile and stationery phase