You can run chloroform on silica-bound C18. I've run dichloromethane on silica-bound C18 to purify carotenes. You can run up to 100% chloroform. The technique is called "non-aqueous reverse phase", referenced in Practical HPLC Method Development,Snyder, Kirkland, Glajch, 2nd edition, p.267.

Essentially, you are using methanol (or acetonitrile, or another alcohol) in place of water and the less polar solvent (chloroform in your example) as your strong, eluting solvent (acetone or ethyl acetate could be used as well). For silica, the C18 is chemically bound and won't wash off or become hydrolyzed with these sort of solvents. A polymeric reverse phase column swells if used for non-aqueous reverse phase and will become ruined, and this is why I emphasized silica based columns.

Wash and store the column in methanol (or acetonitrile or ethanol or 2-propanol) so you can use the column as a normal C18 without being concerned about solvent miscibility.

Here's some work I've done on the subject:

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Facile_Isolation_of_Carotenoid_Antioxidants.pdf

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN58_Non-aqueous_Reverse_Phase_with_RediSep_Rf_Gold_C18.pdf

Please note that I work for Teledyne Isco.

I don't think so...you have to search for another solvent

I would get in touch with the manufacturer before using any new solvents. That said, chloroform is pretty inert - I don't see why you couldn't use it as a mobile phase at 90%, at least.

What i know that chloroform is used in normal phase and not reversed phase chromatography

I have heard it said that you shouldn't run a RP-HPLC column in 100% organic solvent, because all the water bound to the column will be lost, and the characteristics of the column will change. That is in the context of peptide samples and largely aqueous buffers, however. In your case, it sounds like your column will be used with organics only, so maintaining hydration shouldn't be a problem.

I can see no reason why chloroform should harm your column otherwise. Silica-based columns are vulnerable to high pH and high salt conditions in aqueous buffers. Of course, they would also be susceptible to anything capable of reacting with silica or hydrocarbons. That isn't the case for chloroform.

I agree with Marwa Fouad, though, that RP-HPLC seems contraindicated for your sample. In the presence of 90% chloroform, 10% methanol, many analytes will not bind strongly to a C18 column. Unless your sample is very unusual, you might be better off using normal phase HPLC.

If you want to use this high amount of chloroform you should also check the compatibility of the seals used in the hplc chain

I believe C18 is compatible with chloroform, but it is not good to use solvents of higher polarity like DMSO and DMF... not sure about acetone... probably too.

Chloroform (CHCl3) is suitable for Normal Phase Chromatographic (NPC) applications, where the stationary phase is polar and the mobile phase involves non-polar solvents. Chloroform being a non-polar solvent is suited for silica packed columns. Whereas Reverse Phase Chromatography (RPC) comprises of Non-polar (hydrophobic) stationary phase and polar mobile phase. Therefore, for RPC, the solvents used are acetonitrile or methanol in combination with water; while the stationary phase is silica modified with hydrocarbons containing 4 or 8 or 18 carbon atoms. However, CHCl3 can be used for RPC applications also. However, CHCl3 is not miscible with water and on interacting with water, the resulting solution will be turbid. But, CHCl3 is miscible with methanol. I guess, CHCl3 is miscible with acetonitrile also. So, when CHCl3 is intended to be used for RPC, its relative proportion with water and methanol/acetonitrile needs to be optimized so that no turbid solution is formed during the course of chromatographic runs. Or even if turbid solution forms, it must be ensured that there is no formation of precipitate(s) during or due to the formation of turbid solution.

You can run chloroform on silica-bound C18. I've run dichloromethane on silica-bound C18 to purify carotenes. You can run up to 100% chloroform. The technique is called "non-aqueous reverse phase", referenced in Practical HPLC Method Development,Snyder, Kirkland, Glajch, 2nd edition, p.267.

Essentially, you are using methanol (or acetonitrile, or another alcohol) in place of water and the less polar solvent (chloroform in your example) as your strong, eluting solvent (acetone or ethyl acetate could be used as well). For silica, the C18 is chemically bound and won't wash off or become hydrolyzed with these sort of solvents. A polymeric reverse phase column swells if used for non-aqueous reverse phase and will become ruined, and this is why I emphasized silica based columns.

Wash and store the column in methanol (or acetonitrile or ethanol or 2-propanol) so you can use the column as a normal C18 without being concerned about solvent miscibility.

Here's some work I've done on the subject:

http://www.isco.com/WebProductFiles/Applications/101/Poster_and_Paper_Reprints/Facile_Isolation_of_Carotenoid_Antioxidants.pdf

http://www.isco.com/WebProductFiles/Applications/101/Application_Notes/AN58_Non-aqueous_Reverse_Phase_with_RediSep_Rf_Gold_C18.pdf

Please note that I work for Teledyne Isco.

Thanks a lot for sharing valuable Knowledge and Information........this will definitely help me n give me confidence also.

I don't think so.... you have to search anonther organic solvent which would be parallel to chloroform and according to its volatility and solubility. then make mixture of your desire solvent and run them through multi batch chambers.

I enjoy reading this topic, some nice comments.

I might be a little off track here, but if you ate using CHCl3/MeOH as mobile phase why are you not using a "normal" silica column?

Jack: thanks for the references.

Laurent: On occasion, non-aqueous reverse phase works a bit better than silica. My experience is the non-aqueous reverse phase works with very non-polar compounds such as lycopenes and fatty acids esters. Compounds such as tocopherols are borderline non-aqueous reverse phase. My experience is that these non-polar compounds tend to move rapidly in silica with just a little polar solvent; I suspect the silica is so relatively polar that the compound really doesn't "want" to interact with it. I find a better normal phase column for these sort of compounds is diol because it is less polar than silica. Another possibility for silica is the use of toluene as the "strong" eluting solvent although I haven't used it for this purpose yet.

Thanks for your complements.

Old chloroform (opened at least once time ago) oxidizes to fosgen and on contact with moistue (even moisture from the air) hydrolyses to HCl an CO2. Choloform, if too old, can become acidic enough to damage stationary phase or metal parts.

I found the topic discussed here is interesting and the answers are fruitful :-D The question addressed here is like a 'legendary' question.. hahaha..I belief those who have worked with chromatography and separation techniques must have heard about the theory that if we use non-polar solvents such as hexane or chloroform as the mobile phase, it can damage the silica packings in a C18 RP column. However the answers given by fellow researchers seemed that it is possible to use chloroform as the mobile phase, providing that it is coupled with solvents which are miscible with it.

I would like to know if let say I use chloroform/ methanol as the solvents for my HPLC system, is there any possibilities that the the plastic tubings and internal compartments in my HPLC system get corroded due to the presence of chloroform? Thanks :-)

@Chandra Devan,

See http://www.coleparmer.com/Chemical-Resistance for a fairly comprehensive listing of the chemical compatibility of materials. When using strong organics with an HPLC, you typically have to know the composition of all the parts of your system. For instance, some systems might have some plastic parts made of acetal where others would have Kel-F parts (more resistant). Chloroform is certainly hard on some kinds of rubber. It's not too bad for most tubing materials.

Dear Mr. Wes Mcdermott,

Thanks for the link. However I am not sure the type of the tubing that I am using curently for my HPLC system :-P It seems like an ordinary plastic tubing. I guess I just have to contact the supplier (Agilent) and try to get the information from them :-) Thanks again for the link.

Most hplc systems use stainless steel or PEEK tubing at the high pressure end. PEEK tubing is color coded (dimensions), two suppliers being Upchurch and VICI, there should be more of them. The tubing at the low pressure end is easily replaced at a low cost as long as the outside dimensions match.

Chloroform is by far not a good choice for RP. However, in principle it can be used.

You should use chloroform in very less quantity

CHLOROFORM IS USED FOR ANALYSIS OF STEROIDS COMPOUND,YES YOU CAN USE IN C-18 COLUMN.

THERE ARE MANY EG. LIKE http://chromanik.co.jp/old/technicalnote100621.pdf