Is chloroform an appropriate sample solvent for reverse phase HPLC? Will it be harmful to C18 column? The mobile phase is a mixture of acetonitrile and water. Thanks in advance.

It's not going to be harmfull, but you will get ugly peaks if you inject too much.

As mentioned above, use of chloroform per se will cause no harm to your column. However, as a general practice one should never inject the sample made up in an "eluent" more potent than your mobile phase. Chloroform is by definition considerably more potent than 100% acetonitrile so you can expect problems unless: your injection volume is very small or you are using a gradient method (which can refocus your analyte zone in spite of the adverse effects of the chloroform). Frequently, I hear people say that "solvent X is the only solvent I can dissolve the sample in so I have to use this solvent". Generally, this impression stems from the method of sample preparation. At high concentrations, you may well need chloroform to dissolve your sample or your sample matrix components but since you are able to elute these analytes with a water-acetonitrile mixture, this means you're analytes are in fact soluble in water-acetonitrile mixtures. A good way to avoid this problem is to first dissolve the sample in a good solvent such as chloroform and then dilute it in a solvent mixture weaker than the mobile phase (i.e. with less acetonitrile than your initial starting eluent). This is usually sufficient to allow you to produce a sample solution which doesn't interfere with good chromatography.

We frequently use stronger organic solvents (methanol or DMF) as solvents because not only are the analytes soluble in them, but undesired stuff is kept insoluble, and filtered off. The key here, as A. Mouse indicated, is to keep the injection volume relatively small if the mobile phase is "weaker" than the sample solvent. Typically we inject 5 ul using a 2.1 mm i.d. column and 10 ul when using 4.6 mm i.d. columns.

Anon,

We never use Chloroform as solvent in RP chromatography.

My drug is soluble in acetonitrile; DMSO; n,n DMF; dichloromethane and in methanol(methanol in larger qty than the other). Which is the best solvent I should use?

May I know if a sample dissolved in chloroform can be directly injected into ESI-MS (without the LC separation)? What are the solvents compatible with the MS?

To Anonymous April 16:

Assuming that you are using reversed-phase chromatography, MeCN, DMSO and DMF are all usefull solvents, provided your injection volume is small. However, in most cases, the sensitivity of the detection is so large, that the solubility of the sample in a solvent is not an important factor. To avoid strange effects on the peak shape caused by the strong solvent, one usually prefers to dissolve the sample in the mobile phase, if isocratic, or in the starting composition of the gradient, if a gradient is used.

Uwe Neue: what if the mobile phase is a mixture of organic solvent and a buffer and the drug being insoluble in water should we dissolve the drug in the organic solvent separately and then make up with the mobile phase or directly dissolve in the mobile phase. you didn't comment on ACN. can we dissolve the sample in ACN

My abbreviation for ACN was MeCN.

I would try to make up the sample directly in the mobile phase. I think it is simpler than making it up in the organic solvent first and then diluting it. More error possible in the second case.