Can I use chloroform in the RP-HPLC sample preparation in the proportion of 50:50(chloroform:methanol)? Injection volume of sample is 10microliter. Will chloroform spoil the C18 column?
Dear Robin, can you please explain the mechanism of how the chloroform harms the RP column? Thanks =)
As a general practice one should never inject the sample made up in an "eluent" more potent or impact than your mobile phase. Chloroform is 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). 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-ACN 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.
Chloroform is a good organic solvent for sample extraction( In one of my colleague's studies :for extraction of pharmaceutical drugs from plasma the recovery was more than 80% with chloroform), Even if it affects the column it will not be prominent as we used to inject our sample after drying the solvent extract and reconstitution with suitable solvent (It could be mobile phase).
Part of my concerns are already been addressed by Mr Nazmul. Chloroform is not normally injected into the column. But technically, chloroform is just slightly less polar than ACN and MeOH. Low polarity solvent is less likely to harm RP column. With due respect, I still can't figure out how chloroform can damage the column. We not using chloroform as a solvent so often just because of practicality issue, as everybody knows, chloroform is hazardous to health.
@Mr Jagdish, can you please tell us, what is your extraction method in details, and what is your mobile phase composition?
Dear friends...thanks for the suggestion. @ Yung im using 10 ml chloroform followed with sonication for 30 minute & finally makeup the volume to 20ml with methanol.
mobile phase is methanol:THF(90:10)
Dr Jagdish, Robin was truth for suggesting you not to use a 'potent eluent' (strong solvent) as a sample solvent. However, the main reason is not because it can spoil your column, rather, it is because a sample solvent that stronger than the mobile phase will produce a bad peak shape. Since you are using THF (which is also a very strong solvent) as a part of mobile phase and your injection volume is not too large, theoretically, the adverse effect of 'potent eluent' will not be too prominent.
However, will it be possible for you to use other weaker solvent as your sample eluent (eg: 100% MeOH)? You also can try to use mobile phase itself as your sample eluent.
Peace.
Initially for one or two injection chloroform will not harms your column but as the time passed chloroform will gradually solubilises your column stationary phase (as it made of ODS or other material which are more soluble in chloroform) hence it will lead to deterioration of your column and will stop working after approx 100 injections
what about the fact that chloroform is not miscible with water. If you use a MeOH: H2O mobile phase with a C18 column will this be a problem?
If you use CHCl3:MeOH 1:1 as mobile phase, i think it will harmful for you channel (cause it made with some unresistable material), and also effect the coulumn pressure. If you use it as sample solution with small amount like you said 10micromiller, then it is ok
- Badges
- Science topic