Yes, you can use CH3CN or Methanol especially if your compounds are highly hydrophobe and will be retained for long time on C18 for exemple
As they told you before, you can use it without problems. In fact, we ususally use methanol 100% or acetonitrile 100% as mobile phases together with acidified aqueous mobile phases to extract polar and semi-polar compounds from different vegetal matrixes.
Yes you can use 100% organic as mobile phase but 100% aqueous mob phase not recomended as it may reduce coloumn life spam in case of RP-HPLC.
Yes, one can use organic mobile phases. For silica based columns, one can even use solvents such as dichloromethane or acetone, using an alcohol or acetonitrile as the polar solvent. This is called non-aqueous reverse phase. Note that you wouldn't use polymeric reverse phases for this technique as the polymer is likely to swell and cause high back pressure.
For silica, the reverse-phase material is chemically bonded to the silica and these solvents don't bother the stationary phase. IIRC, many analytical columns are shipped in 100% organic solvent. If a column is old, or has been stored with a solvent containing modifier ( such as formic acid, trifluoroacetic acid), the acid may have hydrolyzed some of the C18 from the silica substrate and this may wash off the column during the first use with non-aqueous reverse phase. This can be avoided by washing modifiers off the column prior to storage. Depending on your mixture, occasionally running organic solvents through a C18 column can clean the non-polar material trapped on a column.
See Snyder, Kirkland, Glajch Practical HPLC Method Development, 2nd edition. It works for prep scale too; see:
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
I always use a mixture of organic 100% mobile phase (Phase B) and acidified aqueous mobile phase (phase A) without any problem. It depends on the compounds that you want to characterize...
@ Jack Silver. Thanks sir, but can be get 100 % reproducible results. ?
@Ajay- the results are as reproducible as any other technique, in that your technique and equipment need to be working properly. I have no experience in testing this, but you may find reproducibility is improved by dedicating a column to this technique; I don't know how using a column as regular reverse phase may alter subsequent non-aqueous reverse phase runs as there may be traces of water left on the column.
Yes, you can use 100% of organic solvent but you need to consider if the column has endcapping or not, or if it's a simple cartridge. Anyway, consider the data provided by the manufacturer
As, you mentioned that your mode is reversed phase LC, then It is not recommended that you could use 100% Organic as mobile phase. Typically Carbon C4,C8, C18, C30 etc, used as stationary phase for RPHPLC which are chemically non -polar. 100% non-polar Organic solvent can deform your stationary phase's particle shape or size. It is always best use of a reasonable percent of water , buffer or other polar solvents as combined mobile phase with organic solvent for RPHPLC. Yes, you must see the various column properties which may be compatible for minimum organic solvent constrain properties in some cases.
for RP-HPLC water (or, a little acidified water in some cases) is essential as first solvent
In RP-HPLC, it is always better to use a combination of Organic solvent along with water or a suitable buffer solution. Then you can carry out a gradient elution so as to get a better separation of the compounds.
Normally we used combination of both organic solvent and water as mobile phase with the addition of formic acid to facilitate the ionization of the ions. In addition, gradient mode give better separation compare to isocratic mode. This is because the ion with different polarity will elute out according to the change of composition of solvent A and solvent B.
Normally we use combination of solvent and water RP HPLC. But I think so You can use 100% organic as mobile phase. Because we use 100% solvent like methanol and ACN for column washing so one can use 100% Organics phase. But resolution will good when one will use combination of both.
Reversed-phase high-performance liquid chromatography (RP-HPLC) involves the separation of molecules on the basis of hydrophobicity.solvent and water combination are used in RP HPLC. The separation depends on the hydrophobic binding of the solute molecule from the mobile phase to to the immobilized hydrophobic ligands attached to the stationary phase, i.e., the sorbent. 100% methanol or Acetonitrile (CH3CN), HPLC grade /chromatography grade/analytical grade can be used to run RP-HPLC,
Thank you very much, i had the same query once. yes it could be used 100 % organic based on compound character to be eluted
Combinations of acetonitrile or methanol with water provide a sufficient range of dipolar and hydrogen bonding interactions with solutes to separate a vast number of compounds by reversed-phase chromatography. The use of only organic solvent seems to make no sense. Also, as previously mentioned, organic solvent can deform your stationary phase's particle shape or size.
You also can affect the relative retention of your compounds in isocratic separation, by changing the column temperature.
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