Chromatography Forum

i would like to know why in some gradients,in the m.phase, there is a 5 to 15% isopropanol stable percentage(except the aquas and the ACN/MEOH(usually) portions whose percentage is changing).In addition i would like to ask if large amounts of isopropanol could damage a column.
thanks,

i would like to know why in some gradients,in the m.phase, there is a 5 to 15% isopropanol stable percentage(except the aquas and the ACN/MEOH(usually) portions whose percentage is changing).In addition i would like to ask if large amounts of isopropanol could damage a column.
thanks,

*those such gradients are designed to selectively elute analytes that too hydrophillic to be separated using an iso-m.p.

Adding small amounts of a third component such as isopropanol or THF can give you considerable change in selectivity compared to aqueous/ACN or aqueous/MeOH alone.
Personally, I'd use ternary mixtures only if I absolutely don't get the needed selectivity with binary mixtures. I'm a strong advocate of the KISS principle - keep it simple and stupid. There are already numerous ways to spoil the preparation of a binary mobile phase .

Concerning the use of isopropanol, a standard silica-based column should not have any problems with it, you may even flush such a column with pure isopropanol. Polar columns such as cyano or diol that can be used in reversed-phase and normal-phase mode are usually flushed with pure isopropanol as transition eluent between aqueous and non-aqueous eluents.

Isopropanol is also used in high amounts in the packing solvent to pack RP materials into columns, so it should be not a Problem.

As a matter of fact, today on of my co-workers used a column (stored in hexane but he didn't know) for RP chromatography(H2O/ACN).The analysis was a mess. What happen in a column when NP and RP phases comes together could you tell me?

First of all, what on *earth* would lead someone to store a reversed-phase column in hexane??!!

Hexane is not miscible with ACN/water. This is a situation where IPA can be very useful as a co-solvent: it is miscible with both hexane and with water. If you want to switch from a reversed-phase to a normal-phase system, an extensive rinse with IPA is the way to go.

To pump Hexane on a RP column is one way to kill the packing material bed in seconds. What was the back pressure?
Take the next RP column. The same if he used a NP column, stored in Hexane, and pump ACN/Water on it, it will kill the packing bed.

It wasn't a RP column exactly.It was a silica column(lichrospher Si 60 i think)that was used for NP. But anyway i don't understand why it's such a disaster to be present in a column or tubing together water and hexane. I know it is, but the reason isn't that they are not miscible in each other.What happens? could you help me?

If your colleague was going to use ACN/water on a silica column, it would *not* be reversed-phase chromatography, it would be HILIC (Hydrophilic Interaction Chromatogaphy), which is a form of normal phase. Different mechanism, different elution order, different sorts of compounds.

As to why you don't want to use immiscible solvents: that should be obvious: there is a high probability that droplets of one solvent will "hang up" and come out eventually to cause baseline upsets.

If your AcN/water mixture did not contain any salt I am quite sure you can regenerate the column with IPA.
The standard Merck wash protocol for doing the switch from Hexane to IPA is
Hexane or heptane
Chloroform
IPA
30 column volumes each

But I am sure just flushing the column with IPA (30 column volumes) will clean out the hexane and then you can do your HILIC analysis.
If there was salt in the eluent that will have precipitated and may have blocked frits and what not, to try to fix that wash the column with water after the IPA, but cleaning out salt packed into pores at 200 bar or what your pressure went to may not be possible.

However a 60 Å neat silica will not be the ideal HILIC column, unless you just want to try something I suggest you buy a dedicated HILIC column (says the guy selling them

First of all, what on *earth* would lead someone to store a reversed-phase column in hexane??!!

Hexane is not miscible with ACN/water. This is a situation where IPA can be very useful as a co-solvent: it is miscible with both hexane and with water. If you want to switch from a reversed-phase to a normal-phase system, an extensive rinse with IPA is the way to go.

I've had columns come back with plastic "finger-tight" fittings used as "end plugs." Nothing surprises me anymore

so if i understand well, there is no disaster for the system, the tubing, the valves, the seals if by mistake our m.phase is ACN/H2O/HEXANE (no salts)? The problem will be with the column, right? Of course we"ll have to flush with isopropanol etc. when we find out, but no harm done ,is that right?