Minimize tailing of neutral or acidic compounds

[ahalya]

How do you minimize tailing for neutral or acidic compounds (small molecules; pharmaceuticals and excipients) when using reverse phase LC?

[Uwe Neue]

It is usually not a problem, unless the column is very old. Other causes could be that the injection solvent is too strong, or that the column is overloaded (too much is injected). In the first case, use more water in the sample solvent. In the second case, dilute the sample or inject less.

[ahalya]

Thanks for your input. I'll try and find a specific example and see if you can give me some input on it. I remember a case recently when we noticed a significant tail with a new column. We don't usually use very high solute concentrations so I do not believe overloading is an issue. I'll check into solvent strength.

[Steve]

ahalya

If those comments do not work. You can also look at the unswept dead volumn. 1) minimise the number of connections 2) ensure seal is tight
3) check whether all fittings are tight.

Hope you can solve it

[Chris Pohl]

ahalya,

One other point is that some reversed phase columns designed to give good symmetry with basic compounds actually contain anion exchange sites and will give tailing peaks for acidic solutes even when new. This is also true for at least one commercially available polar embedded reversed phase column. However, if the problem is present with neutral solutes, this (as mentioned earlier) can be indicative of extra-column dispersion issues due to a bad connection. One indication of this type of problem is a strong dependence of the tailing phenomenon on k’ with early eluting solutes tailing the most. If this is your situation, check to make sure all tubing ferules are properly seated so that there is no gap at the tubing end. If you are using PEEK tubing, check to make sure the tubes are cut squarely (PEEK tubing should be cut with a rotary tubing cutter, snapping the tube before the tubing cutter cuts all the way through the tubing will guarantee a square cut without closing off the tubing outlet hole).