I have built a method for a specific component.
The strange thing is that when I change the matris eg have the standard in n-hexane instead of methanol I recive two peeaks? Is this something that always happend in LC??

Can it be due to the fact that it is two similar compounds that is not separated in the first method? or to the fact that the compound is in two different configurations?

What is your injection volume and the diameter of your column? I assume this is a reverse-phase method.

Yes this is a reversed-phase method. The thing is that I inject 5um and the column is 2x150 mm Nova Pak C18 4um particle sise. And it seams that the consentration is not the important thing?
sometimes I wonder if it can be due to a small differense in the molecule? such as a dublelbound in one posission?

If the extra peak occurs when you use hexane as an injection solvent, I would say that that your analyhte is partitioning between hexane and the stationary phase and the hexane is moving a slug of it out ahead of the rest which partitions into the stationary phase.
Is there any concentration step involved before the analysis?

In general, you should try to avoid injecting a sample dissolved in something that's a stronger eluant than your mobile phase because of the liklihood of peak shape problems. Hexane is *extremely* strong in a reversed-phase system. The previous post hit the nail on the head.

If you're interested, there's a good discussion of this topic in the book "Troubleshooting LC Systems" by Dolan and Snyder. You can get it from Humana press (the publishers), from us at LC Resources (click on the sponsor link at left) or (probably) from Amazon.

-- Tom Jupille / LC Resources Inc.

Thanks, this will explain things that I have seen.

Regarding the question about consentration step, I guess that you think of the mobilephas consentration?
Yes I run an gradient starting with 80% MeOH and go to 90% MeOH (The other part is water)in ten minutes.

Regarding the advise to try to not inject a sample that is stronger than the mobile phase.

I use to extract my compounds in different solvents. This solvents is often stronger than my mobile phase ( eg toluene, hexane ) since I normaly use reversed phase. Is there any good ideas how to overcome this problem?

I think asking you about a concentration step simply meant "do you concentrate your extract?"
Regarding your last message, what about evaporating the organic solvent (if you have large volumes, say 10 ml or above, with a rotavapor; if you have smaller volumes, with a stream of nitrogen in a test tube) and redissolving the residue in the same volume of mobile phase (the one to use at the start of the gradient, to avoid the problems pointed out by Anonymous on 26.10.99 and T. Jupille)

I realise that this is a good practise. Unfortnatly time consuming But however I don't have this problem with all my sample.
I shall try to get some understanding for this knowledge.

Thanks again//Olof

If the difference in strength is not too extreme, limiting the injection volume can work (you have to try each specific case to see how much volume you can tolerate). The more general approach is to evaporate to dryness (as discussed above) and then redissolve in the mobile phase.

>I use to extract my compounds in different solvents. This solvents is often stronger than my mobile phase ( eg toluene, hexane ) since I normaly use reversed phase. Is there any good ideas how to overcome this problem?<

You can try to dilute your hexane extract with isopropanol(1:3) if you have enough sensitivity and inject 5uL (you can try 10uL if it works), since your initial methanol content is 80% and IPA is miscible with methanol and hexane.

Thanks for all advise, is it possible for hexan to leave the column? Hexane is not misscible in methanol and shold stay in the column. Shold I wash the column after a sertain injection with IPA to be rid of hexane in the column?