Chromatography Forum

Hello everyone,
Is there any harm of using a method which generate clearly broad (but symmetrical) peaks ?
The method uses 100 ul injection volume of a sample dissolved in Isooctane. Mobile phase is 70% ACN/water and the column is a C18 250x4.6 mm.

Thanks for any comment!

My first thought, without looking at a chromatogram, is that you're injecting a large volume in a very strong solvent. What are the retention times of your peaks?

Thanks for the quick reply!
I have two peaks for the analytes: one at about 6 min and the other at about 8 min (at the 100 ul injection). Both are about 1 min earlier than when injecting 10 ul.

the second one was much broader than the first one.

Is isooctane even miscible with ACN?

Thanks Tom,
Isooctane is NOT miscible with ACN nor with Water!
But the method was OK with 10 ul injections!

Here is a chromatogram




I hope it will help!

Couple things...

Agree with Tom, 100 ul injection is too much, standard injection volume is 20ul on a 4.6 ID...

I would suggest the following....

1) Have you tried increasing the flow rate? standard is 1ml/min for your ID, faster will be the easiest way to sharpen the peak...

2) Increase your organic, faster, rt, peak will be sharper than if it elutes later...

3) Try a smaller particle size, but biggest thing is decrease your injection volume...

Thanks....

Thanks a lot!

Sorry for the error in the photo.
here is the link
http://195.210.38.177:2222//dl/0245c6e2 ... dPeaks.jpg



The flow rate I am using is 1.5 ml/min.

I'd look to dilute your sample with a solvent that's miscible with your mobile phase, and only inject a small volume..

Not sure why you needed to increase injection volumn, but I'd try something like a 1:1 dilution of your sample in iC8 with iso propyl alcohol ( or some other miscible solvent ), and also inject less, maybe 10-20 ul max.

Please keep having fun,

Bruce Hamilton

Thanks Bruce!

I need to use the method for the analysis of very low levels of the analytes and use the large injection volume instead of concentrating extracts.

Do you think the problem is the immiscibility of the sample solvent?

What might be the practical problems in accepting such a peak shape?

If your analytes are not very volatile it´s a snap to get rid of iso-octane or at least reduce it´s volume to ~ 1/10.

When the sample is not miscible with the mobile phase, the chromatography can suffer. I have had good luck dissolving hydrophobic samples in ethyl acetate which is miscible with your mobile phase.

When the sample is not miscible with the mobile phase, the chromatography can suffer.

Thanks Mark!

I agree with you that the chromatography can suffer. In my case, I noticed a huge reduction in plate number (N) of about 90% but with almost no change in symmetry factor. This will clearly affect the resolution. However, resolution is not critical in my separation as the two main peaks are well separated from each other.

I don't usually inject immiscible solvents, but I suspect your peaks demonstrate the profile of large injections of immiscible solvents.

The broadness should decrease with injection volume, and issue may not be the resolution or peak shape, but inconsistent peak retention that varies with sample injection volume.

My other major concern would be that some iC8-soluble components of your sample may not be soluble in the mobile phase, which will cause additional problems, such as backpressure or column degradation.

Your integration parameters should cope with the peak shape, and the peak areas should still be the same, but....

It's like standing in the middle of the road, it's possible, but you're relying on other parameters to keep you safe. Far better to stand on the sidewalk.

I would use a smaller volumes of miscible solvent, eg concentrate your samples, as suggested by H.W.Mueller, unless that brings attendent problems - eg sample insolubility. If you can dissolve your sample in a different injection solvent, then ethyl acetate, as suggested by Mark is good, but if you have to use iC8 then concentrating and mixing with IPA or acetone may be preferable.

Please keep having fun,

Bruce Hamilton

Please keep having fun,

Thanks Bruce and ALL!

This is EXACTLY what I am doing now!

I am trying to see the effect of the immiscibility of the sample solvent on the analysis accuracy and precision.

As you mentioned above, the peak retention was inconsistent and I got linear reduction of retention times with the voulme (ul) injected, BUT .. the %RSD (5 injections) at any injected volume was 10%.

The Area was much more consistent with Only 0.6 % %RSD.

What do you think about the accracy and precision of such a method?