Chromatography Forum

Hello everyone, looking for some input to tighten up a peak here.

Current method:

A: 1% aq TFA
B: 10% A, 90% ACN

Isocratic 60% B, 0.8 ml/min flow

Column temperature 40%

Our system is the Shimadzu Prominence UFLC, using an Ascentis C18 column. Our data system is Empower 2.

Current peak:



Any ideas?

My 1st impression is that the peak looks a bit "fat" i.e. not very efficient.

Could you supply some more info please:

1) Column dimensions
2) Injection volume (and sample diluent)
3) Amount / concentration of analyte

Why don't you just have equal amnounts of TFA in A & B?
i.e. Make MP A simply water + 0.1% TFA and MP B simply AcN + 0.1% B

You might need more retention to get better peak shape - try lowering %B and see how peak shape looks - but 1st supply info in 1,2,3 above for us to answer better for you.

Hey Rob, thanks for the fast reply, it's much appreciated.

Answers to your questions:

1) 3mmx100mm, 2.7 nm pore
2) 16 μL
3) 1 mg/mL, diluted with A

We're going to try altering the solvent proportions first. Do you think 55/45 or 50/50 would be better to try first?

I'm not an expert on these columns - but it could be that you are possibly overloading this column.

First increase your retention. Try varying % B between 30 to 70% to see what you get. If you had a copy of DryLab you could just do the extremes and model in between (or plot it manually).

Second try a injection volume study say 1, 2, 5, 10 and 16 - see how that looks for peak shape.

2.7 nm pores? You mean 27 Ã…? If this wasn't a misprint, then I'd suspect steric hindrance, even for a solute this small. Try a wider pore material.

Andy Alpert

I'm not an expert on these columns - but it could be that you are possibly overloading this column.

First increase your retention. Try varying % B between 30 to 70% to see what you get. If you had a copy of DryLab you could just do the extremes and model in between (or plot it manually).

Second try a injection volume study say 1, 2, 5, 10 and 16 - see how that looks for peak shape.

That's where I'd start, look at the Absorbance scale, over 1000 mAU.

I believe the poster meant to say 2.7 micrometers, NOT nanometers.

Are you sure you have a problem here. A 0.2 min peak width does not seem bad to me at all.

It may be the scale. Usually the time axis would go to, for example, 30 minutes. And on this scale the peak would appear much sharper.

Are you sure you have a problem here. A 0.2 min peak width does not seem bad to me at all.

By "calibrated eyeball", it looks like the baseline width is about 0.15 minutes, with a retention time of 1.5 minutes. That means about 1600 plates, which is abnormally low.

I'd go along with the "overload" hypothesis, although I suspect that it may be more detector- than column-related (1.6 AU is a *lot*!). If it were my problem, I'd take a short-cut version of CPG's suggestion by injecting 2 microliters to see what happens.

There is very little retention, and the column is either over loaded or there is too much extra volume in the system.
Below is data for nicotinamide on Unison UK-C18 (which has more electrostatic interaction and can retain the more polar solutes)

LC-UV: http://www.imtaktusa.com/site_media/fil ... TI268E.pdf
LC-ELSD: http://www.imtaktusa.com/site_media/fil ... TI227E.pdf
LC-MS: http://www.imtaktusa.com/site_media/fil ... TI312E.pdf