Chromatography Forum

I've been having this debate with colleague about the optimum peak height for all HPLC UV peaks. He says that they must all be close to (or more than) 800mAu. Were-as I perfer small peaks with about 100mAu height. Baring in mind that the signal to noise ratio is large, in the 100:1. Is there any preferable technique? What are the pros and cons of large/small peaks, if any?
Thanks, Sloane

At the high end you'll be limited by non-linearity (either stray light or chromatographic overload). Stray light shouldn't be an issue below 1 AU, so if you have no evidence for chromatographic overload (no tailing, calibration plot is still linear), there should be no problem with an 800 mAU peak, but you only have a safety margin of 2X or so on the high end

At the low end, you'll be limited by S/N. A good rule of thumb is that the CV (%RSD) is approximately equal to 1/50 times the S/N* (in other words, a S/N of 100 should provide about 0.5% RSD). If you're seeing baseline noise around 10^-4 AU, then there should be no problem with a 100mAU peak, but you only have a safety margin of 2X or so on the low end.

Pick your poison.

* if anyone's interested, the rationale is described on page 71 of the book Practical HPLC Method Development, by Snyder, Glajch, and Kirkland

1. The height of the largest peaks is usually determined by the signal that you get from the smallest peaks.

2. Injecting a lot of material may overload the column and may make the peaks somewhat wider. This is why I always use the rule of thumb to let no peak get larger than 0.1 AUFS (unless something forces me to do so, e.g. see rule number 1).

why are you using peak height?

I remember having seen in John Dolan's column in LC/GC Europe some discussion on this not too long ago (about 6 months perhaps). I will try and look for the article over the weekend.

Bill, in response to your question I do use area to perform my quantitation. This is really to help settle an arguement. My line of thinking is that having a peak which is around 100mAu high will still hold up to a validation, and will (in fact) perform better.

As long as your s/n, CV, and linearity are suitable for what you want to do, I see no problem with quantifying leaks that are either 100 mAU or 800 mAU. Personally, I try to run about midway between the two so that I have high s/n and can easily calibrate up to 2x of my target (at 400 mAU) and beyond with some impunity. If your coworker can prove his detector is linear to 2 AU and he hasn't overloaded his column, then his method is fine. If your s/n is great at 100 mAU, you're OK, too. I see no real argument here.

CJ

Kolthoff once said "theory guides, experiment decides". Well actually, he said it every time the occasion called for this wisdom. the way to settle this argument is to do the experiment, and please report back so we all can benefit. I am going to place my bet on the higher absorbance giving better precision.

PS there are occasions when peak height will give better results than area.