Chromatography Forum

Hi everybody

What is the minimum resolution that has to be between an internal standard and an analyte?

Thanks

Check your method.
If you have no specification, anything >1.5 is gravy (or a waste of perfectly good mobile phase).

Thanks DR

I am actually developing the method and I got a minimum resolution of 1.5 based on using the half peak width equation

1.18*(Rt2-Rt1)/(W1+W2)

However, I noticed clearly that the valley between the two peaks is not touching the drawn baseline.

I am worry because I think small variation in temperature or %B will affect the separation and, therefore, the quantification.

What do you think the reasons for not seeing the baseline separation?

MDYO,

A value of R= 1.5 is what thye textbooks recommend. However, this is the "minimum" that is acceptable, you would be better off with 2.0, and your "waste" or mobile phase will be low. Values between 2 and 3 would still be good, but then elution times and mobile phase waste may be considerably more than necessary.

If you measure 1.5 and still dont see baseline resolution perhaps your system is not measuring resolution well enough, or, if you are doing it manually, there is too much error in it. In most cases column efficiency or inadequate mobile phase conditions can be the reazon por these observations.

Try to get 2.0, experiment with temperature and mobile phase composition, try a more efficient column (larger number of theoretical plates). A value like 2.0 will give a more robust separation and better results overall.

Good Luck,

josebenjamin

mdyo,

you said you do not see a baseline separation,
what would be your resoluton according to USP?
what is the tailing that you get from both peaks?
you can easily improve resolution if you can improve your peak shapes.
also a peak with a tail bigger then 1.5 is not accurately quantified. after a tail of 2 your deviation in area integration can cause an error of more then 2% on your calculations. do take that as well into account in your method.

Greetings,

We work with an environmental method, EPA 531.1 (carbamate pesticides in water), where this is an issue. The method requires that the resolution between one of the analytes, methiocarb, and the IS be checked before samples are run. The minimum is R > 1.0, and this is with a baseline calculation of resolution.

The method is a C18 reversed phase analysis, with post-column reaction fluorescence detection.

Evan L. Cooper, Ph.D.

A few comments:

1. Rs > 1.5 was originally called "99% baseline resolution". At Rs = 1.5 there is just under 1% mutual overlap between two equal-size Gaussian peaks.

2. If you have perfectly Gaussian peaks, you will get the same Rs values whether you use baseline width or width-at-half-height as the basis for measurement (with appropriate fudge factors, of course). With tailing peaks, the Rs value from width-at-half-height is generally somewhat greater than the value calculated from baseline width.

3. As has been pointed out, if your peaks tail, these calculated resolution values will underestimate the degree of separation between the peaks. This is true regardless of where you measure the width.

4. FDA and ICH recommendations for resolution are Rs > 2.0