determine the best isocratic separation for a set of four analyte compounds.
your four compounds, and the organic solvent (B) that you are to use.
Using the HPLC simulator, determine what the best mobile phase composition is for the separation of your compounds with your given solvent.
Keep in mind that the best separation is the fastest separation with all the compounds being resolved*.
For this lab you will need to report three items:
- The %B for the mobile phase to get your best separation
- The pair of compounds that elute the closest to each other*
- The resolution for those two compounds
To calculate the resolution you can use the data in the table beneath the chromatograms produced in the simulation. The resolution equation is R = (tR2 tR1)/(0.5*(w1 + w2) and a minimum resolution of 1.5 is needed to have properly resolved compounds. tR1 is the first (fastest) eluting of the two compounds, and w is the width of the base of the respective peak.
The table below the chromatogram provides you with the tR values (in minutes), but you need to calculate w, fortunately that is easy to do, simply multiply the ? value by 4 (w = 4? ). When calculating R you need to have the units of time for tR and w the same. Since ? is in seconds, multiply tR by 60 to convert it to seconds as well.
*Note that for some of you it may not be possible to get a perfect separation, you may have a pair of compounds that coelute under all conditions. If that is the case find the best possible resolution between that pair of compounds and report that resolution.
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