Isocratic separation with HPLC Teaching Assistant, an Excel spreadsheet that simulates a reversed-phase liquid chromatographic separation. Download the Excel file from SaplingPlus, macmillanlearning.com, or the link in reference 8. When you open the file, you should see a menu page with Isocratic mode at the top right. If not, click the blue home button in the top left corner. Click on Isocratic mode for this problem. Adjust the image size so that you see the chromatogram with five peaks.

a. The red line labeled t0% is retention time for an unretained compound, which we call tM t_M Record this time. Tip: Excel shows x-^x- and у-^y-values for a peak maximum in the chromatogram when you position your cursor over the top of the peak. Use the slide bar at the top left labeled “%MeOH %MeOH” to vary the methanol content in the mobile phase. The other component of the mobile phase is water. Vary the methanol content. Is methanol the strong or weak mobile phase component in reversed-phase chromatography? Does any peak ever elute before tM t_M?

b. Adjust methanol content in the mobile phase to 70%.70%. Record the retention time of the last eluting peak. What is the retention factor of the last peak?

c. Record the retention time of the last peak at 60, 50, and 40%40% methanol. Calculate the retention factor at each percent methanol. By what factor does the retention factor increase for each 10% 10% decrease in methanol?

d. Plot a graph of log V for the last peak versus percent methanol. Predict the retention time for the last peak at 45% 45% methanol.

e. Adjust mobile phase to 30% 30% methanol. How many peaks are observed? Why are some peaks missing?

f. What percent methanol minimizes the separation time while maintaining resolution ≥1.5 ≥1.5? For ruggedness, a resolution of 2 is desirable so that the resolution remains adequate if there are small changes in conditions or slow deterioration of the column. What percent methanol would provide the most rugged method?